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} | s2 | WATER USE CRITERIA FOR BOATING A CASE STUDY OF WATER-LAND USE CONFLICTS IN GALILEE, RHODE ISLAND
The area of contact between land and water often results in land use conflict, especially between commercial users of land adjacent to recreational users of water, causing disorders of various types. Much inconvenience results in terms of efficiency for commercial and rec-reational users. Physical criteria were established and found applicable to promote maximum efficient utilization of water areas in terms of boat maneuvering and berthing, waterfront safety, and to aid in predicting desirability of land use development. A counterpart to this thesis, A Study of Land Resources Used For ' Boating in Galilee, Rhode Island, has been written by Mr. Donald Sikorski (a classmate). This study, when applied in conjunction with criteria developed in this volume, will reduce conflicts existing between land and water by promoting sound land development methods in relation to efficient water resource use. Research was conducted at the library of the University of Rhode Island and the Graduate School of Design at Harvard University. Letters of inquiry were sent .to agencies in the United States concerned with water resource development and recreational boating. Interviews were conducted with personnel of the Rhode Island Department of Natural Resources, ii
INTRODUCTION Statement 2f the Problem
The zone of contact between land and water offers many areas of study in which complex problems arise. This thesis gives particular attention to boating needs. The problem results from the fact that there are many types of boats and an increasing demand for recreational boating and facilities, and that this demand has resulted in conflicts in waterfront planning. Therefore, the purpose of this thesis is to demonstrate by using Galilee, Rhode Island as the study area that demand and conflicts exist and that criteria can be developed, which if applied, would reduce these conflicts.
Justification of the Problem
The problem is selected for its timeliness and importance to waterfront planning where water-land relationships have been unplanned or haphazardly arranged. Incompatible uses and facilities, and increasing demands for shoreline boating have resulted, in many instances, in inefficient waterfront development. Therefore, it is necessary to establish criteria which will reduce the conflicts of the water-land relationship for boating. To achieve this goal the problem area, Galilee, Rhode Island, serves as an excellent study area because of the marine-land problems which are an outgrowth of haphazard development in mixed land-water uses.
The primary limitations of this study were a lack. of available boating statistics. State officials have said they are in the process of gathering such data and it will be available in the future. The needs in this case were the number, type and size of boats using Galilee, Rhode Island during the summer boating season. Also the write~ found material on water resource planning somewhat limited.
Present Status of the Problem
There have been few contributors to the problem investigated.
The National Association of Engine and Boat Manufacturers, the Outboard Boating Club of America, the American Society of Civil Engineers, and the United States Army Corps of Engineers are the primary contributors to water resource planning. Very little data has been developed by the planning profession, as emphasis is mainly on land use planning.
Organization of the Thesis
The arrangement of the thesis follows. Chapter II, "Inventory of the Problem Area, 11 explains the location and boundaries of the study area; land ownership; existing water and shoreline use analysis; utilities and facilities surveys; and circulation and accessibility.
Chapter III, "Marine Resources Criteria, 11 covers existing facilities inventoried for boating; water area used for boating; necessary marine facilities required for boating; specific marine problems related to boating; and criteria for efficient marine uses involving the type of boating facilities, extent of boating facilities, arrangement of boating facilities, and water-area requirements .
Chapter IV, "Land Resource Criteria, fl is a summary of the land resource criteria established in the counterpart to this thesis entitled flA Study of Land Resources Used for Boating in Galilee, Rhode Island. fl Chapter V, "Application of Criteria to Galilee , Rhode Island, 11 applies the criteria developed in Chapter III and includes schematic designs of recommended water and land use.
Chapter VI is an overall conclusion and summary of the thesis.
Sources of Data and Method of Procedure
Two hundred six letters of inquiry were sent to agencies and individuals primarily in the United States connected with boating and water resources for information related to this thesis. A list of respondents to these letters of inquiry is appended.
Extensive library research was carried out at the University of Rhode Island and Harvard University . Additional material was received from other libraries through the University of Rhode Island interlibrary loan program.
Interviews were conducted with both commercial and sports fishermen, merchants in the Galilee area, Rhode Island Department ·1 of Natural Resources personnel and property owners in the vicinity of Galilee, Rhode Island. Observations were made of marinas and boat launching areas. A forty minute color film entitled "Marinas" produced by the National Association of Boat and Engine Manufacturers was also obtained.
The case study method was used because it describes an existing situation--the water resource problems in Galilee, Rhode Island.
These problems are defined and suggestions are made that will enable the planner to correct them and similar problems elsewhere. because of a lack of funds, has been unable to finance the needs in Galilee. One alternative suggested by fishermen is to establish a 'Port Commission which will have the authority to issue revenue bonds backed by the state to finance development in Galilee. It is understandable that persons living many miles from Galilee unaware of its problems are somewhat hesitant to have their elected representatives vote funds for Galilee when financial needs are so great throughout all of Rhode Island.
Existing Water and Shoreline Uses
Within the study area lies a diversity of land uses. Residential uses tend to be congregative on a small, congested, privately owned parcel of land in the southern portion of Galilee. The commercial uses are scattered throughout the area in both inland and waterfront locations. There are no marine railways in Galilee itself, but this facility does exist on the opposite shore in Jerusalem, Rhode Island. It is not absolutely necessary for such a facility to be located at Galilee as long as there are others in the near area. At present the facilities in Jerusalem are sufficient and are only used when boats need repairs or painting.
Breakwaters or baffles to protect small craft from natural waves or large boat wakes do not exist within th~ Galilee water area. This is probably due to the fact that a small boat area is really not developed.
For all practical purposes, one could say such an area does not exist.
A body of water for small craft does exist, but because no proper facilities are in the area, the writer cannot define it as an accepted small craft area, but only as an unutilized area.
One can purchase fuel in Galilee, but first he has to find it. Existing fuel facilities do not have easy accessibility, are not located at the end of a pier, and would require the patron to seek out the seller at the J. L. Shellfish Company next to the ferry landing, where the only facility is located. No facilities exist to remove sewage from boats at Galilee. The need for such facilities is becoming particularly urgent as the number of craft using the Galilee area continues to increase.
As can be seen, Galilee is deficient in many facilities. T hi s neglect of one of Rhode Island's natural resources necessitates urgent action by the state of Rhode Island and the local municipality.
Water Area Used for Boating
As commercial and pleasure boats gradually increased in number, size and importance, so the need for more spacious accommodation became more pressing and the demand for larger and better harbors more imperative. 13 Such a need for accommodations applies to Galilee, Rhode Island as well as the nation as a whole.
Water resource areas such as Galilee should fulfill the following four main requirements: (1) Provide a tract of tranquil water for protection against wind and waves (2) Permit quick dispatch of the commercial fleet --high point where dry repair or transfer operations can be conducted.
The railway is equipped with a cradle or car on wheels operated by a 20 cable from an engine or motor located near the upper end.
28
Marire railways are as necessary to boats as parking and service areas for automobiles. In time of hurricanes a properly located railway can remove a boat from water to the safety of land. On the other hand, . in the case of a land fire, dry docked boats can be placed in the water for protection. Marine railways are particularly necessary in an area such as Galilee, for the commercial fishing fleet frequently requires repairs that can only be accommodated by such a facility.
The limited depths in the Galilee water area, which is a part of Point Judith Pond, plus the increase in size of the average commercial fishing vessel has kept the boatyards on Point Judith Pond from increasing their marine railways to handle the larger craft. These larger craft are forced to go to Jamestown, Newp~rt, Wickford, and Stonington to be handled and serviced. 2 1 20 Ibid. , p. 14 7.
South Kingstown Waterfront Resources Committee, Point Judith
Harbor--Proposed Navigation Improvements and Hurricane Protection (Rhode Island: South Kingstown Waterfront Resources Committee, 1968), p. 16.
Launching Ramps. The construction of launching ramps aids in eliminating the indiscriminate launching of pleasure boats from adjacent highways. 22 Launching ramps are primarily access sites where recreational boaters can place their trailered boats in the water. Adaqua.cy of launching ramps can become very critical in times of a storm. In such a case inadequacy would find many recreational boaters waiting in rain and possibly rough water to remove their craft.
All launching ramps should be located in such a manner that once the boat is in the water facilities such as fueling, tackle, bait, etcetera should be conveniently located so that the boat operator can proceed from the launching ramp to the dock where he may tie up for a short
FIGURE IV
LAUNCHING RAMP WITH DOCK Breakwaters. The waves have various effects upon boats and these effects can only be described as detrimental, for example, causing the craft to break away from their mooring and drift upon other boats. It is necessary to reduce waves to such small amplitude in small craft harbors that the wave energy transmitted through the breakwater will not form waves that will be damaging. Galilee leaves much to be desired in providing fuel, water and electricity . Improvements have been made and are continuing to be made, but maximum convenience to boaters is lacking. The available pier facilities are constructed on a common design without regard for boat size.
Patrol Boat. A water patrol unit is the effective instrument for enforcing rules and regulations on a water area heavily used by recreational craft. The patrol unit is necessary to protect both life and property and may be used for emergency first-aid. The patrol craft is usually manned by a regular law enforcement officer or constable. Functions of a water resource area may be a storm or emergency haven, a convenience harbor such as one used for overnight stays, or temporary tie-ups commonly referred to as transients, a commercial fishing boat moorage, or a recreation center for pleasure boats for seasonal use. Few water recreation areas will be exclusively any of the aforementioned categories. Most will involve some combination of these functions. In any case the planner, when starting his work, should establish this water resource area function quite definitely and develop his plan so that the functional purpose can best be accomplished.31 In the case of Galilee, Rhode Island the aforementioned categories are all clearly applicable to this area, as shown in A Survey to Determine To be successful in its purpose the location must suit a demand.
As an example, it would usually be in error to locate a deep-sea commercial fishing harbor twenty miles up a narrow river because of time lost traveling to the fishing grounds. On the other hand, that might be an ideal location for a commercial venture or a recreational accessibility from a larger body of water would be most desirable.
Three major factors which have a bearing on access to boating installations from the water are the depth and fluctuation of the water level, existing or proposed bridges, and proximity to open water. A combination of circumstances may occasionally cause extremely high or low tides on tidal waters, but this is usually within the knowledge of local residents. Tide tables and water charts are extremely accurate devices for determining water depth and tide fluctuations. Tides may lower the water level so that deep draft boats cannot use the facilities, but these difficulties can be overcome through proper design.
33 Water areas must be accessible to its users by land. For example, fuel facilities would be located where sufficient water depth for boats existed at both low and high tides. The shallows of a water area are often a zone in which many aquatic activities are concentrated. When activities such as boating and water skiing with high space consumption transgress on activities with low space consumption such as swimming, or scuba diving zoning for certain uses is justified. 46 Water-born wastes have been recognized as one of the major limitations of water recreation areas and can be a hazard to health.
In addition many accidents have occured because of persons swimming where boats were passing by. Accidents could also occur when pleas- The boating uses in Galilee, Rhode Island are c ommercial fishing, ferry service to Block Island, sports and charter fishing, and recreational or pleasure boating. These boating types result in many problems .
The fishing industry is quickly becoming outmoded by change i.e . its position as a competitive market will remain dubius unless expansion and planning occurs in the near future .
The ferry transportation service lacks a good location and area for automobile parking and boarding . The charter fishing boats are a magnet for many enthusiastic fishermen who come to Galilee every summer; however, their parking areas are insufficient in size . Also there appears to be a lack of adequate berthing facilities which are desirable for these enthusiasts. 1. Stores should be relatively close to berthing areas to insure easy access for boaters.
2.
A sufficient amount of land should be retained between the retail establishments and slips so that access to boats is not impeded, spectator areas are provided, and landscaping is planted.
3. Preferably the establishments should be constructed on relatively level land to reduce the cost of construction and facilitate easy access.
4. The center should be in such a location that it is easily accessible from the main entrance to the facility and other parts of the site.
5. The establishments should be visable and readily accessible from the highway.
6. Stores should be grouped according to selling power, visual access, requirements, and service needs.
57
The land area needed for facilities has been established and tested by Mr. Charles Chaney. When many activities exist such as in larger marina operations with boat handling areas, sales rooms. restaurants, and the like, one and one-fourth to one and three-fourths acres of land are needed for each acre of water. However, for the smaller facility, which basically receives fishing traffic and trailered boats, one acre of land is needed for each acre of water.
The needed facilities are dictated to a large degree by the demands placed upon existing facilities. However, in all probability commercial establishments will be constructed as more people enjoy visiting such water areas to partake in and view the boating activities. Therefore, it is important to establish districts where commercial activities will result in efficient utilization of the existing land area.
ALL LAND USES ALONG THE WATER SHOULD BE ARRANGED TO MAKE THE BEST USE OF THE SUR-FACE WATER.
Industrial uses of waterfront locations are for four reasons: 1. Availability of raw water for manufacturing or processing purposes.
2, Need for disposal of wastes created in the manufacturing process.
3. Convenience in the production and maintenance of water-related products such as fishing equipment.
4. Capitalization of various resources from the sea as in commercial fishing .
In Galilee the utilization of land for these purposes is focused primarily upon the commercial fishing industry for unloading an ex- It is necessary to provide boaters with a parking area close to the docks so that gear may be transferred easily from automobile to boat and vice-versa. The trailer parking area needs about 600 square feet of maneuvering and parking space for each vehicle with trailer and a design that permits the vehicles to move efficiently from ramp to parking space. Parking requirements to be considered are: 1. One space for each transient boat .
2. One and one-half spaces for permanently moored boats .
3. One space for each three spaces on a sightseeing or party fishing boat.
4 . One space for every two employees working in a fish processing plant. The Galilee water area is primarily a commercial fishing port.
Boatyards and suppliers catering to the commercial fishermen are active year-round industries. The area includes extensive, although not ideal, facilities for commercial fishing, sport fishing, recreational boating, and swimming. The principal year-round activity is commercial fishing which includes the fish dehydrating plant and the fisherman 1 s cooperative which processes and exports edible fish from Rhode Island.
Not only is Galilee a commercial fishing port, but the area is a center of pleasure boating and sport fishing for Rhode Island and nearby Connecticut and Massachusetts. Approximately 150 large sport fishing boats come to Galilee each year for the annual Atlantic Tuna Tournament.
A large number of charter boats operate from Galilee primarily for recreational fishing parties and cruises.
It is obvious that Galilee functions as both a commercial fishing port and a center for recreational boating; and due to its location on the 62 ocean, Galilee is both a convenience harbor and an emergency haven.
The growth of recreational boating and commercial fishing activities in Galilee suggest the need that Galilee be developed as both a commercial and a recreational area . The area is undergoing rapid deterioration and has suffered from poor planning as will be shown in the following pages. Island boat count, as sailboat owners are not required to register their boats if without motors, the writer feels that state officials are aware of these problems and are attempting to gather more accurate data.
Site selection for facilities in Galilee must be accomplished without recreational boat statistics. Fortunately, the commercial boat information is available. It is the writer's contention that commercial facilities should be planned in accordance with known statistics, and that The preceeding boat types and percentages could be of some aid in determining space requirements by giving the planner some insight as to the type of boats for which he must provide facilities. Table I on page 24 indicates water space requirements for pleasure boats. Table II Existing water space utilization at Galilee is inefficient, but efficient utilization of such space is possible. The schematic design on page 79 is meant only as a guide for suggested water use areas by boat types based on all the criteria contained in this thesis.
IN ACCOMMODATING TRAILERED BOATS ONE LA UNCH LANE SHOULD BE PROVIDED FOR EACH FORTY BOATS USING THE FACILITY
Galilee presently has one launch lane. No official statistics are available on its use, but the writer interviewed merchants in the area and the general consensus was that from 40 to 60 boats used the facility during summer days. On this basis it is suggested that Galilee have two boat ramps to accommodate the recreational boater during the summer season. While two ramps are somewhat more than is presently needed, one ramp is not sufficient as boaters frequently have to wait in line to use the existing facility. It is recommended that the ramps be loca~ed as shown on page 79 because of ease of access, adequate space for parking and maneuvering. The writer was unable to determine why the existing facility is located in its present area for any other reason than access from Great Island Road .
. The ramps should be constructed with an optimum slope of 12 per cent, minimum slope of 10 per cent, and maximum slope of 14 per cent. If the slope is too shallow, the trailer will have to be backed far out into the water before the boat itself can be floated from the trailer.
If the slope is too steep, there is danger that the car will not be able to get sufficient traction to pull the trailer back out of the water.
Adequate turning area at the boat launching ramp is necessary ao that an experienced driver can easily back his trailer into the water. 60 A single ramp should have an optimum width of 12 feet and should not be less than 10 feet. Finally, a small dock or pier should be provided near 60 charles C. Stott, Evaluating Water Based Recreation Facilities and Areas, Bulletin No. 70 (Washington: National Recreation & Park Association, 1967), p. 34. Ideally to assure orderly traffic control and to reduce vandalism one waterside access to recreational boating facilities would be desirable .
Such a measure is possible at Galilee and could serve a two fold purpose, the other being one of protection for small craft within the area.
To implement this criteria the writer suggests a breakwater to be constructed in a southeasterly direction from the point of Little Comfort Island approximately 400 feet; then an easterly direction for approximately 175 feet, as shown on page 79. Also an alternate breakwater, as shown cm pagG 79 may offar greti.teJ? p;rot@etion to all graft in thiB wator area. The breakwaters would not only provide control, security, and safety for small craft, but small recreational craft would be separated from the commercial fleet, an advantage discussed earlier in this study.
However, the writer suggests that an engineering study be conducted to Galilee has particular need for zoning because of competing users of water area and facilities. Water zoning may be of three types, namely, area, time and space. In area zoning various water areas are established for certain kinds of boating activity. Time zoning involves setting aside some portion of the day for one activity and a different portion for conflicting activity. Space zoning is designed to minimize the conflict between fast and slow boats by providing a barrier of space around the slow boats. A boat thus approaching a fisherman would be required to slow to a no-wake speed until it is the required distance away from the fisherman. 67 The writer feels area zoning is the greatest need in Galilee. Because of the vast water area available to the recreational boater in Salt Pond, the Harbor of Refuge, and the ocean, space and time zoning are not an immediate problem as they might be on a lake where water zoning is limited.
The schematic design on page 79'shows suggested water uses for Galilee. The commercial boat area is located close to both fish proc- It must be stated that implementation of such water use patterns in Galilee would be expensive; yet it must be realized that due to the extensive deterioration of facilities in Galilee improvements are necessary.
IN PLANNING WATER RESOURCE AREAS FOR BOAT FACILITIES THE WATER AREA SHOULD BE COMPLI-MENTED BY AT LEAST AS MUCH LAND .
Land equal to the water area used for boating facilities exists in Galilee, yet much of it is not directly related to boating. Many of the structures in Galilee are deteriorating and empty. Efficient utilization of the land requires that some of these buildings be removed. Galilee has a great potential as a boat harbor . Realization of such potential depends upon adherence to criteria developed in this and the counterpart to this thesis. The land adjacent t o the water area must be developed so that harmony between the two may be achieved. Activities such as marine railways, road3and driveways, parking areas, service and sales should be located with respect to c onvenience and efficient utilization. In order that this may be achieved land facilities require as much space as the water area to be served. While detailed statistics are not yet available for Galilee, one could find solace in the fact that so many boaters do not require permanent facilities. However, if more facilities at a reasonable cost 78 were available, many boat owners might be enticed to use them. As stated, due to the absence of data for Galilee the writer has recommended that pleasure boat areas be planned for 60 per cent capacity and developed in stages as demand increases .
VI SUMMARY AND CONCLUSIONS
In the preparation of this thesis the writer's desires were to develop criteria to r.educe conflicts in areas where the land and water meet. Such criteria were developed and shown how they could be applied in Galilee, Rhode Island. It was found that if the criteria in this thesis were applied to Galilee, benefits to boaters, both commercial and recreational, would be realized.
Criteria developed and applied to Galilee could result in a much safer water area for its users. Segregated use areas could increase efficiency for the commercial fishing establishment and allow the recreational boat enthusiast to enjoy adequate facilities that presently do not exist.
At present ther~ is a shortage of material available to guide planners in dealing with water-use and boating recreation. The writer feels that this thesis will contribute to an understanding of water resource planning by showing the mistakes that have been made as a water area developed, and ways of avoiding these same errors.
Considerable time was spent gathering material that w6uld aid in planning water areas. It is felt this thesis will guide others to these 80 same sources and aid them in solving water resource planning problems as they may arise.
The criteria in this thesis will enable planners to approach a water area with an insight as to the needs and possible potential of that particular area. It is the writer's feeling that this thesis will help to determine the most appropriate use of water resources. It will also aid in determining ideal locations for recreational water facilities, obtaining maximum potential utilization of water resources that will result in safety and convenience to the public, and further expose the need for such planning. The criteria in this thesis aid in determining clearances necessary for boat maneuvering and berthing and sizes and capacities of berthing areas. In addition the planner will have an awareness of how wind, tides, currents, water depth, and littoral drift may influence his decision making.
Galilee's problems are mainly financial, and a Port Authority would probably solve such problems. The authority would establish rules and regulations for the safe and efficient operation of the area; fix standards of design, construction, safety and sanitation. The authority should be able to float bond issues for such needs that exist and pledge revenues of the port to pay these bonds. 68 One factor that cannot be neglected in Galilee is the need for current data on recreational boating. At present the size and number 68 united States Department of Commerce, Economic Development Administration, .Q.E.· cit., pp. 14-15. of recreational craft that use Galilee either seasonally or for short stays is not known. The ·writer recommends that a survey be conducted to ascertain such data in order that facilities may be provided with more accuracy.
There is every indication that the improvements recom,mended for Galilee are economically justified. The navigational improvements suggested by the Corps of Engineers have a benefit-cost ratio of 1. 6 to 1. 0. 69 Necessary facilities of the types recommended for Galilee are similar to those found in marinas in Rhode Island. Nothing has been suggested for Galilee that is not in present demand or has not been provided in other municipalities. Due to the fact that Galilee is partially a commercial fishing port the Federal Government would bear over 50 per cent of navigational improvements ..
Criteria Summarized
When developing water areas, such as Galilee, Rhode Island, the primary reason for this development must be its function. Such functions are recreational area, commercial area, refuge area, convenience harbor, and transportation center.
[, The location of water resource areas to be planned should clearly relate to function. Location must consider the distance to fishing grounds for commercial fishermen, aesthetics particularly for recreational users, water quality for both commercial and recreational 69 united States Congress, House, Committee on Public Works, 2.E.• cit, I p, 64 o users, and must be accessible by land and water.
Water site selection for boating facilities should be determined by the number and size of boats to be accommodated and should be accessible by land and water. Depth requirements increase as boat sizes increase. Channels should offer safe accessibility to facilities . Sufficient water area is necessary to avoid congestion.
£13
Water resource space requirements for berthing and maneuvering should be determined by the number and size of boats to be accommodated .
The demand for pleasure boat facilities inc reases 10 per cent per year.
The present trend is towards larger craft. It is necessary to provide facilities for transients .
In accommodating trailered boats one launch lane should be provided for each forty boats using the facility. One facility per forty boats is adequate to prevent delays in launching. Launching facilities must be protected from wind and waves. Launching facilities ·should include a dock to aid in handling the craft.
In planning water resource areas there should be only one waterside access to recreational boating facilities . One access point aids in boat tr-affic control. Also, one access point is a security factor to aid in preventing vandalism .
In order to exercise control of marine traffic harbor master facilities should be so located as to be able to observe and regulate this traffic and also provide security for moored craft . Ideally the harbor master must be able to observe craft both entering and leaving the harbor. Harbor master facilities should be readily available to boaters seeking information and berthing instructions .
In planning berthing areas, such as slips for boats of all types, clearances between opposite slips should be beyond the beam (width) and length of the boats. Inadequate clearances between slips can be a fire hazard should conditions require boats to be moved. Adequate clearances insure maximum convenience to boat operators.
The entrance to a harbor or berthing area should be so located and wide enough to permit the speedy and safe passage of boats in time of storms, fire or other emergency. Channel width should be five times the beam (width) of the largest boat expected to use the channel. Proper c hannel width will reduce the possibilities of boat collisions and congestion.
Breakwaters and floating baffles will protect small craft from natural waves, waves from larger craft and floating debris . Breakwaters and floating baffles will create a buffer to separate recreational craft from other water uses such as the commercial fishing industry .
Water resource planning should include zoning of water areas with respect to function such as commercial or recreational boating. Space zoning is designed to minimize conflict between different types of water users. Time zoning involves allocating a portion of the day to different water users.
In planning water resource areas for boating facilities the water area should be complimented by at least as much land. An equal amount of land is necessary to support water activities. An equal amount of land will increase harmony between land and water by providing adequate shore facilities.
In planning water resource areas mooring and berthing areas should be so located as to permit quick evacuation in case of fire or other emergency. Berthing areas should provide for convenient maneuvering space and should be accessible to open water areas.
In providing facilities on water resource areas planners should assume that 50 per cent of the pleasure craft will be launched, hauled and stored by the individual owners. It is not necessary to provide seasonal berthing facilities for all recreational craft in a particular area. It must be realized that adequate, economically priced facilities would attract persons who normally haul their craft out of the water I after each use.
The writer in this thesis has tried to show the reason water use deserves equal concern and why zoning should also be a tool for water use planning.
The counterpart to this thesis, "A Study of Land Resources Used for Boating in Galilee, Rhode Island," has established criteria, which are summarized in Chapter IV, to reduce the problems as they pertain to land resource uses. These criteria if applied provide visual and physical access to the water, solve land use problems where land is associated ----with boating functions and simplify the decision making process in determining facility needs.
86
Water and land uses may enjoy a degree of harmony provided certain factors are realized. Those factors are the criteria developed in this and the counterpart to this thesis. It is hoped that other planners may benefit from this research as the writer has in conducting the project.
--(Sample of letter of inquiry) 96 Gentlemen: I am in the beginning stages of writing my thesis in the Department of Community Planning at the University of Rhode Island and would greatly appreciate any information you might be able to provide related to my problem.
The title of my thesis is "A Study of Marine Resources Applied to Galilee, Rhode Island. 11 The problem which I have chosen to investigate is the demand for boating and that this demand has resulted in conflicts in waterfront planning. Therefore, it is my intention to demonstrate by using Galilee as the problem area, that the demand and conflicts in the water-land relationship exist and that criteria can be developed, which if applied, would reduce these conflicts. Specifically, the c_ riteria will relate to the type of boating facilities, extent and arrangement of these facilities, and water-area requirements. Any information you may be able to give me would be greatly appreciated.
Sincerely yours William R. Onosko . Post Office Box 15 Wakefield_, Rhode Island | v3-fos |
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} | s2 | Contribution to the study of inheritance of laying performance and feed efficiency in the fowl under hot climatic conditions
Hens of four sire families and two genotypes for a plumage color gene (Ii and ii) were distributed equally at months of age in two climatic rooms with individual cages, one " hot " (27 °C at " night ", 3 ¢ °C on " day "), the other " temperate " (constant 20 °C). In the same families segregation took place also, independently from the I locus, at the P (pea comb) and O (blue egg shell) loci. Production, egg traits, feed and water intake were recorded during three periods each of 2 8 days. Results are as follows: i. As expected, a depression of production traits and feed intake is caused by the " hot " environment; water intake and body temperature are increased. z. Significant sire family effects appear for most traits, without any detectable interaction associated with the other sources of variation. 3. Genotypes at the I locus show no difference for production traits or feed consumption and no interaction with treatment. On the other hand, pea-comb (Pp) hens as compared to their single comb sisters (pp) have a significantly lower feed intake at high environmental temperature. A similar effect is observed for the Oo genotype (P and 0 genes being linked). 4. Some correlations between weight of the whole egg and its components (yolk, albumen, shell) seem to differ for the Ii and ii genotype, and between environments.
Introduction
Laying stock developed by breeders in temperate climates has been improved for laying performance and egg quality traits in these climates, but in general not necessarily for production in hot climates.
On the other hand, the aim of poultry breeders is not only to maintain high production, but at the same time to minimize its cost, especially feed consumption ( * ) Permanent address: Faculty of Agriculture, Cairo University, Egypt. of birds which represents about 6 0 p. 100 of the total cost. However, feed efficiency is not only a biologically complex character, but also time-consuming to measure in a selection program. Few recent works investigated this character, and even fewer concern the effect of single genes (ME RA T and B ORDAS , 1972 , I974 ; ME R AT, ig75)! Performance in hot climatic condition, and particularly feed efficiency, can be considered as a trait correlated with the same trait measured in temperate conditions. M A T H E R and A HMAD ( 1971 ) pointed out, on dwarf (dw) and normal hens, that heat had a depressing effect for both genotypes for feed and ME intake for immature and mature birds. It seems that no other single gene has been investigated with respect to its possible relation with heat tolerance of laying hens.
This work, therefore, had the purpose of detecting possible differences between the two genotypes Ii and ii, and on the other hand between families, for laying performance and feed efficiency in a temperate condition ( 20 °C) and in a hot condition ( 34 °C during day). In addition, on part of the data, possible effect of genotype at loci P (pea comb) and 0 (blue egg) was investigated. Moreover, correlations between performances within each environment were compared, concerning especially egg characteristics.
Material and methods
. -Experimental birds and conditions
The birds used were issued from our experimental flock segregating for several marker genes. Breeding birds ( 4 sires, z 5 dams) were chosen so as to realize the cross Ii X ii at locus I responsible for plumage color: about half the progeny were of the Ii genotype (suppression of black pigment in the plumage), half were ii (presence of black). The colored ii birds had a black-red plumage owing to the presence of the E, e + or e b alleles for extension of black and of the s allele for red pigmentation.
The I locus had been chosen because of a previously slight but significant difference between genotypes at this locus for feed efficiency of laying hens in a temperate environment (ME RA T and B O RDAS, 197 2).
On the other hand, some families were segregating (independently of the I gene) at the P and 0 loci. The types of crosses realized were respectively Pp x fifi, giving half pea-comb (P!) and half single comb (fifi) progeny, and Oo X oo, giving half blue-shell egg (Oo) and half non-blue egg (oo) daughters. The P and O genes (H U TT, 1949 ) were linked in coupling in some families where they segregated both with their recessive allele; in other families only one of them segregated.
Birds were hatched in september 197 6. The female progeny of chosen breeding birds were raised on the floor till 1 6 weeks of age. Then they were placed in individual cages and distributed in equal numbers in two climatic rooms (q.8 cages in each), both with 14 hours light and 10 hours darkness per 24 hours. From about 7 months of age (beginning in April rg 77 ), one of these rooms (temperate room) was kept at constant 20 °C, the other at 27 °C during the dark period, raising from onset of lighted period to 34 °C, this temperature being maintained till the onset of darkness. Percentage humidity was kept at 50 percent as far as possible. Each sire family, and as far as possible each single locus genotype within families, were distributed equally in the two environments. All birds received a 16 per cent protein and 2, 52 o kcal /Kg M.E. ration given ad lib. in the form of pellets. The experimental period consisted of three consecutive periods each of 2 8 days (April to June 1977 ). Several laying and egg traits were recorded during this time, as well as feed intake, water intake and morphological and physiological parameters. Egg number and percent cracked eggs are recorded for the total test period. Individual average body weight, variation in body weight, total egg mass and feed consumption were measured for each of the three 2 8 day periods. Daily water consumption was an average based on 14 consecutive days in the last period. Mean egg weight corresponded to the last 2 8 day period, Albumen height and shell thickness were average values for eggs collected during two weeks in this same period (6 to 21 june). One egg per hen (in June) was broken to determine the weight of its components, and another egg was used to measure the weight of shell membranes after washing and drying at room temperature during 4 8 hours. The temperatures were read to the nearest o. i °C with a thermocouple connected with two different probes, one for rectal and another for surface temperatures. Respiration rate per minute was estimated only on birds kept in the &dquo; hot &dquo; cell. Temperatures and respiration rate were measured, for each individual, once in the third week of each 2 8 day period, around 2 p.m. Hematocrit was measured once at the end of the experiment.
For each individual, only the average value is considered for traits with repeated measurement and only birds having all measurements recorded are considered. Table i summarizes the definition of observed traits and abbreviated symbols designating them in the following tables. 3 .
-Statistical analysis
For each trait, analysis of variance was done including all birds, with environment, sire family and genotype at the I locus (Ii vs ii) as controlled sources of variation.
For the comparison between P! and pp or between Oo and oo genotypes, it is restricted to pairs of full sisters, on which a t test is made for the different traits.
Finally, phenotypic correlation coefficients are estimated for some traits within environments and also within genotypes at the I and 0 loci.
Results
Tables 2 , 3 and 4 show the means and analyses of variance for the traits studied with respect to the effect of treatments, genotypes at the I locus and sire families. Tables 5 and 6 give respectively the means and t tests for comparison of Oo vs oo and Pp vs pp genotype for all traits. For most of the phenotypic correlations between traits, it is preferred, because of the limited numbers available, to pool them for several experiments in a separate work. However, correlations including egg weight and weight of egg components, which are specific to the present experiment, are considered here. Table 7 contains these correlations within the Ii and ii genotype within each treatment, then compared for the two environments with genotypes pooled on a within environment basis, and finally for the two genotypes grouping environments on a within genotype basis. Discussion I . -Treatment and family differences As in shown in tables 2 to 4 , the higher environmental temperature, as compared to constant 20 °C, has a significant to highly significant depressing effect on feed intake, egg mass produced, body weight and weight gain, average weight of the egg and its components, .shell thickness, and hematocrit value. Egg number is depressed and percent cracked eggs is increased but not significantly. Water intake is significantly higher, and the same for water/feed ratio and for surface and internal body temperatures. These results are in concordance with the generally observed effects of high ambient temperature in the literature (e.g. review by SMITH and OLIVER, 1971 ). On the other hand, weight of shell membranes and albumen height are not significantly affected by treatment. Significant sire family differences are apparent for most traits, but no interaction including families is significant for any trait, suggesting that breeding for these traits in one environment may be satisfactory for improvement in the other. 2 . -Differences associated with single genes Tables 2 , 3 and 4 show that no effect is associated with genotype at the I locus, except a significant but slight difference for comb and shank temperature.
No interaction appears between genotypes at this locus and treatment or sire family, with an exception for the trait &dquo; albumen height &dquo;, with no obvious interpretation.
In particular, the slight but significant association found previously between plumage color (Ii vs ii) and &dquo; residual &dquo; feed consumption is not found in the present study.
On the other hand, in spite of the limited numbers available, tables 5 and 6 show respectively several significant differences associated to the 0 and P loci in the &dquo; hot &dquo; environment: For both Oo and Pp genotypes as compared to their recessive counterpart, total feed intake (F) and egg number (EN) are lower: the same holds for egg and albumen weight for Oo hens.
As 0 and P are linked in coupling in part of our data, this effect might be attributed to either of the corresponding loci. It is interesting to note that pooling with other pairs of sisters, one with peacomb and blue egg shells (Pfi Oo), the other with single comb and shells without blue pigment (!oo), submitted to similar &dquo; hot &dquo; conditions during the same 3 month period of the year in 1974 and 197 6 (unpublished data), a significantly lower feed intake appears for P! Oo birds: on 41 pairs of individuals on the whole, averages of the two genotypes for feed intake per 2 8 days are respectively 2 344 g and 2 5 7 6 g (P ! 0 . 02 ). For egg mass the corresponding figures are 5 32 g and 6 22 g (P -0 . 10 ).
It may be suggested that the small comb and wattles associated with the P! genotype are responsible for depressed feed intake and egg production by reducing heat dissipation rate in the head region. One way of partially checking this hypothesis will be comparing genotypes at the P locus independently of the 0 gene.
. -Correlations between egg characters
From table 7 it may be seen that on the whole, for the Ii genotype, the correlation between egg weight (EW) and albumen weight (AW) is significantly higher (P < o.ooi) than for the ii genotype. One can note also that the YW-AW correlation is higher for the former genotype, though not significantly so. This suggested difference in correlations between egg weight and weight of some egg components may have some analogy to that already mentioned by OsW Dnx et al.
(I977) ! On the other hand, the !W-AW correlation is somewhat higher in the &dquo; hot &dquo; environment than in the &dquo; temperate &dquo; condition. This may correspond to the lower egg weight, on the average, in the former environment.
It may be mentioned in addition that a similar comparison of correlations between weights of the whole egg and its components in the Oo and oo genotypes does not show any significant difference associated with these genotypes. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Effects of processing and cooking on PBB residues.
To study the effect of processing on polybrominated biphenyl (PBB) levels, milk from four dairy herds containing less than 0.3 ppm (fat basis) of physiologically incorporated PBBs was presented individually into cream, skim milk, butter, and stirred curd cheese. Pasteurized and freeze-dried whole milk, skim milk, and cream, spray-dried whole milk and skim milk, and condensed whole milk were also made. PBBs were concentrated in the high-fat products. Spray-drying reduced PBBs in whole milk and skim milk while pasteurization, freeze-drying, aging of cheese, and condensation were not effective. To study the effect of cooking on PBB levels, thigh meat, thigh skin, drumstick and breast (with skin) from half of chickens fed PBBs were analyzed raw, and pieces from the other halves were analysed following separate pressure cooking. The level of PBBs expressed as parts per million on a solids basis was lower in the cooked sample than in the corresponding raw piece and part of the PBBs lost were found in the drip. Recoveries of PBBs in cooked tissue and broth ranged from 68.1% in the thigh skin to 84.6% in the drumstick, with approximately two-thirds of the recovered PBBs found in the cooked meat itself. Therefore, pressure cooking resulted in a loss ranging from 36% for the drumstick to 53% for the thigh skin.
Introduction
Polybrominated biphenyls (PBBs) were mistakenly used in place of magnesium oxide in dairy feeds in Michigan in May, 1973 (1). Subsequent cross feed contamination led to their occurrence in milk and eggs as well as meat of beef, swine, sheep, and poultry. Although precise information concerning toxicity of PBBs at very low levels is not known, research was initiated to assess the possibility of reducing PBBs by processing and cooking because of possible chronic effects of continued assimilation and accumulation of these compounds in body fat.
Processing and cooking have been found to have some potential for reducing levels of chemically similar compounds such as the chlorinated hydrocarbon pesticides and polychlorinated biphenyls. Condensation has shown to be effective in the reduction of telodrin in milk (2), freeze-drying significantly reduced lindane, dieldrin, p,p'-DDT, and o,p'-DDT-DDD from whole eggs (3), heating skimmed milk decreased PCB residues (4), and spray-drying promoted losses of chlorinated hydrocarbon pesticides (5,6). Cooking has reduced these lipophylic compounds from such products as poultry (7)(8)(9)(10)(11), sausage patties (12), bacon (13), pork loins (14), pork muscles (15) and beef loaves with texturized soy (16). Most of the losses were attributed to fat rendering or leaching out during cooking-the more severe the rendering the greater the loss. Different levels of success in elimination of the contaminants were found depending upon the compound studied, the levels of contamination and the tissue from which the extraction was accomplished.
Methods
Processing Milk with less than 0.3 ppm (fat basis) was obtained from four dairy herds identified by the Michigan Department of Agriculture. The milk from each herd was separated into cream and skim milk fractions followed by pasteurization of the whole milk and skim milk at 62.8°C/30 min and cream at 71.1°C/30 min. Cream was churned in an institutional mixer to make butter. A stirred curd Cheddar type cheese was made from pasteurized whole milk following the procedure of Kosikowski (17). Por-tions of the pasteurized cream, whole milk, and skim milk were placed in freeze-drier trays giving a depth of approximately 6.5 mm (1/ in.) and frozen overnight at -23°C. These were further freeze-dried in a Virtis REPP freeze-drier, model FFD42 WS, for 24-30 hr with a system pressure of 5 x 10-3 torr. The temperature of the platen was 65.6-71.1C, giving a final product temperature of 48.8-51 .6°C. Milk from two herds was spray-dried and condensed. The pasteurized whole milk of each herd was prewarmed to 54.5°C and then condensed in a research model Rogers vacuum pan to approximately 35% solids. Pasteurized whole milk and skim milk were heated to 48.8°C and spray-dried by using pilot plant equipment with an air outlet temperature ranging from 76.6 to 98.8°C.
Pressure Cooking
Fifteen White Leghorn hens, approximately 9 months of age, were fed a standard cage layer ration for 5 weeks which was contaminated with 0, 30, 45, 60, and 90 ppm FireMaster FF-I (three hens/feeding level). At the end of 5 weeks the first three groups (0, 30, and 45 ppm levels) were slaughtered. The remaining two groups were placed on clean feed for an additional 8 weeks and then slaughtered. The hens were picked, eviscerated, and chilled overnight in ice water before being carefully dissected to provide breast pieces, drumsticks, thigh meat, and thigh skin from the right side for raw analyses and those from the left side for cooking and subsequent analyses of cooked meat and broth. A 3.8 liter aluminum pressure sauce pan was used to pressure-cook each piece separately for 15 min under 15 psi in 500 ml deionized water.
Analyses
PBBs were extracted and cleaned-up in duplicate by using the general AOAC procedure for chlorinated hydrocarbon pesticides (18). Moisture and lipid determinations were carried out in duplicate by drying at 100 and 70°C, respectively, under vacuum of 711 mm. Lipid samples were evaporated, and aliquots of the petroleum ether extracts used for PBB analyses.
Gas chromatographic analyses were carried out by using a Tracor 560 GLC instrument equipped with a 63Ni electron-capture detector and interfaced to a Digital PDP-8-Pamila GC data system. The column for GLC was a Pyrex column, 1.83 m long x 4.0 mm id, packed with 3% OV-1 on Chromosorb W 80/100 mesh HP. The carrier gas was nitrogen with a flow rate of 40 ml/min. Temperatures at the injection port, column, and EC detector were 270, 240, and 300°C, respectively. Standard PBB (FireMaster BP-6, lot No. 5143, Michigan Chemical Corporation, Chicago, Illinois) solutions were prepared in petroleum ether and inijected at the beginning of each run, after every five or six samples, and at the end of the run. Quantitations were based on the peak area of the standards (hexabromobiphenyl peak). PBB residues were confirmed by ultraviolet spectral and mass spectrometric analyses.
Results and Discussion
Processing The lipid content and levels in the raw milk and in most of the manufactured products are shown in Table 1. Examination of PBB levels on a wet weight basis in the total sample shows that the PBB concentration follows the fat in these dairy products. Analysis of variance revealed the PBB contents (fat basis) of these products were not significantly different. Pasteurization appeared to reduce slightly the PBB content of the whole milk and cream. Pasteurization has been reported to have very little effect on the DDT levels in milk (19). Skim milk, buttermilk, and cheese whey contained slightly higher PBB levels than did pasteurized whole milk, per fat unit, suggesting some PBB may be associated with lipoprotein and/or may be soluble in the serum of milk. This nonproportionality to the fat content has previously been observed (6) in studies with several chlorinated hydrocarbon pesticides. The freezedried whole milk and cream showed slightly higher PBB contents than the pasteurized whole milk and cream, but these results were primarily due to extraneous interfering peaks present only in the freeze-dried products. Aging of Cheddar cheese for approximately 2 months showed very little effect on the PBB levels. Condensation was not effective in the removal of the PBBs from whole milk. Significant differences (p < 0.05) were found among high-fat and low-fat content products when the PBB levels were expressed on a total weight basis. Butter, cheese, and freeze-dried cream had higher PBB levels than buttermilk, cheese whey, and cream, respectively, reflecting the preferential distribution of PBBs in the lipid phase of the products.
Spray-drying appeared to promote losses of PBBs from whole milk and skim milk at the specified conditions ( Table 2). Significant losses of PBB of approximately 30-36% from whole milk and 61-69% from skim milk of herd 2 were observed. Although the levels in the spray-dried products from herd 1 were less than the levels in the pasteurized products, the differences were not significant.
Losses of PBB from the spray-dried skim milk (61-69o) were greater than from the spray-dried whole milk (30-36%). These results suggest that PBBs were more easily removed from low-fat content products and/or when the levels in the original product were low. It is possible that some PBB is distributed in the serum phase of milk and that this quantity could be more easily removed than the PBB associated with the lipid phase. Moreover, the greater surface area formed during spray-drying of the liquid product and the relatively high temperature at which the formed particles are exposed could facilitate the volatilization of the PBBs.
Pressure Cooking
When the chicken pieces were pressure-cooked, the level of PBBs in the wet tissue decreased slightly (Fig. 1). Part of the PBBs lost was recovered in the broth. The level of PBBs in the broth of breast pieces was higher than that in the broth of the other pieces. Since the amount of cooking water was constant, greater amounts of PBBs in the breast broth resulted from a larger amount of fat rendered from the larger piece.
Since rendering of fat is the major mode of removal of these lipophylic compounds, PBBs were also expressed on a fat basis (Fig. 2). Average values in the fat of cooked pieces were slightly higher than those in the fat of the raw pieces. Thus, while rendering of fat is undoubtedly an important mode of PBB reduction, the amount of PBB reduced is not directly proportional to fat removed.
Total weights of PBBs in the cooked chicken and broth were compared to the level in the respective raw chicken piece to calculate the percentage recovery. No significant differences occurred among the percentages of PBB recovered in any of the four pieces; percentage recoveries were 68.1% in thigh skin, 75.8% in the breast piece, 83.9% in the thigh meat, and 84.6% in drumsticks. Recoveries, however, did tend to be higher in the chicken pieces which contained less fat (i.e., drumstick and thigh meat) even though these pieces had lower percen-32 F 30 - tage meat yields than did the higher fat breast piece or thigh skin. The percentage of recovered PBBs in the meat did not differ significantly among the four pieces evaluated and ranged from 65.5% in the cooked thigh skin to 72.9% in the cooked drumstick (Fig. 3). The distribution of the recovered PBBs between the cooked meat and broth is illustrated in Figure 4. The proportion of the recovered PBB in the cooked meat is considerably higher than that found in previous studies. Recovered PCBs were about equally distributed between the cooked meats and broth (7), while only 1/4 to 1/3 of recovered lindane, dieldrin, and DDT compounds occurred in the cooked hen pieces (8). The bulkier size and higher molecular weight of the PBB molecules may contribute to a smaller proportion of the recovered material being found in the broth. Moreover, Stadelman et al. (20) reported that feeding high levels of several pesticides resulted in greater residue persistence in eggs and abdominal fat of hens than was found when low levels were fed. Thus, the high levels of contamination may have influenced this distribution. For pieces from control hens with 0.02 to 0.07 ppm (wet basis) of PBB, the proportion of recovered PBBs in the meat was slightly less, ranging from 39.8% in the drumstick to 52.7% in the thigh meat.
Considering ingestion of the cooked meat only, pressure cooking brought about a total loss of PBBs from that which occurred in the raw meat of 44% for the breast piece, 36% for the drumstick, 42% for the thigh meat, and 53% for the thigh skin. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Somatic mosaicism in plants with special reference to somatic crossing over.
Plant systems in use for the detection of environmental mutagens appear capable of detecting all types of genetic effects which can be studied in animals. The study of somatic mosaicism, however, is better developed in plants than in higher animals. A case is presented here which shows the ability of plant systems in analyzing a host of genetic end points, including chromosome aberrations like deletions, somatic crossing over, numerical inequality, gene conversion, paramutations and point mutations. The systems in general use utilize certain varieties of Tradescantia, Glycine max, Nicotiana tabacum, Antirrhinum majus, Petunia hybrida, and Arabidopsis thaliana. Heterozygous plants or their homozygous counterparts with gene markers affecting chlorophyll development or anthocyanin in floral parts are exploited in these studies. Mutagens produce different frequencies of different types of spots typical of the mode of action of the agent. Analysis of these parameters may be used to predict, at least qualitatively, the kind of genetic damage that might be produced in man. Besides, one can test the validity of interpretation by traditional progeny tests of plants raised from tissue culture from sectors as in Nicotiana and/or by precursor analysis as done in Antirrhinum. The study of mosaicism in plants offers quite inexpensive, rapid, and reliable tests of mutagenicity at least as a preliminary eukaryotic test system. ImagesFIGURE 1.FIGURE 1.FIGURE 2.FIGURE 9.
Introduction
A variety of plant and animal species express mosaicism which is either a natural phenomenon under the normal control of the genetic system of the organism or is induced as an aberration resulting from the influence of environmental factors. We are interested in the latter as a tool in the study of environmental mutagenesis.
Mosaicism, mostly as spots on leaves or petals or as variant cells in stamen hairs, has been exploited in such studies. A great advantage is that the sectors showing alterations are being studied against the "native", in situ background. This situation minimizes the effect of variations due to cultural conditions. Also, one is scanning thousands of cells when one looks at a spot and its surrounding tissue. Another great advantage is that the affected cells do not enter a stringent competition as is visualized for other tissues of the plant. Most of the colonies of cells expressing as spots are only a few generations (22) somatic crossing over and other chromosomal processes leads to mosaicism on leaves (23)(24)(25)(26) (27), or simple point mutations (23,28). Thus, an increase in the frequency of double or simple spots on these heterozygous leaves may reflect the capability of an agent for inducing somatic crossing over or gain/loss of a partial or complete chromosome. An increase in the frequency of light green spots on the ylly11 leaves (Fig. 2) will point to the induction of mutation at the y11 locus. The system, therefore, has the advantage of testing new mutagens and, also, of detecting the various modes of action of these mutagens, all by one treatment. In order to define the "discriminatory sense" of the system I shall consider the chemical mutagens and radiations tried according to the spectrum of changes these produce. The case of NaN3 may be a sector is similar to that of Y,,y1, leaf and hence the interpretation that these sectors originate by mutation of Yii-*Yi.
Only certain chemicals can induce sectoring on yI ylI leaves.
starting point. As seen in Figure 3, the chemical is capable of producing several-fold increase in the frequency of dark green spots and an equivalent effect on the production of yellows. However, in every sample analyzed, the frequency of twin spots was barely above that found in the control (28). The yllyl1 leaves showed a general lack of effect in the production of light green sectors. However, a few spots, very dark green in color, could be seen on the dark green leaves of Y11Y,1 plants. These data do not conform with the interpretation that these spots originate due to somatic crossing over, point mutations or segmental losses. Also, NaN3 has been well tested in plant material to negate its capability in inducing chromosome aberrations (29). The tentative conclusion, therefore, remains that this chemical can cause nondisjunction so that Y11Y1,y11 sectors are dark green and y,1y11 Y1 sectors are near yellow (28). Most of the monosomic lines are supposedly incapable of competing with the trisomic ones in growth and are hence lost. We have not been able to find similar action of any other mutagen that we have tested. Chemicals like caffeine produce a dramatic increase in the frequency of all types of spots (29) (Fig. 4), that for twins being most prominent. The mitotic recombinagen mitomycin C (MC) has effects which closely resemble those produced by this oxypurine. The experiments conducted with mitomycin C even at concentrations as low as 3.25 ppm for 24 hr leave no doubt about its effectiveness. The highest relative increase is that for double spots, as if it were the major effect of MC treatment (Fig. 5). Thus it was concluded (23,24,28,(30)(31)(32) that ucts of somatic crossing over or chromosome aberrations like exchanges between nonhomologs, deletions, etc. are considered responsible for single spots. The idea of somatic crossing over having occurred is supported strongly, not only by the type of effect on Glycine but also for similar results obtained with tobacco (9, 10, 33, 34), Ustilago, and Saccharomyces (25) as well as the quadriradials formed in human (25,35) and plant (25) chromosomes after MC treatment. A regression coefficient for the rate of increase of each type of spot on the leaves of Glycine max and Nicotiana tabacum calculated by Evans and Paddock (6) demonstrates that whereas both MC and x-rays are capable of inducing dark green and yellow spots on the leave of heterozygous plants, mitomycin C clearly has advantage over xrays in the production of twins (Table 2). Similarly, in Nicotiana (10), this chemical has been found to be most effective in producing twin spots when compared with similar effect of radiations and some other chemicals (Table 3).
A third type of effect is produced by agents like 3H and y-rays. In this instance, there is an increase in all three types of spots on the heterozygotes (36). However, the highlight is an overabundance of yellow spots produced in these treatments (Fig. 6). This is an indication of the agent being able to produce somatic recombination and segmental losses of the chromosome carrying Y1. The Y,, deficient cells will produce yellow sectors but those deficient for y11 may not be phenotypically different from the Y11yll area surrounding them. These data, and those obtained with NaN3, fall in line with the quantitative pattern of inheritance for the Yl, locus.
The radiations, discussed above, also produce light green sectors on YllY,, plants indicating to their effectiveness in inducing point mutations of yii-*Yii type. These specific locus mutations increase, in general, in frequency with dose and in parallel to the frequency of spots on the light green leaves (Fig. 7). Besides, methyl methanesulfonate (MMS) (26), ethyl methanesulfonate (EMS) (26), caffeine (30), diepoxybutane (39), and trenimon (39) also produce light green sectors on yellow homozygotes. Another way of looking at comparative effects of some mutagens is shown in Figure 8 reproduced from data obtained with EMS and MMS (26). Yellows outnumber the dark green and twin spots in these experiments. This is true also of y-ray-treated dry or wet seeds. Not only are differences in effectiveness of the two alkylating agents in producing spots on the leaves shown but also the effectiveness of the two chemicals is differentiated in production of relatively more twin spots by MMS than EMS. Also, clearcut differences appear between the relative efficacy of the chemicals in producing Y11y1l sectors on y, y1y plants. 236 FIGURE 7. Parallel between the frequency of light green spots on YllYil leaves and total spots on Yllyll leaves of Glycine max from material treated with 3H20 or y-rays.
Lg. In y11y1 Twin Spots in Other Systems The occurrence and induction of twins as well as single spots on the heterozygous background is not unique to the Glycine system. Similar situations have been found in Nicotiana tabacum for the genes al, a2, t, and su (9,10,33,34) and in Antirrhinum majus for the genes inc and pal ( Fig. 9) (2). In these cases, the frequency of twin spots, as also those of singles, has been increased by administration of solutions of caffeine as in case of Glycine. The increases, for example, considering the Eos, Nivleos, niv, heterozygotes has been 53-fold in the frequency of twins and 38-fold in the frequency of singles, for 0.25% caffeine treatment applied for 24 hr. Similarly, increases have been observed by Deshayes and Dulieu (9) in the frequency of all types of spots on tobacco leaves treated with y-rays and mitomycin C. Besides, the well known case of stamen hairs of Tradescantia so exhaustively studied by Sparrow, has been found to be highly sensitive to radiations and chemical mutagens. Since the details of this system are being provided in two papers in this symposium, I shall add only that besides other chromosomal aberrations (15)(16)(17)(18), somatic crossing over has been invoked as a mechanism ofproduction of paired sectors observed on stamen hairs (19). Another plant species expressing twin sectors is .r concentration--FIGURE 8. Frequency of spots/leaf on Glycine max as function of concentration of MMS and EMS. MMS is more effective in the production of all types of spots than EMS, at equivalent concentrations; the increase of twins is more pronounced than those of single spots in case of MMS, but not EMS. Also, notice the slope of the curves for the two chemicals. Petunia hybrida. In this case no spots are observed on the leaves of plants heterozygous for genes a+/a but can be induced with radiation, e.g., 10,000 R of y-rays (Vig and Dulieu, unpublished data). Similarly, Arabidopsis thaliana and Lycopersicon esculentum have been subjected to the induction of twin spots by chemicals. As a matter of fact, all the species listed in Table 1, with the exception of Hordeum and Zea for which no data for the induction of twin spots exists, show such paired sectors either naturally or under the influence of mutagens. The frequency of somatic crossing over, or twin spots to be more precise, even though subject to fluctuations from experiment to experiment, may be roughly comparable for organisms widely diverse on evolutionary scale. As Table 4 indicates, the frequency in higher plants and fungi, when tabulated on log scale, is within an order of magnitude, most values being in the neighborhood of 10-5 events/ cell. When data become available it would be interesting to compare these values with those found in mammals. In case of the housefly, however, sectors are not found in control populations whereas twin spots can be induced with a frequency of 2.7 x 10-2 with 1000 R of x-rays.
Mutagens Requiring Metabolic Activation
Over the last few years, increasing attention has been focused on mutagenic action of chemicals which need metabolic activation. The S9 fraction of the liver homogenate is the common component used to activate promutagens into mutagens. Recent studies have indicated that liver is not the only system which has enzymatic machinery needed for such activation. It was interesting to note that Veleminsky and Gichner (41), in 1968, could show the mutagenic activity of some promutagens in the plant systems without requiring the addition of S9 fractions.
In soybean, we treated the seed with aqueous solutions of a nitrosoamine, dimethylnitrosoamine, with concentrations as low as 1.25 ppm for 24 hr. Even at this dose a 2.8-fold increase in the frequency of twin spots, a 2.6-fold increase for dark greens, and 1.7-fold increase for yellows was observed, testifying to the ability of a plant system to activate this chemical. However, one series of treatments using concentrations between 60 ppm and 500 ppm, produced a response indicative of maximal conversion of the chemical into a true mutagen. As Figure 10 shows, such saturation effect was not observed for a related nitrosoamide, methylnitrosourea, which does not require metabolic activation. However, methylnitrosourea seems to be much more toxic in that an adverse effect of leaf expansion, and hence on spot frequency, is observed at 125 ppm doses than that for those of dimethylnitrosoamine which is tolerated by the plant at doses as high as 500 ppm (38).
With the data obtained with Glycine system and other plants used in the experimental induction of mosaicism, one may list the mutagens found effective in various systems. Table 5 is such a compilation, which in addition indicates whether the frequency of twin spots was less than, equal to, or more than frequency of 1/2 of both types of single spots. In case of Glycine, thus, caffeine, colchicine, methyl methanesulfonate, and mitomycin C are the only agents which have so far been found to in- ppm. MNU appears toxic at concentrations of between 60 and 125 ppm, whereas DMN, perhaps because of its limited conversion to true mutagen is tolerated up to 500 ppm concentration. Bars show total spots x control. (6) crease the frequency of somatic crossing over producing twin spots more than all other phenomena together working for the production of single spots.
Evidence of Genetic Block by Precursor Analysis of Spots
Harrison and Stickland (42) have provided interesting insight into the precise genetic block induced by mutagens on the petals of Antirrhinum majus. In this case the niv gene blocks all flavonoid synthesis, inc blocks the flavanones, pal causes a late block after flavonon synthesis, and eos locus controls the synthesis of pelargonidin (eosleos) or cyanidins (Eos/-). The insertion of specific precursors into the system can permit the synthesis of pelargonidin or cyanidin. Thus the production of color by dihydrokaempferol in Pallgenotype permits the conclusion that the block was at the niv and/or inc locus. Administration of dihydiroquercetin can permit synthesis if the block is at niv or inc loci, but permits further differentiation between Pal or palrec alleles. The administration of dihydroflavone, naringinin, will permit polargonidin development only if the block is at the niv locus and also if the Eos locus carries the recessive allele, (eos). In case of block at niv but with Eos gene, cyanidin will develop. Since nirengenin cannot lead to production of the end product in an inclinc, neither dihydroquercetin nor naringenin can initiate synthesis in a flower homozygous for pal allels. Thus the "aberrant sectors produced in plants heterozygous for niv, inc, eos and pal can be analyzed by precursors" (2) (Fig. 11). To the best of my knowledge this is the only use of precursors in determining induced variability in plants.
Controlling Elements
In the case of instabilities resulting from the activity of controlling elements, the frequency of mutational events has not been always affected by exogenous factors like chemical mutagens and x-rays, nor has any effect of palrec unstable genes on the frequency of somatic crossing over in Antirrhinum (43) been found. Also in the snapdragon, Harrison and Carpenter could not increase. the frequency of instabilities induced by nivrec and palrec genes by application of EMS, MMS or x-rays (43). However, Culella and Gavazzi (44) have successfully induced changes in the degree of instability expressed by paramutable R2nc gene in corn by treating the seeds with x-rays and EMS. This may point to the fact, first reported by Linden in 1963, that the paramutation process is sensitive to physical and chemical agents (45). These studies, however, have not been carried on to the extent of making paramutational systems useful in study of environmental mutagenesis.
Interpretations
The interpretation of most data in cytological terms is admittedly only conjectural at this point and would remain so unless techniques are developed for tissue culture of spots, or some flanking markers become available in the species under investigation. Both these conditions have been, however, met with some success in Nicotiana tabacum. In such early attempts, Deshayes and Dulieu (9) demonstrated the regeneration of mesophyll explants confirming genetic variations involved in the production of spots. Thus variations of nonreciprocal type, i.e., single spots on a,+/a, background were determined to be due to deletions, point mutations, and gene conversion (9). The twins were the result of exchanges between homologous loci as well as between nonhomologs (33). On the other hand, Carlson's data (10) from plants regenerated from induced spots indicate that 11/12 twin spots resulted from true somatic crossing over, and only one was the result of nondisjunction. An analysis of data from progeny of chimaeric branches in Arabidopsis thaliana (4) can be interpreted to support the idea of somatic or premeiotic recombination, and Ahnstrom, Natarajan, and Veleminsky (3) have attributed induced spots in this species to a phenomenon like somatic recombination. Ample opinion therefore exists that sectors in these systems, as well as those in Tradescantia (19), originate from somatic crossing over. Additionally, studies in tobacco (33) and cotton (7) have indicated possible homeologous exchanges giving rise to twin spots.
Single spots may result from more than one cause. Segmental interchanges between homologous or nonhomologous chromosomes, deletions, and additions of the segment or chromosome carrying the gene in question, particularly where the quantitative pattern of inheritance is clear, may result in single exchanges. Another phenomenon involved in origin of such spots may be the failure of one of the original two components of a twin spot from developing, and point mutations. Moizonnier and Cornu (46) have provided evidence that mosaicism on petals of Petunia results from phenomena like breakage-fusion-bridge cycle. Hagamann's studies (47), showing that somatic conversion in tomato may result from localization of Sulf locus close to heterochromatin, provide additional evidence of genetic basis for mosaicism. A tabular representation of some interpretations for quantitative genes controlling chlorophyll development has been given elsewhere (24).
Advantages of Plant Systems in Study of Environmental Mutagenesis
No one can claim that quantitative data obtained from plants can be utilized for making risk assessments for man. However, one may not neglect the efficiency of plant systems with which they can determine the mutagenic capability of an agent with only a fraction of the cost of such experimentation in animals. More than 99%o concordance has been found between plant and animal species regarding their qualitative response to mutagens. This has been demonstrated in a recent NIEHS workshop. A few exceptions, however, among chemicals are the anthracyclines, cytosine arabinoside, and maleic hydrazide, which may affect the genetic system of one kingdom and not the other. One may also need to consider the rapid growth, availability and control of cell stages as well as mitotic, premeiotic, meiotic and post meiotic cells available for treatment. Plants can be vegetatively regenerated, in some instances at least. Somatic mosaicism in such cases has a special place since the advances in tissue culture leading to regeneration of the whole organism may not become possible in animals of routine use, at least in the foreseeable future. Some other advantages, besides those discussed in Introduction, are possible study of storage effects, dosimetry, artificial germination of pollen, etc. In order to study mosaicism as caused by changes in environment, one may look for perennial angiosperms of suitable genotypes and compare results from year to year.
The plant systems, in my opinion, have a place in at least the preliminary screening of new chemicals being poured into the environment. The wonderful ability of plants to activate promutagens may be of great interest, particularly in view of the fact that all of us ultimately have to consume plant materials, many or perhaps all of which have been treated with a variety of agents. From this point alone the study of plant systems including mosaicism should be seriously considered. | v3-fos |
2019-03-30T13:10:17.953Z | {
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} | s2 | Fungus diseases of field bean in Finland during 1975 — 1977
During 1975 1977, studies were carried out on the fungus diseases of field bean and attempts were made to find, through seed-dusting trials, suitable methods for their control. The fungal flora in a total of 43 seed lots containing 140 200 seeds each were determined. In addition, plant samples collected from farms where field beans were under cultivation and field bean cultivar trials in seven localities were analysed. The weather conditions in the growing seasons of 1975 and 1977 were rather exceptional, the former being dry and warm and the latter rainy and cool. Ripe crops were obtained from the control experiments, in which the seeds of 3—4 field bean cultivars were dusted with thiram and benomyl preparations, only in 1975 and 1976. Ascochyta fabae Speg. was rare in all the seed samples apart from those from LänsiHahkiala, where the degree of infection varied, depending on the cultivar, from 1.3 20 %. Botrytis fabae Sard, was rare in the seeds, B. cinerea Pers. ex Fr. quite common in 1976 and especially in 1977. The frequency of the Fusarium fungi were very great on the seeds in 1977, F. culmorum (W. G. Sm.) Sacc. and F. avenaceum (Fr.) Sacc. being the most common. A. fabae was quite common in the stand samples collected at Länsi-Hahkiala in 1975 and 1976. The chocolate spot-causing agents, B. fabae and B. cinerea, were relatively rare in all years except 1977, when some cultivars were heavily infested by B. cinerea.
Introduction
Cultivation of the field bean (Vida faha L.) has greatly increased in the Nordic countries since the last decade. Attention has thus been focused on the fungus diseases affecting the cultivation of this plant and on the possibilities of controlling them.
The cultivation of field bean in Finland is restricted by the length of the growing season. Almost all the foreign cultivars require too long a growing period to be grown under the conditions prevailing in Finland. Breeding work using old native varieties from Southeast Finland was therefore started in 1969 at Hankki] a's experimental station in Anttila (Hovinen and Kivi 1976). As a result, one improved cultivar is already on the market the early and small-seeded Mikko. The aim of this study is to determine the species of fungi causing diseases in stands and seeds of field bean under Finnish conditions and their practical importance.
Material and methods
During 1975 -77 a seed-dusting experiment was carried out on the Viikki Experimental Farm. The field bean cultivers Mikko and the native variety Pirhonen were grown throughout the duration of the experiments, the Swedish varieties Sving and Primus in 1975only, and Aria in 1976. Dusting was carried out with benomyl and thiram preparations, in 1975 with the latter only. The dusting trial was combined in 1975 with a cultivar trial which included the cultivars Aria, the German Herz Freya and Hankkija lines 70006 and 70028.
When the seeds were checked for healthiness the number of seeds in each lot was found to vary from 140 to 200. Untreated and surface sterilized seeds were used in 1975, but in subsequent years surface sterilized seeds were used almost exclusively. Seed treatment and incubation was carried out using the same methods as described earlier (Ruokola and Kössi 1977), with slight modifications. The seeds were soaked for 10 min. in NaCIO-(1 % clorine) solution to destroy saprophytic fungi. In addition to cellulose discs, the seeds were also incubated on weak CM-(12 g/I corn meal (Difco) + 300 ppm streptomycin/1) agar (WCMA) to induce growth of the pathogenic fungi. Plant samples were taken from 7 different localities ( Fig. 1), most of them, however being from the experimental field of the Institute of Plant Pathology. Sampling took place during different growth stages. The microbes were isolated from infected parts of the plants using the method described earlier (Ruokola and Kössi 1977). CMA, PDA (Difco) and Czapec-Dox agar were used as the growth media. Most of the transfers were soaked for one hour in streptomycin solution (300 ppm 1) to inhibit bacteria.
Weather conditions
In 1975 the growing season at the southern sites, i.e. Viikki, Tikkurila and Tuusula, was on average very warm with only slight rainfall. The summer was cooler in Länsi-Hahkiala than at the other localities. Total precipitation here during the growing season was lower than normal.
In 1976, the growing season, apart from in May, was cooler than normal.
The monthly precipitation sums were also generally lower than normal. The rather heavy rain in Länsi-Hahkiala in July was exceptional.
In 1977, the start of the growing season at Viikki and Tuusula was warmer than normal and less rain than normal fell in Viikki. The rather cool period which started in the middle of the summer continued up until October and precipitation, especially in July, was heavy.
Seed-dusting trials
A combined seed-dusting and cultivar experiment was set up in Viikki in 1975. A total of eight field bean cultivars were used, four in the dusting experiment and four in the cultivar experiment.
Owing to the severe dry period which occurred during the growing season, the early cultivar, Mikko, was harvested already at the beginning of August and the rest during August. Of these, Primus was the latest, its growing period lasting for 103 days. Seed yields were overall rather low, varying from 1 128 to 1 977 kg/ha, which is rather a lot less than the normal yield of 3 000 kg/ha. There were no significant differences between the yields, weight of 1 000 seeds, volume weight and plant density of the dusted and corresponding undusted test units.
The growing season in 1976 was favourable for the development of Vida faba and hence the seed yields of two of the cultivars used in the dusting experiment, Mikko and Pirhonen, were rather good. The seed yields for Mikko ranged from 4 722 to 5 026 kg/ha and for Pirhonen from 4 085 to 4 511 kg/ha. However, no reliable differences as regards yields were found between different treatments. No crop was obtained from Aria owing to the poor germination of the seeds. The weather conditions prevailing at Viikki in 1977 were unfavourable for the development of field bean since ripened seeds were not obtained from any of the cultivars used in the experiment.
Seeds
Altogether a total of 43 seed lots comprising 5 040 seeds were analysed.
A total of 3 315 fungus determinations were carried out on these seeds, either the species or at least the genus to which the fungi belonged being determined (Table 1). The fungal species and the frequency of occurrence of certain species of fungi varied considerably depending upon site type, cultivar and experimental year. In the analyses carried out in 1975, the most diverse microflora were found in the seed lots representing the yields obtained in Länsi-Hahkiala and Viikki ( Table 2). The most pathogenic fungi, especially A. fabae, were found in the samples from Länsi-Hahkiala. It was most common in the native variety Pirhonen.
Hardly any A. fabae or B. fabae fungi were found in the sample lots from Viikki in 1976 and 1977 ( In 1977, the Phoma species, P. herbarum Westend and P. eupyrena Sacc., were also isolated from very many samples, in one lot of Mikko seeds about 40 % of the seeds were infected with P. eupyrena.
When seed lots with germination percentages of less than 20 were excluded, the germination percentage of the seeds varied from 25 to 100. In 1975, germination clearly decreased as the proportion of seeds in the seed lots from different cultivars which were infected with fungi or bacteria increased, r = 0.718*. In 1976, the correlation between germination percentage and the number of bacteria and fungi isolated from the different cultivars was similar to that found the previous year, r = -0.712*. The seed lots, apart from Mikko ones, were 100 % infected in 1977.
Plant samples
Altogether 39 different species of fungi were isolated from the field bean stands, but part of them were classified only at the genus level. The number of isolates of the most important fungi was 130 (Table 4). In 1975, leaf sample transfers were taken in the proportion of 2.5: 1 to other types of sample (stem, root and pod); in other years the proportion was 3: 2. AUernaria alternata, which is considered to be a saprophyte, was frequently found together with pathogens causing spot diseases. Sometimes, however, it also' grew as the only fungus transferred from stems or leaf spots.
In 1977, when the weather conditions prevailing during the growing season were rather unfavourable for the ripening of field bean, the condition of the plants in the dusting experiment was estimated at the end of August using the scale 0-5. 0 indicated a healthy plant and 5 a completely infected plant. Of the three cultivars, Mikko was the most severely infected with Botrytis fungi and Aria the least. However, these values do not give a completely reliable picture of the susceptibility of these cultivars to disease, because the soil of the different experimental plots was not sufficiently uniform. Mikko clearly did not grow as well as the other cultivars, thus presumably increasing its disease susceptibility. On the other hand, this cultivar was found to be heavily infected by Botrytis fungi at two sites where it was under cultivation, at Tuusula and Sipoo.
The relative proportions of B. cinerea and B. fahae in the infection of plants in the dusting experiment was determined by randomly selecting one leaf (5 7 leaflets) from each of the experimental plots and placing the leaves on moist filter paper in petri dishes (0 13 cm) to induce sporulation. The disease was then partly in the chocolate spot phase and partly in the aggressive phase. which appeared as large necrotic spots especially along the edges of the leaves.
The leaf samples were examined under the microscope after 6-B days. They were found to be almost completely covered with conidiophores of B. cinerea; B. fabae was found only occasionally. B. fahae occurred, however, in every one of the experimental units where Mikko was grown, almost every one of the Pirhonen ones and on one Aria only. B. fabae fungi were also found in the leaf and pod samples taken from sites at Tuusula where field beans were under cultivation. Rather many conidiophores of this fungus developed in these samples.
A. fahae fungus occurred at five different localities. Fungus damage was the greatest at Länsi-Hahkiala in 1975 (cf. Neither of these fungi were found in the samples taken at Tikkurila in 1975 and, but almost a quarter of all the Fusarium isolates were obtained from these samples. Mildew was found on the leaves of Primus cultivars at the end of the growing season in 1975 and proved to be caused by Erysiphe polygoni DC. fungus. Other less important fungi which caused spot disease were as follows: Cercospora cruenta Sacc., Helminthosporium sativum Panun., King & Bakke, Phyllosticta viciae (Lib.) Cooke, Septoria viciae Westend and Stemphylium botryosum Wallr. Pathogenicity of the fungi Ascochyta fahae Speg. Spegazzini (1899) was the first to isolate A. fabae from field beans in Argentiina in 1899. Sprague (1929) showed that the fungus he isolated from field bean was a form of A. pisi Lib., which resembles A. fahae. According to the literature, A. pisi, which is a parasite on pea, can also infect field bean (Anon. 1969). A number of researchers (Ratschlag 1930, Beaumont 1950 have named the new fungus Ascochyta sp. which is a parasite only on field bean. According to present-day knowledge, there is no doubt that the Ascochyta fungus in question is identical to that found by Spegazzini. The disease occurred at the end of the summer. Small brown spots appeared on the leaves of field bean, which gradually enlarged, turned light grey brown, often circular and were separated from the rest of the leaf tissue by a dark brown margin (Fig. 2a, b). Black pycnidia were formed in the center of the lesions and during damp weather great masses of conidia escaped from the ostioles in the pycnidia causing secondary infection in neighbouring parts of the plant. The field bean pods in Viikki were only slightly infected by the primary infection derived from the infected seed used in sowing. It was slightly more common in Länsi-Hahkiala.
On the germination medium the fungus initially covered the infected seed with a white mycelium which later became slightly pink or olive-green.
On CMA the colonies of A. fahae were initially white but turned light olive-green as the mycelium aged. On PDA the mycelium was tinted slightly orange and yellow.
The size (m) of the pycnidia and conidia of A. fabae were as follows: Botrytis cinerea Pers. ex Fr., Botrytis fahae Sard. Sardina (1929) was the first to describe B. fahae on field bean. Paine and Lacey had already earlier (1923) isolated this fungus from field bean, but considered it to be a secondary one. They postulated that the actual disease-causing agent was the bacteria Bacillus lathyri Manns. & Taubenh.
Very many researchers have since published information about the occurrence of B. fabae and B. cinerea on field bean (e.g. Ogilvie and Munro 1947, Äkerman 1955, Gerlach and Rudnick 1972, Sundheim 1973. The large numbers of reddish-brown spots, of from one to a few millimeters in diameter, found on the leaves of field bean (Fig. 3 a) and also later on on the stem and pods are characteristic of diseases caused by Botrytis species. The center of the spots were light brown, the edges dark chocolate brown. These chocolate spots represent the harmless stage of the disease (Sundheim 1973). Under certain conditions favourable to the fungus the disease can develop into the aggressive stage, when the plant may be quickly killed-off. In general. however, growth of the fungus stops at the maturation stage (Sode 1969).
B. fabae produced conidia on the surface of the seeds on the germination media and in pure culture, although only in small numbers (cf. Sundheim 1973). In the leaf spots the conidia developed only after being transferred on to moist filter paper.
The fungus initially formed a hyalin, later brownish, loosely packed mycehum on CMA. The conidiophores produced single-celled, oval, colourless conidia (Fig. 3 b). According to Menzinger (1966), the shape, size and occurrence of septa in the conidia varies greatly in this fungus. The sclerotia formed by B. fahae on CMA were smaller and more irregular than those formed by B. cinerea.
F. avenaceum (Noll 1939) been found to cause wilt disease. In general, these disease forms occur together or else root rot alone. No attempt was made in this study to determine which of these two disease forms was present. A total of 41 Fusarium isolates were obtained from the stands of field bean, mainly from the stem, especially the lower part. The isolates were divided into the following species on the basis of the systematic division of Booth (1971Booth ( , 1977. The relatively abundant occurrence of F. oxysporum in the samples, although it was not found at all in the seeds, indicates that Fusarium infection has mainly been soil or air-borne infection. F. oxysporum occurred most frequently on the base of the stems of yellowish plants, sometimes on the roots. F. avenaceum and F. culmorum were likewise isolated from the reddish-brown elongated lesions (Fig. 4), which were sometimes surrounded by a violet ring, at the base and also higher up the stem. The leaves of these infested plants were often completely black when sampled in late summer/early autumn.
Discussion
Spot disease on field bean caused by A. fabae is rather dependant on the air humidity (Beaumont 1950). The total precipitation during the 1975 and 1976 growing seasons at Länsi-Hahkiala was higher than that recorded at the more southerly localities (Fig. 1). A. fabae were also more abundant there than in field bean plants from other places. For instance, the Primus cultivar in Viikki was, owing to the dry period in 1975, only slightly spotted although the seeds used in sowing were severely infected with A. fabae. Secondary infection did not take place that year. However, it is interesting to note that even under such dry conditions the fungus was able to infect the plants via the seeds. The over-wintering of A. fahae in infected plant residues remaining in the ground does not appear to be possible in Finland (cf. Sundheim 1973).
B. cinerea and B. fahae spread in epidemic proportions in the rainy summer of 1977 and caused more damage than A. fahae. Botrytis species can overwinter both in seeds and in infected decaying plants (Sode and Jorgensen 1974). However, no connection was found between the degree of infection of the seed and the seed crop obtained from them. In fact, according to Harrison (1978), B. fabae originating from the seeds used in sowing can destroy field bean seedlings, but no significant differences have been found under field conditions between the spottiness of plants developed from seeds infected to differing degrees with this fungus. Besides, B. fahae was no longer present in 9-month old commercial seed.
In this study seed dusting was not found to have any significant effect on the yield level, which is in good agreement with, for instance, the study carried out in Denmark (Noddegaard and Hansen 1972). Despite the fact that according to the Danish and other studies (Maude et al. 1969) satisfactory results can be obtained in laboratory tests against A. fahae, no compound is available at the present time which would give full protection against diseases spread through the seeds used in cultivation. For this reason the use of clean seed is of prime importance. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | 0 | [] | 1978-04-01T00:00:00.000Z | 17135919 | {
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} | s2 | Absence of plant uptake and translocation of polybrominated biphenyls (PBBs).
Studies of polybrominated biphenyl (PBB) uptake by plants have been conducted in hydroponic solutions and in greenhouse experiments with soil. Autoradiograms of corn and soybean seedlings grown in hydroponic solutions showed no translocation of 14C-PBB from 14C-PBB-treated solutions to plant tops or within the leaf from 14C-PBB-treated spots on the upper leaf surface. A significant portion of the 14C-PBB associated with the roots was removed when the roots were dipped in acetone. Three root crops (radishes, carrots, and onions) were grown in two soils, each treated with a mixture of FireMaster BP-6 (PBB) and 14C-PBB to achieve final concentrations of 100 ppm and 100 ppb. All roots showed more PBB when grown in the soil with the lower clay and organic matter content than they did when grown in the soil with more clay and organic matter. In the latter soil (clay loam) no PBB was detected in any roots from the 100 ppb treatment. More PBB was associated with roots of carrot than of radish or onion. Corn leaf whorls containing dust from a PBB contamination soil and washed radishes from a heavily contaminated garden showed no PBB. ImagesFIGURE 1.FIGURE 2.FIGURE 3.FIGURE 4.
Introduction
PBBs have entered Michigan soils from manures of farm animals fed PBB, from disposal of milk, carcasses, and other produce, and from effluent and dust discharges of the PBB manufacturing plant. Since most insoluble halogenated hydrocarbons are not taken up or translocated by plants, one would predict PBB to behave similarly. However, because of the importance of this conclusion to the quality of future Michigan food, we felt it prudent to thoroughly investigate this question.
In our preliminary greenhouse studies we had shown no PBB in tops of orchard grass and carrots grown in soils amended with high levels of PBB (1). We did, however, find traces (2040 ppb) of PBB associated with carrot roots. We also found no detectable PBB in plants collected from the most highly contaminated Michigan fields (2).
This study was undertaken to evaluate PBB translocation from roots and leaves by using * Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824. '4C-PBB, to more thoroughly evaluate the association of PBB with root crops (carrots, radishes, and onions), and to determine if PBB could be found in root crops grown in a PBB contaminated garden or in dust on plant tops.
Materials and Methods
Corn and soybean seedlings were grown in a coarse sand-vermiculite mixture with Hoagland's solution. At 3 weeks of age the seedlings were removed, the adhering particles washed off the roots, and the plants placed in Hoagland's No. 1 hydroponic solution. For root uptake studies, 14C-PBB (hexaand heptabromobiphenyl isomers only) (2) was added to the hydroponic solution to achieve a final PBB concentration of 100 ppb (1.41 ,uCi/l). To determine translocation from leaves, a 0.5 1,u water drop containing 17 ,ug 14C-PBB (0.25 AOCi) was spotted on the upper surface of a mature leaf. In the first trial, plants were exposed to PBB for 3 and 7 days, while in the second trial, plants were exposed for 4 and 8 days.
After the indicated exposure periods the plants were removed from the hydroponic solutions and the roots were washed by dipping them into distilled water. In the second trial, part of the plant roots that were exposed to '4C-PBB were quickly dipped five times into an acetone bath and then washed with distilled water. All plants were then immediately frozen with crushed Dry Ice, freezedried, and later examined by autoradiography. Portion of the soybean and corn leaves and roots were macerated and the 14C quantitated by liquid scintillation counting.
Radishes, carrots, and onions were grown in the greenhouse on Brookston clay loam (3.27% organic C) and Spinks loamy sand (1.31% organic C) soils amended with a mixture of 100 ppm of FireMaster BP-6 and 50 ppb '4C-PBB (0.68 uCilkg soil). The soil was prepared by mixing 4.9 g of FireMaster BP-6 and 2.35 mg '4C-PBB (33.1 uCi) in 100 ml acetone with 300 g of air-dried soil in a 500 ml brown glass bottle. The soil was gently blown with N2 to remove excess acetone then mixed in a twin shell dry blender overnight. The PBB-treated soil was diluted with 48.7 kg of untreated soil in a mixer overnight to achieve the desired concentration of 100 ppm. A 48-g portion of 100 ppm soil was further diluted with 48 kg of untreated soil to achieve the 100 ppb concentration. From our previous experience this procedure gave a satisfactory distribution of PBB in soil.
PBB-amended soil (5 kg) was placed in a plastic container lined with a polyethylene bag. The radish, carrot, and onion seeds were planted approximately 1.25 cm below the soil surface and nutrient solutions were added as needed. The moisture levels of the soils were maintained at 29 and 13% (approximately field capacity) for the Brookston and Spinks soils, respectively, by weighing the container and adding the required amount of distilled water daily. After 6, 9, and 10 weeks the radishes, carrots, and onions, respectively, were carefully removed from the container. The edible roots or bulbs were washed with tap water, then dried with paper towels. A few of the plants grown on the 100 ppm PBB-treated soils were immediately frozen with liquid nitrogen, freeze-dried, and later examined by autoradiograph.y.
Plant roots were cut into small pieces and extracted with hexane-acetone (1:1 v/v) in a Waring Blendor equipped with a glass mixing container. Three successive 100 ml portions of hexaneacetone were used to extract each plant sample and then combined. The hexane-acetone was then dried with anhydrous Na2SO4 and concentrated to 10 ml on rotary flash-evaporator. This concentrate was passed through a Florisil column to remove interfering components. The PBB was eluted with 200 ml benzene-hexane (1:1, v/v). Concentrated extracts from the above samples were analyzed on a Beckman GC-5 gas chromatograph equipped with an electron-capture detector (3) and a 2% SE-30 on 100/120 mesh Gas-Chrom Q column operated at 250°C with a carrier flow of 40 mi/mn. Minimum detectable PBB concentration in plant material was 0.3 ppb on a wet weight basis.
Since dust on leaf surfaces might result in PBB contamination of plant tops, corn leaf whorls, which commonly show a concentration of dust, were collected from a corn field which had a high concentration of PBB in the soil (102 ppb). Approximately 5-cm sections of leaves, including the whorl, were cut from the lower leaves of stalks, and 50 g of the plant tissue plus dust was extracted and analyzed as above.
In our survey of PBB concentrations in Michigan field soils (2), we found a garden planted in the contaminated field which contained the highest PBB concentration encountered (371 ppb). This garden was also heavily amended with and located near a highly contaminated manure pile (1650 ppb). From this garden, 50 g of radishes were collected, washed, extracted and analyzed as above.
Results and Discussion
The hydroponic study and greenhouse experiment were designed to favor the greatest uptake of PBB. Corn and soybeans were selected for the hydroponic studies since both are major crops in Michigan. The results of the 14C uptake studies are shown in Figures 1-4. Autoradiograms of corn and soybean seedlings grown in the presence of 14C-PBB showed no translocation of PBBs. PBB was found concentrated at the roots, but no PBB was translocated to the plant tops. This was confirmed by liquid scintillation counting of macerated plant tissue. Due to the insolubility of PBB in water, we expected that the PBB would be primarily associated with the root surface. A significant portion of the '4C-PBB was removed when the roots were dipped in acetone (see Figs. 2 and 4), as shown by the lighter autoradiograms. The autoradiograms also showed no movement of PBB within the leaf from the site of '4C-PBB application.
Three root crops, radishes, carrots, and onions, were grown in two soils which differed greatly in organic matter and clay content. The extent of PBB uptake by edible roots or bulbs was determined by autoradiography and by gas chromatographic analysis. No PBB uptake was shown on autoradiograms; however we did find trace amounts of PBB associated with the edible portions of each crop by gas chromatography (Table 1) amount of PBB found on roots was probably associated with root surfaces. Iwata et al. (4) found 97% of PCB residues in carrot roots in the peel. This observation has also been found for DDT and other organochlorine pesticides in soil in which carrots were grown. Carrot roots showed more PBB than radish or onion ( Table 1). The plants grown on the sandier, low organic matter Spinks soil showed higher PBB uptake than ones grown on the Brookston soil which had more clay and organic matter. This finding is consistent with the report of Filonow et al. (3) who found that the adsorption of hexa- bromobiphenyl by soils increased with increasing soil organic matter.
Both the corn-dust sample and the radish sample from the PBB contaminated garden showed small gas chromatographic peaks (<10 ppb) with retention times similar to and identical to, respectively, the major hexabromobiphenyl isomer in PBB. However, neither peak was destroyed by a 20 or 60 min exposure to ultraviolet light (5). When the samples were spiked with a small quantity of PBB, larger peak areas were noted but only the portion attributable to the spike was removed by a 20-min ultraviolet treatment. Thus, the peaks present in these samples were judged not to be due to PBB.
These findings are particularly significant, in that they probably represent the worst possible cases. The dust was from one of the most heavily contaminated fields (102 ppb), and the concentration of dust relative to plant biomass was far higher than animals would normally encounter. The garden with the radishes was from the most heavily contaminated farm encountered in our survey. Though we did not collect soil from the garden, we estimate the PBB concentration of the garden soil to have been between 500 and 1000 ppb [based on the PBB concentrations found in the manure (1650 ppb) and the surrounding field soil (371 ppb)]. However, in neither plant sample could the presence of PBB be confirmed. The fact that we did find peaks with the retention time of PBB points out'the importance of using a confirmatory procedure.
From these results plus our previous results of greenhouse (1) and field studies (2) in which we found no PBB in plant tops, we conclude that little if any PBB will be transferred from contaminated soil to plant tops. Thus, recontamination of animals from feeds grown on contaminated soil will likely not occur. Although some root crops from very highly contaminated soil might contain traces of PBB, much of this PBB could probably be removed by peeling. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | 0 | [] | 1978-04-01T00:00:00.000Z | 11191633 | {
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} | s2 | Effects of PBBs on cattle. IV. Distribution and clearance of components of firemaster BP-6.
Sixty dairy animals were utilized in seven experiments to determine aspects of the distribution and clearance of FireMaster BP-6. Experimental protocols of various studies provided daily exposures from 0.25 to 25,000 mg, exposures for 1 to 202 days, and total study periods from 10 to 1100 days. Necropsy of 28 animals provided information on residue concentrations in 35 tissues, and the excretion in milk was determined in 15 animals. These studies showed that the major brominated biphenyls of this commercial mixture were absorbed from the gastrointestinal tract and appeared in the blood plasma within 4 hr. With continued exposure to the residue plasma concentrations reached a steady state by 15 days. Free PBB was not detectable in urine. During PBB feeding feces was the major route of excretion, representing approximately 50% of the amount fed to animals not displaying signs of toxicosis. Following a withdrawal of PBB, fecal concentrations declined to 1 to 2% of concentrations during dosing, yet, feces remained the major excretory route in nonlactating animals. In contrast, in post-exposure lactating animals milk fat became an important excretory route removing three-times the quantity of residue cleared in feces. Following parturition, concentrations of PBB in milk fat declined approximately twofold in 6 days. Thereafter, the residue concentration in milk fat was approximately 0.4 that in depot fats. PBB had a predilection for lipid tissues with similar concentrations in various depot fats. Concentrations of the residue were notably low in tissues of the nervous system despite the high content of lipid material. Liver contained residue concentrations that were disproportionately high when compared to the lipid content of the organ. Calves born to PBB-exposed cows had similar distribution of residues in body tissues although concentrations were less than those of the dam.
Introduction
An understanding of the distribution and clearance of xenobiotic residues in food producing animals is critical for evaluation of possible subacute toxic effects on exposed animals and subsequent transmission of the residue to man. This information is vital for the interpretation of residue concentrations in samples of body tissues or excretions used to regulate the exclusion of animals and/or food from marketing channels.
Development of most of the information on distribution and clearance of polybrominated biphenyls (PBB) was necessitated by the impact of inadvertent mixing of FireMaster, a commercial flame retardant, into cattle feed (/). This accident resulted in exposures to animals that have varied greatly in amount and duration. A variety of nonspecific toxic responses were reported by cattle accidentally exposed to high concentrations of the flame retardant (2). Claims that many of the same toxic symptoms have occurred in cattle exposed to very low levels of PBB have been made but could not be verified by a field study (3). An understanding of the distribution and clearance of FireMaster at acutely toxic levels and below was necessary to understand toxic responses and to aid the establishment of regulatory tolerance levels in food producing animals. Like commercial preparations of polychlorinated biphenyls (PCB), the PBB in Fire-Master consists of a mixture of halogenated biphenyls (4). By analogy to PCB, each of these specific brominated biphenyls may behave differently in vivo. Studies with PCB have shown that the number of chlorines generally affects absorption (5), excretion (6), and toxicity (7). However, the behavior and toxicity of some chlorinated biphenyls is influenced by chlorine position on the biphenyl in addition to the halogen content (8). An understand-ing of the kinetics of each of the brominated biphenyls in FireMaster was needed. This paper reports an overview of several experiments conducted over a three year period designed to elucidate mechanisms of clearance and toxicity of polybrominated biphenyls in dairy cows and calves.
Methods and Materials
Animals Sixty dairy animals have been involved in seven studies to determine the distribution, clearance, and health effects of FireMaster BP-6. The protocol for these experiments with cows, heifers, and calves are in Table 1. Detailed descriptions of methods used for experiments: 1, 2, 3 (9); 4 (10); and 7 (11) have been reported. Experiments 1, 2, and 3 were designed to carefully monitor the short-term distribution and clearance of FireMaster. In these studies close interval sampling of blood and excreta, complete collections of excreta, inert plus radiolabeled tracers, and necropsies were employed to determine the fate of the residue. Experiment 4 provided information on residue distribution when from 0 to 25 g was given for 60 days to pregnant heifers. Samples of blood, body fat, and excreta were collected frequently throughout the dosing period and currently extends beyond the second parturition for many of these animals. In order to determine the distribution of PBB in 35 tissues, animals from each group were allotted for necropsy immediately following the dosing period and 10 days postpartum. All moribund animals were also necropsied. In experiment 5, the residue concentrations in calves from experiment 4 have been studied. Experiments 6 and 7 were designed as toxicity studies; however, additional information on residue dynamics was obtained from blood, body fat, and excreta samples.
Doses of FireMaster were administered to animals in gelatin capsules with balling guns in order to insure complete consumption of the residue and minimize cross-contamination. Separate equipment was used for handling and management of experimental groups in order to further reduce chances of cross contamination.
Analyses
The procedures used for the analysis of PBB in this laboratory have previously been reported in considerable detail (9,12). In experiments 1, 2, and 3 only the major hexabromobiphenyl (peak 3, Fig. 1) was quantitated. The position of the bromines on this biphenyl have been identified as 2,2',4,4',5,5' (4,13). In experiments 4 through 7 all six peaks ( Fig. 1) were quantitated, although chromatographic conditions were optimized for peak 3. Unfortunately peak 1 could not be accurately quantitated in all samples: therefore, all data for this peak has been omitted from this paper. Peaks 1 and 2 have been identified as pentabromobiphenyls, peaks 4 and 5 as hexabromobiphenyls, and peak 6 as heptabromobiphenyl (4). Hereafter the components of the mixture (Fig. 1) will be referred to as: peak 2, PtBB2; peak 3, HBB3; peak 4, HBB4; peak 5, HBB5; and peak 6, HpBB6.
Results and Discussion
The two lots of FireMaster BP-6 used in our studies, 158-RP and 6244A, differed slightly in the relative concentration of the brominated biphenyls (9). These differences were minor and probably have no significant effect on interpretation of the kinetics of PBB. In the studies in which lot 158-RP was used only HBB3 was quantitated. Since HBB3 represented greater than 60% of lot 6244A, primary emphasis has been placed on determining the concentration and kinetics of this component of Fire-Master in tissues and excretions of cattle.
The minor brominated biphenyls of the mixture, having varying halogen content and/or position, provide valuable insight into the kinetics of PBB in cattle. Therefore, quantitation of these components was made, although GLC conditions were optimized for HBB3. The minor components were not quantitated as parts per million of that compound because they comprised minor quantities of a commercial compound which still has not been fully identified. Changes in vivo of inert, nonhalogenated biphenyl or other biphenyl components could cause profound errors in reported concentrations of these minor components. In order to minimize these errors, yet quantitate the behavior of the minor components, samples from experimental animals were April 1978 I L L I I quantitated against a standard of FireMaster BP-6 (lot 6244A) in which the concentration of each component was assigned the value of 1. Thus absolute concentration could not be determined, but the relative concentration of each component could.
PBB Concentrations in Feces
When FireMaster BP-6 was fed to dairy animals, feces was clearly the most important excretory pathway during exposure. The rate of passage of unconjugated PBB through the gastrointestinal (GI) tract was much the same as the flow of alimentary contents. The appearance and decline of HBB3 in feces of animals given single 3-g doses of Fire-Master were significantly correlated with passage of a chromic oxide (Cr203) marker (9). Presence of both PBB and Cr2O3 were detected in feces approximately 12 hr after administration, reached peak concentrations between 20 and 36 hr after dosing, then declined to about 2% of peak concentration during the subsequent 48 hr. Approximately half of each single dose administered was accountable in feces by day 8 (9).
The concentration of HBB3 in feces of heifers fed 250 mg/day of BP-6 for 60 days reached steady state by day 10. Following withdrawal of the PBB the concentration of HBB3 declined, and in 10 days had decreased to approximately 1% of the concentration present during exposure.
Following the initial decline, the residue concentration in feces remained stable until parturition. Total collections of feces were not made during this period so daily excretion can only be estimated. However, assuming 20 kg of fecal output daily, these animals were clearing only 0.7 mg of unconjugated HBB3 daily.
In experiments in which heifers were given 250 mg of FireMaster for 202 days, concentrations of HBB:, in feces during exposure were virtually identical to animals given the same daily dose for 60 days. Presently, direct relationships between the amount of PBB fed and fecal concentration cannot be made. The animals given 25 g daily displayed toxic effects which included diarrhea, anorexia, and dehydration (12). Concentrations of PBB in samples of feces from these animals sometimes were greater than 40,000 ppm. In contrast, PBB concentrations in feces from heifers receiving 0.25 mg daily were often at or below the sensitivity limit of our analytical procedures (14).
Few studies with PBB have reported fecal excretion. Detering et al. (15) -and Cook et al. (16) studied cows that had been environmentally contaminated (2) 7-9 months prior to study. The authors estimated exposures of between 200 and 400 g of PBB over a 2-week period. Concentration of PBB in feces at the time of their study averaged 0.22 ppm with a daily excretion of 10 mg. Animals in our studies which received similar daily doses of PBB became moribund during the dosing period, and thus post-exposure clearance was not determined. Animals that received 250 mg daily had PBB concentration ratios in body fat to feces of about 750:1, which correspond with ratios reported for these environmentally exposed animals (15); however, the feces to plasma ratio of 0.7:1 in our studies does not correspond with reported ratios of 4.2:1 (16).
Free (unconjugated) residues of PBB have not been detected in urine of cows in our experiments. This route of excretion must not be overlooked as a possible route of clearance of minor or metabolized brominated biphenyls. For that matter, hydroxylated, conjugated, or otherwise bound PBB may escape detection in feces. During our initial study a '4C-labeled tetrachlorobiphenyl was used as a tracer compound and administered with the Fire-Master BP-6 (9). Total radioactivity accumulated in urine accounted for 24.4.% of the 14C dose, none of which was ether-extractable.
Although a-large proportion of '4C from the PCB with four halogens was recovered, presumably in conjugated form, this route still may be of little importance in PBB exposed cows. Most of the major biphenyls in FireMaster contain five or more halogens, thus reducing the chances of metabolism and excretion (5). Further, if HBB3 were efficiently conjugated and excreted it would not be likely that tissue PBB concentrations would persist as have been observed in our studies.
Whereas feces was the most important excretory route in nonlactating animals, milk became the major route in lactating animals. Animals that previously received 250 mg/day for 60 days had a clearance of PBB via milk that was three times that in feces. This clearance in milk was only about 2 mg/ day.
When FireMaster was fed to lactating cows, HBB3 was detected in milk 13 hr post-exposure (9), and milk concentrations were stable by 20 days (19). Upon withdrawal of the PBB source, Fries and Marrow (19) reported a 71% decline in the concentration of HBB3 in milk fat within 15 days, and thereafter the decline had a half-life of 58 days. Animals in our studies exposed to PBB prior to parturition exhibited similar initial declines in milk residue concentrations postpartum. Perhaps accumulations of PBB in the mammary tissue may have contributed to these initially high concentrations. Thereafter, the reported 58 day half-life does not agree with our data. During peak lactation, when our experimental animals were metabolizing body fat, concentrations of HBB3 increased in milk fat and again declined when animals were gaining weight. Whereas the estimate of a 58 day half-life may be applicable for animals not changing weight, a more complex model is necessary to describe excretion of PBB in milk throughout a complete lactation.
PBB Concentrations in Tissues
Understanding the transfer of PBB to and from plasma is critical to an evaluation of distribution, clearance, and potential target organ toxicity. Following administration of FireMaster to dairy cattle, the major brominated biphenyls were readily absorbed from the GI tract and were detectable in plasma within 2-4 hr. In single-dose experiments, the concentration in blood plasma was maximal 24 hr after exposure. When multiple doses were administered, plasma concentrations were at equilib- When dosing with FireMaster was terminated, concentrations of HBB:, in plasma declined approximately 50% in 10 days and 66% by day 20. Thereafter, plasma residues do not fit a consistent decline model. To describe plasma changes in PBB concentration among environmentally exposed animals mathematically is difficult to achieve with accuracy. Changes in plasma concentrations of HBB:1 among experimentally exposed animals have indicated that corrections for pregnancy, lactation, and body weight (fat) changes will be necessary. Table 2 shows mean concentrations of HBB3 in plasma and major tissue groups of heifers and their calves that were necropsied when moribund, at the end of PBB exposure, or 10 days postpartum. Tissue:plasma residue concentration ratios are also presented. The relative tissue concentrations in various tissues were not unlike those found in necropsied tissues of cows and calves environmentally and experimentally exposed to PBB (9,17,18). Basically PBB has a strong predilection for fatty tissues with the notable exception of tissues of the nervous system. Whether the latter effect reflects failure of the residue to cross the blood brain barrier or simply reflects the inability of PBB to accumulate in the phospholipids, glycolipids, and sulfolipids of nervous tissues has not been demonstrated.
Most of the tissues of the body will fit nicely into one of the five tissue groupings in Table 2 having similar concentrations of extractable lipids and therefore similar PBB content. Bile, liver, and mammary tissues would not fit these categories, as they contained disproportionately large residue concentrations.
Where residue concentrations were above the limits of analytical sensitivity, the tissue:plasma ratios were remarkably consistent for nervous tissues (spinal cord, pons, and cortex) contractile tissues (smooth, striated, and cardiac) and liquid tissue (allantoic, amniotic, CSF, and synovial). This relationship probably represents an equilibrium between blood and the liquid tissues. However, it is April 1978 unknown whether an equilibrium exists between nervous or contractile tissues and the blood or whether the constancy simply reflects bound residue resulting from the initial exposure.
The grouping of 10 glandular tissues (adrenal, spleen, pancreas, kidney, thymus, thyroid, ovaries, parotid, lymphatic, and lung) ( Table 2) was the most heterogenous group. Due to the widely varied function of each of these organs the residue accumulation of each merits further study.
High concentrations of PBB in liver tissue were not surprising in light of the detoxification and excretion functions of the liver. Among the animals directly exposed to the residue, greater efficiency of accumulation of HBB:, at a very low level was clearly evident, particularly in liver. Ratios of the residue concentration in liver or fat tissue to the total dose consumed by the animals were calculated. Exposures of 25 g and 250 mg/daily resulted in liver:total exposure ratios of approximately 0.25. The ratio was 17 for heifers in the 0.25 mg daily feeding group.
While not as pronounced as in liver, the accumulation pattern in fatty tissues was similar, with ratios of 1.7 for the higher exposures and 10 for animals receiving 0.25 mg daily.
Excretion in bile was probably the major source of PBB in feces following withdrawal of the residue. Unfortunately, the low numbers of samples of bile obtainable and unknown daily bile production did not allow quantitation of biliary excretion.
The concentrations of HBB3 in the various depot fats of the body were quite similar. Within animals receiving the same exposure, the coefficient of variation was approximately 25%. Subcutaneous fats appeared to be the most variable, and concentrations in this pool were particularly affected by body weight changes of animals. Figure 2 shows the HBB3 concentrations in subcutaneous fat samples of animals given 0, 0.25, and 250 mg for 60 days or 25 g until moribund. This figure clearly shows the biomagnification or concentration of residues in fatty tissues of animals in contact with very small amounts of PBB. By design, to obtain the toxicological data for this experiment, all animals were housed in the same facility. Despite a meticulously designed protocol to minimize cross-contamination, the animals not experimentally exposed to BP-6 accumulated detectable concentrations in body fat. Based on tissue concentrations observed in animals receiving 0.25 mg daily (a total of 15 mg), we estimate that the experimentally unexposed animals consumed approximately 7 mg. Considering that more than 3 million mg were excreted in the facility by other animals, the experimental protocol appeared very successful. Animals given 25 g daily lost weight, became moribund, and were necropsied with 33 to 66 days (10,14). Concentrations of HBB3 in subcutaneous fat from these animals ranged from 1100 to 6000 ppm. Among animals given 0.25 or 250 mg daily there was a 40% decline in HBB3 by 20 days post exposure (Fig. 2). Fat samples were not again collected until parturition, when an increase in the residue concentration had occurred. The concentration in subcutaneous fat continued to increase during heavy lactation when the animals were mobilizing depot fats. Biopsies were again collected in late lactation, and significant declines in residue concentrations had occurred. The latter effect probably reflects the redeposition of subcutaneous fat and a reduced body burden of PBB resulting from excretion of the residue in milk.
Distribution of Minor Components of FireMaster
The quantities of hydrocarbon represented by the minor penta-, hexa-, and heptabrominated biphenyls in FireMaster are quite small. In order to understand the significance of each of these components when accumulated in the mammalian system, additional studies will be needed. The positive identification of each biphenyl is especially needed.
Environmental Health Perspectives
Despite very low concentrations of these components and less than ideal conditions for quantitation, a comparison of the relative concentrations of the minor biphenyls has provided insight into the behavior of PBB in vivo. The best comparisons of concentrations for the minor peaks were obtained from samples from heifers given 250 mg/day of FireMaster.
During the period when doses were being ad-.ministered the relative concentration of pentabrominated biphenyl (PtBB2) in feces was decreased by about 45% and elevated in plasma approximately 50%. It was also slightly elevated in bile of the two necropsied animals. At the same time the relative proportion of heptabrominated biphenyl HpBB6 in feces was elevated approximately 15% compared to the FireMaster standard. HpBB6 was proportionately decreased in plasma and bile by 50 and 80%.
Ten days after the last dose was given and the concentration of all of the FireMaster components had declined, the relative concentrations of each had changed. The relative concentration of PtBB2 increased sharply, nearly doubling in feces and plasma; HpBB6 declined by 45 and 90%, respectively. Following initiation of lactation, very high relative concentrations of PtBB2 were detected in milk fat, and the HpBB3 was virtually undetectable.
These relationships indicated that bromine content influenced the absorption of PBB from the gut and subsequent transfer from plasma to depot fat. The pentabrominated compound was apparently more efficiently absorbed during feeding and obtained equilibrium between plasma and tissues at a higher relative concentration than found in the FireMaster that was fed to the animals. The opposite occurred with the heptabrominated biphenyl as a large proportion was apparently excreted in feces during the dosing period. Changes in the accumulation and excretion of the minor hexabrominated biphenyls also occurred; however, these differences cannot be easily explained without positive identification of the bromine position. The position of the halogen on the biphenyl has been shown to influence the distribution and clearance of polychlorinated biphenyls (5).
Although these relationships have suggested an effect on halogen content absorption of PBB, the possibility of biotransformation or degradation of one or more components of FireMaster cannot be ruled out. Transformations could greatly alter the relative concentrations of minor components.
General Discussion
These studies have provided pertinent information on the distribution and clearance of the compo-nents of FireMaster when dose levels and durations were varied. Animals from nearly all stages of productive life have been utilized. The data from these studies are being used to develop pharmacokinetic models which can be used to predict the behavior of PBB residues in environmentally contaminated livestock. The variety of protocols used for these studies provided data that have clearly demonstrated the simple kinetic models are inadequate to describe the long term behavior of this residual compound in dairy cows. The data from these studies has demonstrated that growth, fat deposition, fat mobilization, lactation, pregnancy, and level of exposure have important effects on the kinetics of PBB in cows. The true behavior of FireMaster in vivo is further complicated by the nature of the compound. Since it comprises of a mixture of compounds with differing halogen content and positional isomers, each component has a different biological behavior.
Surprisingly high concentrations of PBB can be accumulated in tissues of cows without apparent signs of toxicity (10,14,20). Claims that PBB causes toxic responses and death among dairy animals environmentally contaminated with trace levels of the compound must be seriously questioned, as we still have healthy animals on experiment with PBB concentrations up to and exceeding 30 ppm. Along with the information on the distribution of PBB in cattle, we have developed a broad base of information on the clinical (9, 10, 20, 21), pathological (14,22), and target organ (11,23) effects of exposed animals. These studies are continuing to develop a data base of residue and clinical information for the lifetime of remaining experimental animals. | v3-fos |
2016-05-16T17:38:24.046Z | {
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} | s2 | Progeny group size for evaluating natural service bulls using AI reference sires
Use of on-farm beef performance recording for evaluating NS sires might be a means to improve the efficiency of beef sire selection. However, the validity of ranking sires from a large number of herds remains restricted as long as systematic connections between herds have not been realized. This study determines the sizes of the progeny groups to be tested in a system where these ties result from the wide diffusion of AI sires.
Introduction
For about the last twenty years, rational selection of beef cattle in France has mainly been intended for AI bulls. This trend has led in particular to the progressive setting up of integrated selection schemes of males used for either terminal crossing or producing breeding females. In the latter situation, a rather large fraction in the population, variable according to breeds from 8 5 p. 100 for Charo-(') N.S.. A.I. : natural service and artificial insemination respectively. lais to 30 -5 0 p. 100 for Limousin and Blonde d'Aquitaine breeds is still bred by natural service. The value of this population both for selection within itself and for the diffusion of selected genes after progeny testing on breeding qualities remains limited, however, as long as the breeding value of NS sires has not been calculated objectively.
To that end, the data gathered from field recording of growth to weaning may be used with the aim of evaluating sires on their progeny. However, the immediate use of these data will suffer from a lack of exchanges of bulls between herds. In fact, because of the very low number of service bulls used in a herd, the mean genetic level of the sires may vary considerably fom one herd to another. An evaluation of sires based on the classical contemporary comparison does not appear in these conditions to be relevant since a sire compared to sires of contemporaries of a high genetic level will be disadvantaged relative to another compared to sires of a lower level. Srrx! and FREEMAN ( 1977 ) have emphasized, in relation to this, the value of correcting for herd effects including environmental effects only (and not the genetic effects) especially if bulls have been evaluated on a small number of progeny.
For that purpose, different types of design of connections between herds can be imagined. A large scale diffusion of AI sires appears to be an attractive and realistic solution. This system is already (or will be) applied in the main countries possessing specialized beef herds. With respect to the progeny numbers to be recorded, some standards have already been supplied such as those of the Beef Improvement Federation in the USA (Arrorry M OUS, 1972 ). However in our opinion, the justification given seems to be incomplete from a theoretical point of view; therefore the aim of this study is to discuss this problem with a more rational approach.
A. -Generalities
This study is based on the assumption that sires are evaluated by BI,UP procedures of HE ND E R SO N ( 1973 ). BI,UP, the predictor with minimum mean square error of prediction in the class of unbiased linear predictors maximizes the probability of a correct ranking on true genetic values (si), when individuals are ranked on predicted (!t) values (H!ND!RSOrT, 1973). Therefore, we shall discuss the numbers of progeny to be recorded by referring to the minimum E(!, -S ,) 2 criterion or equivalently to the maximum coefficient of determination (CD).
The model used is the following : where: IL is the population mean, Sí the effect of the ith sire (i = r, 2, . h jk the combined effect of the j th herd and the k th year (j = 1 , 2 , ..., q; k = 1 , 2 ,..., t) e 2t xt the residual effect for the l th progeny (I = 1 , 2 , .. , nij k ) of sire i in herd-year jk.
In addition, we assume that : -the si are independent with expectation zero and variance 0 2 ,, -the h lkl are fixed effects, -the ec!r!a have a homogeneous variance a 2 e and are at first, independent.
In these conditions, it has been shown that: i) BLUP solutions are obtained by solution of where -A is a symmetric (!, !) matrix relative to the p sire effects which is a func-2 tion of the progeny numbers and of the ratio À = cr 2 e . 0 2 8with xoj k = Lnij k i * s is the vector of BLUP solutions for the p sires to be evaluated, * B the right-hand side vector of the system defined by : ii) The variance-covariance matrix of prediction errors is given by: Hence, in particular the expression of the coefficient of determination for sire i is w i being the i th diagonal term of A-'.
B. -Formulation of the simple case with only one reference sire It is useful to begin by discussing the simple situation where connections are made by one only AI sire.
In addition, the form of the matrix A is simplified under the following hypotheses: -NS sires have progeny in only one herd and the different sires of a given herd have the same number of progeny; -the AI reference sire has progeny each year in all herds and the proportion of calves derived from AI(6 j ) is constant from year to year in a given herd j.
Under these conditions, the use of ( 3 ) enables the matrix A to be written in partitioned form, with the NS sires arranged by herd (!'j bulls in herd j) in one part, and the reference sire (index r) in the other part.
where A j , R j , are square blocks and vectors of size p' j respectively such as: nj k , xzj k indicate the numbers of progeny for each NS sire and for the reference sire in the herd-year jk respectively and n o px represents the progeny number in the herd-year jk and n j , the total progeny number in herd j.
Results
A. -Calculation of the accuracy An algebraic expression of the inverse of matrix A defined in 6 can be obtained by inverting A by blocks. Details are given in appendix r. The part of matrix A-1 relative to p'! NS sires of herd j is where Ip'; the unit matrix of size !)'j. jp'j the square matrix of size P' j with all elements one defined as -:j -0!n'o 6!!+X x In most practical situations, the last term of ( 7 ) can be neglected. Hence, we obtained the following expression of the term w j (variance of prediction errors for a NS sire of herd j ) The coefficient of determination can then be written, in simplified notations as follows where : -n, m are the numbers of progeny per herd of a NS sire and of the reference sire respectively, -N the total number of recorded progeny in a herd -À = ! -1; h2 being the heritability of the trait considered.
It is to be noticed that assuming the existence of a sufficient number of sires in the evaluation program (at least 8 5 ) the accuracy depends only on the characteristics of the herd using the sire to be evaluated.
B. -Numerical application If (a, !', n and m) are known, it is possible using formula (8) to calculate the accuracy of the predictor s<. In fact, a more interesting goal is to try to optimize the progeny numbers (n, m) according to the level of accuracy desired. For a fixed size N of herd and number !' of NS sires, there is an optimum distribution of the numbers (n, m) maximizing the CD. This is an optimization with one only variable since n, m are related by (io). A graph is given in figure i showing for h 2 = 0 . 2 the curves giving maximum accuracy according to N and !'. The graph also shows the iso-n and iso-m curves. Using this graph, it is possible to determine for a given CD the number N of calves to be recorded and the optimum distribution (n, m) of the latter knowing the number of NS bulls used. Thus, for instance, in herds having two NS sires, it is necessary to test a total of 100 calves to get a CD of 0 . 5 , these 100 calves being distributed into 2 n = 68 and m = 32 .
In the practical situation of French beef breeds subjected to performance recording, growth and conformation traits at weaning exhibit a heritability of about o.2 2 (M OLINUEVO and V ISSAC , 1972 ) and the number of NS bulls used per herd varies between i and 3 . In these conditions, table i gives the optimum numbers of progeny of the reference sire (m) and per NS sire (n) for various levels of accuracy (CD = 0 . 30 ; 0 . 35 and 0 . 40 ). Thus, on the basis of a CD of 0 . 40 which may be considered as a reasonable threshold of accuracy for evaluating this type of sire, it is necessary to test the following numbers of progeny (n, m) per herd : (25, 25); (2 3 , i 9 ) and ( 21 , 12 ) where herds use one, two or three NS bulls respectively.
In addition, it will be noticed that a certain range of variation is allowed for these numbers of progeny without affecting the accuracy too much as shown by results in table i indicating for the same total number N the two combinations (n, m) providing 90 p. 100 of maximum accuracy. In particular, it is possible to substantially reduce the number of progeny m derived from AI (naturally by increasing n per contra) without reducing the accuracy too much. Thus, for example with 2 sires t6 be evaluated in a herd, 6 5 progeny divided into m = 19 and 2 n = 4 6 are needed in order to get an accuracy (CD) of o. 4 . Recording again a total of 6 5 progeny, it is possible theoretically to adopt a distribution into m = 7 and 2 n = 58 without reducing the accuracy by more than 10 p. 100 . It is important to emphasize this possibility because in practice obtaining a sufficiently high rate of AI appears to be one of the most limiting factors for the application of such an evaluation system.
! Discussion
A. -Comparison with previous results Contrary to the american data (N IELSEN , r 97 q.) the number of progeny (m) required for the reference bull decreases when the number !' of NS sires increases or, in other words, when the size (N) of herd increases. The following explanation can be given on account of the model adopted: when p' increases, the amount of information useful for a given sire and which comes from contemporaneous calves of the herd derived from NS (i.e. intra block information) also increases. As reasoning is based on a fixed total accuracy, the part of information needed from AI progeny (i.e. inter block information) decreases accordingly.
B. -Utilization of several AI reference sires A design using only one reference sire has the advantage of being simple. Moreover, it is theoretically ideal, at least in the absence of a marked sire x herd interaction. For a concrete estimation of the magnitude of this interaction and for giving a choice to the breeders rendering the programm more attractive, it is necessary to suggest several AI reference sires.
Thus, in some associations several reference sires are available for the breeders who are required to use two of them in their herd, one being chosen freely by the breeder. NWZS!rr ( 1974 ) clearly showed the value of such a measure where the total number of reference sires available is limited to 4 in comparison with an unplanned program where the overall number of AI sires ( 10 in his study) and the number used in each herd ( 5 to 10 ) are much higher. In any case, it is very important to create a minimum of spatial and temporal links between these AI sires. With respect to the former, the examination of the particular case where 2 AI sires are available for the breeders shows that a large latitude of utilization is possible in the distribution of progeny of these two sires provided the standards of offspring are followed (see appendix 2 ). Concerning connections between years, it may be suggested like the Beef Improvement Federation to use each AI sire at least two years in the program so as to calculate the prediction from a common fixed genetic basis. Besides, within herds, repetitions of one same AI sire at least should be made from one year to another, particularly when obliged to wait several years before reaching the numbers of progeny necessary to evaluate a sire.
C. -Effect of considering a common c 2 environmental effect With the aim of chosing sires from different herds and consequently for a potential use of these bulls in different herds, it is important, not only to correct accurately for the effects of herds, but also to take into consideration that, even adjusted, the offspring of the same sire reared in the same herd are generally more alike than when kept in different herds. This effect called common c 2 environmental effect implies in model (i) that the residual variables e 2s xi are no longer independent. Various structures of covariances may exist. That generally adopted (the most simple but not necessarily the most realistic one) corresponds to a common environmental effect within a herd year jk as shown in the following model: ' where: -the C ij k are independent with expectation zero and homogeneous variance a 2 C , -the es5x are independent among themselves, independent of ci;x and have variance 6 2 E .
The system of equations giving the predictions of s i in this new model, after absorption of t L , h jk and c jk can easily be deduced from that of the previous model (see 2 and 3 ) by replacing (H!Nn!xsonT, 1974). This means that the number of progeny in a herd-year is replaced by half the harmonic mean between this number and p, becoming the upper limit of this mean.
Accordingly if (n * , m * ) are the annual standards deduced from model i, the corresponding numbers (n * c , m*!) in the presence of c 2 effects in ( II ) are obtained by If assuming like some authors (NW!,s!rr, 1974 ) that a2c may be of the same magnitude as G 2 ,, it appears that its effect on the accuracy of ?< is far from negligible especially in herds using i or 2 bulls (Table z). Thus, for these herds, it is necessary to increase the progeny groups recorded by 70 to 8 0 p. 100 if one desires the same accuracy (CD = 0 . 40 ) as previously and assuming that «2c equals 5 p. 100 of the variance a 2 y. For the evaluation of one NS sire, the standards (n, m) change from (z5, 25) to ( 45 , 45 ); with two sires in a herd ( 40 , 30 ) are needed per sire instead of ( 23 , zg). c 2 effects probably exist in French beef herds in particular because of birth grouping, feeding and choice of dams. However, its magnitude has not been determined due to lack of sufficient exchanges of bulls. It is probably necessary to distinguish with respect to this, selection from commercial herds where more homogeneous management between paternal offsprings is expected. As sufficiently accurate estimates of c 2 effects are lacking at the present time, it is necessary, on the one hand to demand a rigorous design in order to limit them as much as possible and, on the other hand, not to fix standards which are to low. The model used assumes that all sires including reference sires are randomly sampled from the same population. This hypothesis may appear unrealistic.
As the objective is to compare NS sires, it may be thought that introducing a group effect for those sires does not appreciably modify the results obtained here. In fact, the number of NS sires being very high relative to the number of reference sires, the estimate of this group effect will be little different from the general mean and of similar accuracy. With different objectives, if in particular the aim is to compare NS sires to certain reference sires, a specific study of the accuracy and of the progeny numbers should be undertaken although, in this case, the value of grouping would be also questionable on account of the large number of progeny per reference sire. b) Distribution of sires according to herds The differences between mean genetic values of sires used in different herds are largely due to a sampling of a very small number of bulls used per herd-year.
It may be that these differences are also affected by a choice of sires within herds.
According to HE ND E RSON ( 1973 ) and F IMLAND ( 1975 ) the bias due to this type of selection is eliminated by considering herds as fixed like in model i.
Besides, if :his sire selection is related to the herd effect h, the model should be changed because of this correlation between s and h effects. It might be assumed that if the NS sires are produced and used in the same herd, a relationship will appear between s and h effects since h includes the genetic level of stock females. However, a rather large number of breeders buy bulls from outside and the criteria of their choice may be rather different from those considered in the evaluation so that the relationship (h, s) is much less obvious than assumed a priori.
E. -Value of criterion R 2 -Conclusion The first element to be discussed is the value of choosing the criterion R 2 to plan this selection program. This criterion is in fact well adapted to provide an overall appreciation of the validity of ranking sires relative to their true breeding values. If we are interested in particular comparisons between sires, we must take into account not only the variances but also the covariances between prediction errors.
The covariance relative to 2 sires of the same herd j can be obtained from formula ( 7 ) i.e. For 2 sires used in different herds j and j', this covariance is On account of the low value of this term (see appendix r), the variance of prediction errors of the difference between these 2 sires is practically equal to the sum of approximate variances of prediction errors (see 8). Reasoning at constant level of CD for all sires leads to a constant variance of error for the difference between 2 sires of different herds. Thus for h 2 = 0 . 2 and the progeny numbers defined for CD = 0 . 4 , this variance equals 6. 32 X IO -2 a 2 e (i.e. a standard deviation of o.25 6e ).
As regards the error committed when comparing 2 sires within the same herd, it is naturally lower. In our model, this variance is 2 a 2 /(n + À); for the optimum progeny groups of 1 8 and 23 given in table i, its value then varies between 4 .8 and 5.4 X jo-l a 2 e' Furthermore as well demonstrated by Rors!xTSOrr ( 1957 ) the determination of the size of progeny groups should result not only from consideration of R 2 but also from the parameters R, i (selection intensity) and L (generation length) which are influencing the expected genetic improvement.
The use which will be made of the indices for selection purposes will determine the efficiency of such an evaluation system as we know that the power of decision is not centralized but rather atomized among different categories of breeders. In addition as the means of recording are limited (size of herds, number of cows bred per year to one bull, AI rate practised) obtaining a certain level of accuracy may imply progeny testing over several years. Here we have to find a compromise again between accuracy and generation length.
The procedure adopted here consisting fixing progeny group size for a given level of accuracy appears to be incomplete. Its main purpose is to show clearly to the breeders'associations the constraints of applying such a system and accordingly to avoid an anarchic setting up of the latter.
More generally, the model used in this study may seem excessively simple. However, it seems to us to provide a concrete basis for an initial discussion with breeders of the possibilities of using such a system under French conditions. Reçu pour publication en livrier 19 7 9 .
Thanks are also expressed to Mrs Kirsten R ERAT and Dr Jehr!s for their valuable assistance in translating the manuscript.
Les incidences de différents facteurs tels que le nombre de taureaux de connexion, les effets de milieu commun et le mode d'échantillonnage des taureaux sont abordés dans la discussion. | v3-fos |
2017-07-29T04:25:16.461Z | {
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} | s2 | Sensitivity of overall economic gain and correlated responses to variation in economic weights in an aggregate genotype for pigs
terms of goods rather than in money terms. Taking account of inflation the average cost of borrowing in real terms in the U.K. over the past 32 years (1945-197 6) is estimated as 2-3 p. 100. This also provides the best available estimate for discounting future returns, and it brings the opportunity cost rate into line with the social time preference rate often proposed for public investments. ,. The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years. On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competitors and they are often at high risk of getting no returns. Implications to the form and amount of investment in animal improvement are discussed. For the aggregate genotype used in the Dutch national pig breeding scheme effects of variation in economic weights are studied. The effect of adoption of « false » economic weights on the economic response to index selection is small. The contribution of changes in different traits to this economic response varies considerably, however. Using examples from sheep populations, the relative importance of male, female and slaughter traits is discussed in relation to different ways of obtaining replacement animals. Three types of population are distinguished, namely nucleus populations with self-supply of breeding animals of both sexes, subnucleus populations with self-supply of females only, and commercial populations buying all breeding animals. Nucleus populations which supply various other populations with breeding animals are also considered. The examples given indicate that the appropriate breeding objectives need not be identical for both sexes, even if the animals selected are to be used in the same population. The way in which replacement is recruited influences the relative importance of the various traits to be considered within the same sex as well as the differences in appropriate breeding objectives for the two sexes.
in terms of goods rather than in money terms. Taking account of inflation the average cost of borrowing in real terms in the U.K. over the past 32 years ( 1945 -197 6) is estimated as 2 -3 p. 100 . This also provides the best available estimate for discounting future returns, and it brings the opportunity cost rate into line with the social time preference rate often proposed for public investments. , . The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years.
On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competitors and they are often at high risk of getting no returns. Implications to the form and amount of investment in animal improvement are discussed. Using examples from sheep populations, the relative importance of male, female and slaughter traits is discussed in relation to different ways of obtaining replacement animals. Three types of population are distinguished, namely nucleus populations with self-supply of breeding animals of both sexes, subnucleus populations with self-supply of females only, and commercial populations buying all breeding animals. Nucleus populations which supply various other populations with breeding animals are also considered.
SENSITIVITY
The examples given indicate that the appropriate breeding objectives need not be identical for both sexes, even if the animals selected are to be used in the same population. The way in which replacement is recruited influences the relative importance of the various traits to be considered within the same sex as well as the differences in appropriate breeding objectives for the two sexes. Dpt of Animal Breeding, Agricultural College, S. 750 0 7 Uppsala, Sweden An investigation concerning the economic influence on the production cost of various traits in pig breeding has been carried out. The terms " economic influence " or " economic value " of a trait in this work have been used to indicate the effect on the production cost per kg lean meat. To compute the production cost an equation was constructed, where the traits were included as variables. Using this equation the production cost per kg lean meat can be computed at different levels of the variables. To obtain the proper mathematical expression of the financial effect of changes in the variables, the partial derivative of the equation was computed for each of the traits referred to. These expressions directly show the financial value of each trait per unit of change at different levels of the variables. The stability of the economic weights has been investigated. The frequence and points of time of the monetary returns from breeding as well as the influence on the genetic gain of different or incorrect economic weights have been discussed. Hybridisation programmes are mostly realized by means of three levels of herds where breeding animals used in elite herds are deciding. In the consequence of the high reproduction rate in the pig it is possible to produce 700 -8 00 final hybrids per sow and year in elite herds. In Czechoslovak conditions 73 p. 100 of the total cost is expended on the own fattening, 21 p. 100 on sow breeding in commercial herds (i.e. 94 p. 100 on the third level of final hybrids production), 5 p. 100 on multiplier herds and i p. 100 only on elite herds. It is therefore possible to evaluate the effectiveness of the hybridisation programme on the basis of results achieved in the last production level.
ESTIMATION OF ECONOMIC VALUES OF PERFORMANCE CHARACTERS IN
In the sheep with much lower reproduction rate is the situation qualitatively different. In a specialized large scale unit for lamb production within the closed system of breeding about 20 p. 100 of the total cost is expended on the breeding of pure lines, 45 p. 100 on the breeding of F t females and 35 p. 100 on the own fattening. The calculation of a profit function is here therefore much more complicated. An example of the construction of a multifactorial function is presented. It includes four groups of basic production parameters and five categories of animals only compared with the practical situation where 22 categories are necessary.
THE USE OF PRODUCTION SYSTEMS ANALYSIS IN DEVELOPING SELECTION GOALS AND METHODS
J. W. WILTON Department of Animal and Poultvy Science, University of Guelph, Guelph, Ontario, Canada Production systems analysis is described, its present applications are discussed and its uses in determining selection goals and methods are discussed. The key features of systems analysis are the statement of a well-defined objective, the accurate representative of real-life production programs and the use of alternative procedures by decision makers. Measurements that have been suggested to describe selection goals are discussed and compared with the objectives specified in systems analysis. Selection procedures that have been used for these various measurements are also discussed and compared with methods that could be used in systems analysis. L'objectif d'une sélection est un ensemble complexe de nombreuses exigences exprimées sous forme soit de pondérations liant les caractères, soit de gains génétiques à réaliser pour certains caractères ou ensembles de caractères. Un index doit représenter le meilleur compromis possible | v3-fos |
2019-04-03T13:06:01.753Z | {
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} | s2 | Nitrate, ammonium and urea nitrogen as fertilizers for wheat and rye in a field experiment
In a five-year field experiment on a well-limed sandy soil five nitrogen fertilizers were compared. They were urea, ammonium sulphate, calcium nitrate, calcium ammonium nitrate and a mixture of ammonium sulphate and calcium nitrate. The crops in successive years were spring wheat, winter rye, winter wheat, spring wheat and winter rye. The average yield level was not different because of different fertilizers, but in individual years some differences were found. Average contents of nitrogen and calcium in grain and straw, however, showed a slight superiority of calcium nitrate to ammonium sulphate. Other fertilizers did not deviate significantly from either of these. Placement vs. broadcasting, application time and rate of fertilizer nitrogen were also investigated. These factors did not affect the differences between fertilizers. The soil-acidifying effect of the fertilizers decreased in the order: ammonium sulphate, urea, mixture of AS and CN, calcium ammonium nitrate. Calcium nitrate had no effect on soil acidity.
Introduction
In Finland calcium ammonium nitrate is the most common straight nitrogen fertilizer. Calcium nitrate has formerly been widely used for agricultural crops, but in recent years its importance has decreased and today it is rarely used. The utilization of urea has, on the other hand, increased to a remarkable extent. In addition to these, small amounts of other nitrogen fertilizers are used, among others ammonium sulphate. Ammonium sulphate is produced in Finland as a by-product of the cobalt industry in quantities many times in excess of its present utilization as fertilizer. However, the main part of fertilizer nitrogen, about 75 per cent, is given in multinutrient fertilizers together with phosphorus, potassium, and in some of them, also other nutrients.
The nitrogen in these fertilizers is both in ammonium and in nitrate forms in varying ratios. Thus, from this point of view it is also important to know the differences in the effects of those nitrogen forms.
Different forms of fertilizer nitrogen have been compared earlier by several investigators, in Finland e.g. by Salonen andHonkavaara (1954, 1970) and Pessi et ai. (1971 a, b). However, the above-mentioned forms of fertilizer nitrogen, i.e. nitrate, ammonium, ammonium nitrate and urea have not all been compared together in the same experiment. The aim of this study was to perform this on a well-limed sandy soil under cereal growing. Because winter cereals are an important object in the use of straight nitrogen fertilizers they were grown in three out of five years of the experiment.
Material and methods
The fertilizers compared in this stydy were 1. Urea, 46 % N totally in amide from 2. AS = ammonium sulphate, 20.5 % N totally in ammonium form 3. AS + CN = mixture of ammonium sulphate and calcium nitrate, 17,5 % N as ammonium and nitrate nitrogen in equal amounts 4. CAN = calcium ammonium nitrate, 26-27.5 %N as ammonium nitrate 5. CN = calcium nitrate, 15.5 % N, mainly in nitrate form. Content of ammonium nitrogen 1 per cent.
The urea, calcium ammonium nitrate and calcium nitrate were usual cornmercial products. The ammonium sulphate was a by-product of the cobalt industry containing 350 mg/kg Co. The mixture of ammonium sulphate and calcium nitrate was composed by mixing the fertilizers immediately before application in proportions of 40 and 60 per cent, respectively.
The soil on the experimental field was a sandy soil with the following characteristics: The plough layer was 25 cm deep. The subsoil sample was taken from the layer between 25 and 35 cm.
Incubation experiment. In order to get an idea of what happens in the soil to the nitrogen derived from the different fertilizers an incubation experiment was performed. Lots of 200 g air-dry soil representing the plough layer of the experimental field were weighed into 0.5 1 plastic pots. Amounts containing 100 mg of nitrogen of each fertilizer were weighed into eight pots and mixed with the soil. In addition eight pots were left without fertilizer as controls. Four pots of each treatment were watered with 50 ml of water, the other four received double amounts water to get low or high soil moisture levels, respectively. The low moisture corresponded to about one bar tension, the high moisture corresponded to about 0.01 bar. The pots were covered with paper which was occasionally removed to prevent anaerobic conditions. The pots were watered when necessary to keep the moisture at the initial level.
After a period of 24 days the pH and the ammonium and nitrate nitrogen were determined in the soil. For the pH determination 10 g of wet soil in an airdry state was suspended with 25 ml of 0.01 M CaCl 2 solution. For ammonium and nitrate determinations the same amount of soil was shaken with 100 ml of 0.5 N K 2 S0 4 solution for one hour. Ammonium and nitrate were determined in the filtered solution potentiometrically by means of an ammonia and a nitrate electrode, respectively.
The field experiment was established in 1972 on the soil whose characteristics are given above. The field was divided into four blocks. Each block consisted of three main plots containing five subplots. Between and outside the main plots there were in all fifteen control plots, which received no nitrogen fertilization. The size of the subplot as well as the control plot was 3.5 m by 20 m. The treatments A, B and C were randomized among the three main plots of each block. For the last experimental year the randomization was repeated with the treatments a, b and c. The placement was done to a depth of 8 cm in rows 16 cm apart. The times of nitrogen application for winter rye in 1976 were 30. 11. 1975 (a), 9.5. 1976 (b), and 25.5. 1976 (c). The treatments arranged for the subplots were the five sources of fertilizer nitrogen listed above. These treatments were randomized within each main plot.
The experimental crops were sown and harvested at the following dates: An annual basal dressing of 500 (years 1972 74) or 600 (years 1975 76) kg/ha of ammoniated PK fertilizer containing 2 % N, 7.4 % P and 12.5 % K was broadcasted and mixed into the soil by harrowing before sowing.
The plots were harvested with a combine harvester to a stubble height of ca. 10 cm. The harvested area varied in different years between 50 and 60 sq.m per plot. The threshed grain was weighed immediately. The straw was baled with a pick-up baler from every plot separately and weighed. Immediately after weighing grain and straw samples were taken from each plot. The dry matter content was determined in a subsample for calculation of the dry matter yield of each plot. The main part of the sample was allowed to dry to an air-dry state. Total nitrogen was determined by Kjeldahl digestion. Potassium, calcium and magnesium were determined by atomic absorption and phosphorus colorimetrically in an acid extract of ashed (at 500°C) plant material.
In addition to the yearly sampling of grain and straw, the plant stand of the first experimental year was sampled twice at one-month intervals from the sowing. The plants were cut just above soil surface. The sampled area was 0.5 sq.m on each plot and consisted of two couples of sowing rows from the opposite ends of a plot. The samples were allowed to dry to an air-dry state, and were weighed and analyzed for nitrogen, potassium and phosphorus like the grain and straw samples. Every sample consisted of at least three subsamples taken from different parts of the plot. The samples were allowed to dry to an air-dry state in the laboratory (ca. 20°C), sieved through a 2 mm sieve and homogenized. In the samples taken in 1972 the pH CaCl was determined as in the incubation experiment. In 1976 the pH was determined in water suspension at a soilwater ratio of 1: 2.5 on volume basis. The results were tested statistically by analysis of variance. The significance of differences between individual means was tested with Tukey's procedure.
Incubation experiment
When incubated for 24 days in dry or wet soil at an application rate of 500 During the incubation period almost all the ammonium nitrogen applied in the fertilizer had been nitrified when the soil was kept dry. The only exception was ammonium sulphate, of whose ammonium nitrogen one third was left.
Because the soil was extracted only once all applied nitrogen could not be recovered. Accordingly it seems likely that only very small amounts were lost or in other forms than ammonium or nitrate. The pH values followed the nitrification being the lower the more ammonium had been converted into nitrate. In wet soil no nitrification occurred. Part of the applied nitrate nitrogen was lost, probably by denitrification. No decrease in the pH was observed. Application of urea led to a pH rise probably because of its hydrolysis to ammonia.
Field experiment, period 1972-73 The grain and straw yields of spring wheat grown in 1972 and winter rye grown in 1973 are given in Table 1. Without nitrogen fertilization winter rye gave better grain yields than spring wheat. The grain yield of spring wheat responded to the nitrogen application very clearly. The grain yields of nitrogenfertilized wheat were higher than those of rye. Irrespective of the treatment the rye gave more straw than the wheat.
In the first year raising the nitrogen rate from 75 to 150 kg/ha significantly increased the grain yield. The straw yield was not affected. Broadcasting instead of placing the fertilizer seemed to reduce its effect on the grain yield a little, but not significantly. The different fertilizers did not differ in their effects.
In the second year when winter rye was grown, nitrogen fertilizers were given only to treatment A to reach the total amount of 150 kg/ha N in the two years of experiment for all treatments A, B and C. Nitrogen application to rye (treatment A) gave a marked yield increase. A slight residual effect of nitrogen given in the previous year (treatments B and C) seems likely. It was observed that the broadcasted nitrogen had had a somewhat larger residual effect than the placed one, but the difference was not statistically significant.
Ammonium sulphate given to rye was slightly superior to calcium nitrate in grain production. Urea gave more straw than the mixture of ammonium sulphate and calcium nitrate. Other treatments did not differ from either of these. The residual effect of broadcasted nitrogen on rye grain was greater when its source was calcium nitrate or calcium ammonium nitrate than urea or a mixture of ammonium sulphate or calcium nitrate.
The total grain and straw yields during the two years of wheat and rye responded clearly to the 150 kg/ha of nitrogen given to these crops. The total grain yields were not significantly dependent on the year or the way the nitrogen was given. The total straw yields were higher when the applied nitrogen was divided for both years than when all was given in the first year. Different fertilizers did not differ in their effects.
The nitrogen contents in the grain and straw of spring wheat (1972) rose at least as much on the nitrogen rate being increased from 75 to 150 kg/ha as when it was increased from nil to 75 kg/ha (Table 2). It made no signifi- The fertilizer was placed 2 ) » » * broadcasted The means of treatments A-C as well as nitrogen sources within the same treatment are compared according to Tukey's test. Yields in each column not followed by a common letter differ significantly (P = 0.95). cant difference whether the fertilizer was placed or broadcasted. The nitrogen given to rye in 1973 likewise clearly increased the nitrogen content of its grain and straw. No residual effect of the nitrogen applied in 1972 could be observed. Different forms of nitrogen did not differ in this respect.
The uptake of nitrogen by spring wheat was clearly increased with increasing nitrogen fertilization (Table 3). The amount of nitrogen in the grain yield rose in the same manner. The uptake after broadcasting was less than after placement of the fertilizers. The winter rye grown in the following year apparently utilized some of the residual fertilizer nitrogen, especially after having it broadcasted. The amount of nitrogen in the grain yield of rye responded to nitrogen fertilizers far less than did that of wheat. Thus, the total amount of nitrogen contained in the grain yields of both the crops was less in treatment A than B and C. In all these treatments the same amount of nitrogen had been given, but in treatment A half of it was given to rye while in the others the whole amount was given to spring wheat.
No marked differences could be observed between different fertilizers in their effects on the nitrogen uptake of spring wheat and winter rye. However, there was some tendency for the nitrate containing fertilizers to give a greater uptake than urea.
During the growth of the first crop, i.e. spring wheat in 1972, the accumulation of dry matter in the tops and the uptake of some nutrients were followed by sampling the plant stand at monthly intervals. The nitrogen was taken up in the main plot treatments as follows (kg/ha): The final nitrogen uptake was clearly raised by increasing the nitrogen rate from 75 to 150 kg/ha, though not so much as by the increase from 0 to 75 kg/ha. With broadcasting the uptake was less than with placing. All these differences were apparent already at the earlier samplings, but some of them were not significant. There were no differences between the fertilizers in the crop's nutrient uptake except on 17.7. when 150 kg/ha N was placed. Ammonium sulphate then differed from the other fertilizers in the nitrogen and potassium uptake: The means of treatments A-C as well as nitrogen sources within the same treatment are compared according to Tukey's test. Values in each column not followed by a common letter differ significantly (P = 0.95).
In the first two-year period the uptake of phosphorus and potassium was affected by the treatments only insofar as the yield was affected. This was also the case with calcium and magnesium which were determined in 1973 only. There was no difference between the fertilizers. Field experiment, period 1974-75 In this period the same nitrogen rate was given to all plots except the controls. The winter wheat in 1974 received 75 kg/ha nitrogen, the spring wheat in 1975 100 kg/ha. The residues from earlier treatments did not cause any differences. Thus, only averages over the main-plot treatments are presented in Tables 4 and 5.
The nitrogen fertilization increased the grain and straw yields of both winter and spring wheat very clearly (Table 4). Only the straw yields of winter wheat were affected by differences in fertilizers. Ammonium sulphate was inferior to calcium ammonium nitrate. In addition, it was observed that nitrate tended to be superior to ammonium in general.
The nitrogen contents of winter wheat grain and straw as well as the uptake figures (Table 5) confirm the superiority of nitrate to ammonium in fertilizer.
A similar tendency was observed in spring wheat 1975, as well as in the total uptake of nitrogen by the four crops grown during the years 1972 75, when totally 325 kg/ha of fertilizer nitrogen had been given. Urea was rather similar to ammonium sulphate in this respect.
During this period the phosphorus and magnesium uptakes did not respond to the differences in the nitrogen source. In the year 1974 the following uptakes of potassium and calcium by winter wheat were recorded: The change in the potassium uptake follows very closely the change in the straw yield. No differences in the potassium contents of grain or straw between the fertilizers could be observed. However, the change in the calcium uptake exceeded the change in the yield caused by a different source of nitrogen. The yields in each column not followed by a common letter differ significantly (P = 0.95) according to Tukey's test.
Field experiment, year 1976
The grain and straw yields of winter rye grown in the fifth year were increased by nitrogen fertilization (Table 6). It was most effective when given in late May. Application in early May did not differ from the application in late autumn the previous year. Only the grain yields differed, but not the straw yields. Between the fertilizers no differences were found.
Differences similar to those in the yields were observed in their nitrogen contents and nitrogen uptakes. The latest application gave grain richest in nitrogen. Also the uptake of nitrogen was highest when it was given late. There was no difference between the two earlier applications. At the first application in late autumn in the previous year, calcium nitrate gave a higher content of nitrogen in straw than ammonium sulphate or calcium ammonium nitrate. The uptake of nitrogen by plants fertilized with urea in late May was lower than when they were given calcium nitrate. Ammonium tended to be inferior to nitrate at both application times in May.
The uptake by the last crop of phosphorus, potassium, calcium and magnesium did not change with the different source of nitrogen.
The field experiment as a whole Table 7 represents the total yields of grain and straw of the five experimental crops, their average nitrogen contents, the uptake of nitrogen over the whole five-year period and the total amount of nitrogen in grain yields harvested. The yields were very markedly increased by the 400 kg/ha of nitrogen given during the five years.
There were no differences in yields between the fertilizers. The nitrogen contents of both grain and straw were also increased by nitrogen applications. The superiority of nitrate to ammonium was evident. Urea did not deviate from the ammonium-containing fertilizers. Differences similar to those in the nitrogen contents existed also in the nitrogen amounts in grain yields and in the total uptakes of nitrogen. grain grain + stra\ Urea did not differ from the ammonium containing fertilizers. At the sampling one month after application the differences were very clear in the uppermost soil layer of 8 cm. Ammonium sulphate and urea acidified the soil. The layer below 8 cm was not affected. At the end of the experiment the differences were similar, not larger in spite of the bigger amounts of fertilizer nitrogen given to the soil. However, at the final sampling the nitrogen given in the first four years hade been mixed with the whole plough layer of 25 cm while at the first sampling the applied nitrogen was distributed in the thin surface layer only. The amounts of nitrogen given to the soil until the first and final soil samplings were 150 kg/ha and 400 kg/ha, respectively. At the final sampling subsoil samples were also taken. Average pH value was 6.5 without any differences due to treatments.
Discussion
The fertilizers compared in this study contained a wide range of soluble forms of nitrogen: from pure ammonium to almost pure nitrate as well as urea. When incubated in wet soil taken from the experimental field the ammonium contained in any of these was not nitrified in 24 days. However, on the field the soil only very rarely remains for a longer period as wet as during this incubation. When the soil was kept dry during the incubation all the ammonium of the different fertilizers except some of the ammonium sulphate was nitrified in the 24 days. The slower nitrification of ammonium from ammonium sulphate depends, partly as least, on the large decrease in the pH after the application of this fertilizer. Although a decrease of the pH in field conditions probably is always smaller and not so important, differences between fertilizers in their nitrification speed are still expected. Denitrification that was found to occur in wet soil may also account for the different behaviour of fertilizers in soil and for differences in their effects on crop growth. The field experiment was performed on a sandy soil with cereals as experimental crops, which clearly responded to the nitrogen fertilizers. The amount of nitrogen given to the crops during the five-year period was in all 400 kg/ha. The average annual amount, 80 kg/ha N, was higher than in the experiment reported by Salonen and Honkavaara (1970), but lower than in the experiments of Pessi et ai. (1971 a, b). However, owing to the long experimental period of Salonen and Honkavaara (1970), the total amount of nitrogen given during their experiment was higher than in the present one. All the experiments referred to above were performed on clay soils.
Although the differences in the behaviour of the fertilizers during the incubation were rather clear the crop growth was, in general, independent of the source of fertilizer nitrogen. The average differences over the whole experimental period were clearest in the nitrogen and calcium contents of the yields and in the soil pH. Differences in grain or straw yields were found only in some years and with some methods of application. It was not possible to conclude whether it was different weather, different crop or different application method that accounted for this. Ammonium nitrogen was on an average inferior to nitrate nitrogen but only slightly and not every year. In 1973 ammonium sulphate applied to winter rye gave an even larger yield increase than calcium nitrate. After ammonium application the yields mostly contained less nitrogen and calcium and, in some instances, were lower than when nitrate had been applied. The differences observed here were not as clear as those reported by Salonen andHonkavaara (1954, 1970). In their experiment ammonium was very much inferior to nitrate. The difference was reduced by liming the soil (Salonen and Honkavaara 1970). In the present experiment the soil was initially well limed and not even the plots given ammonium sulphate became so acid that the crop growth would have been affected. However, the acidity in the soil immediately surrounding the fertilizer granule may have increased appreciably reducing the availability to plant of the fertilizer. Between the calcium ammonium nitrate and the mixture of ammonium sulphate and cal-cium nitrate no difference was found, although the lime contained by the former led to the hypothesis that it would acidify the soil less than the latter.
The soil pH in the plough layer was decreased by 0.3 -0.4 units by the addition of 400 kg/ha of ammonium nitrogen. This is in rather good agreement with figures given by Fogh (1972), Alessi andPower (1972) andWatson et al. (1973). All these experiments were on similarly textured soils but in different climatic conditions. Results of Salonen and Honkavaara (1970) also agree quite well, although their experiment was performed on clay soil. Urea was as good as or only slightly inferior to calcium nitrate. Similar results have been published by Devine and Holmes (1963 a, b), Pessi et ai. (1971 b) and Klausen (1974). In its effect on crop yield or on soil pH urea did not deviate significantly from ammonium sulphate.
A different method or time of fertilizer application did not cause any marked change in the behaviour of different fertilizers. One reason for this may he found in the fact that conditions in the years when the method and time were studied were favourable for the efficiency of all fertilizers. However, some obtained evidence pointed to calcium nitrate when broadcasted having had a larger residual effect than ammonium containing fertilizers or urea. The differences in the effect of the fertilizers on crop were, even in extreme cases, very small. This was due partly to the fact that the soil was only very slightly acid. The acidity did in no case increase up to a detrimental level. If ammonium sulphate is applied continuously a marked decrease of soil pH may be expected. To prevent soil acidification due to ammonium containing fertilizers liming at regular intervals is necessary, otherwise crop growth and nitrogen fertilizer efficiency will decrease. | v3-fos |
2017-07-29T04:25:16.345Z | {
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} | s2 | Effect of population type on the definition of breeding goals
. The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years. On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competitors and they are often at high risk of getting
in terms of goods rather than in money terms. Taking account of inflation the average cost of borrowing in real terms in the U.K. over the past 32 years ( 1945 -197 6) is estimated as 2 -3 p. 100 . This also provides the best available estimate for discounting future returns, and it brings the opportunity cost rate into line with the social time preference rate often proposed for public investments. , . The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years.
On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competitors and they are often at high risk of getting no returns. Implications to the form and amount of investment in animal improvement are discussed. Using examples from sheep populations, the relative importance of male, female and slaughter traits is discussed in relation to different ways of obtaining replacement animals. Three types of population are distinguished, namely nucleus populations with self-supply of breeding animals of both sexes, subnucleus populations with self-supply of females only, and commercial populations buying all breeding animals. Nucleus populations which supply various other populations with breeding animals are also considered.
SENSITIVITY
The examples given indicate that the appropriate breeding objectives need not be identical for both sexes, even if the animals selected are to be used in the same population. The way in which replacement is recruited influences the relative importance of the various traits to be considered within the same sex as well as the differences in appropriate breeding objectives for the two sexes. Dpt of Animal Breeding, Agricultural College, S. 750 0 7 Uppsala, Sweden An investigation concerning the economic influence on the production cost of various traits in pig breeding has been carried out. The terms " economic influence " or " economic value " of a trait in this work have been used to indicate the effect on the production cost per kg lean meat. To compute the production cost an equation was constructed, where the traits were included as variables. Using this equation the production cost per kg lean meat can be computed at different levels of the variables. To obtain the proper mathematical expression of the financial effect of changes in the variables, the partial derivative of the equation was computed for each of the traits referred to. These expressions directly show the financial value of each trait per unit of change at different levels of the variables. The stability of the economic weights has been investigated. The frequence and points of time of the monetary returns from breeding as well as the influence on the genetic gain of different or incorrect economic weights have been discussed. Hybridisation programmes are mostly realized by means of three levels of herds where breeding animals used in elite herds are deciding. In the consequence of the high reproduction rate in the pig it is possible to produce 700 -8 00 final hybrids per sow and year in elite herds. In Czechoslovak conditions 73 p. 100 of the total cost is expended on the own fattening, 21 p. 100 on sow breeding in commercial herds (i.e. 94 p. 100 on the third level of final hybrids production), 5 p. 100 on multiplier herds and i p. 100 only on elite herds. It is therefore possible to evaluate the effectiveness of the hybridisation programme on the basis of results achieved in the last production level.
ESTIMATION OF ECONOMIC VALUES OF PERFORMANCE CHARACTERS IN
In the sheep with much lower reproduction rate is the situation qualitatively different. In a specialized large scale unit for lamb production within the closed system of breeding about 20 p. 100 of the total cost is expended on the breeding of pure lines, 45 p. 100 on the breeding of F t females and 35 p. 100 on the own fattening. The calculation of a profit function is here therefore much more complicated. An example of the construction of a multifactorial function is presented. It includes four groups of basic production parameters and five categories of animals only compared with the practical situation where 22 categories are necessary.
THE USE OF PRODUCTION SYSTEMS ANALYSIS IN DEVELOPING SELECTION GOALS AND METHODS
J. W. WILTON Department of Animal and Poultvy Science, University of Guelph, Guelph, Ontario, Canada Production systems analysis is described, its present applications are discussed and its uses in determining selection goals and methods are discussed. The key features of systems analysis are the statement of a well-defined objective, the accurate representative of real-life production programs and the use of alternative procedures by decision makers. Measurements that have been suggested to describe selection goals are discussed and compared with the objectives specified in systems analysis. Selection procedures that have been used for these various measurements are also discussed and compared with methods that could be used in systems analysis. L'objectif d'une sélection est un ensemble complexe de nombreuses exigences exprimées sous forme soit de pondérations liant les caractères, soit de gains génétiques à réaliser pour certains caractères ou ensembles de caractères. Un index doit représenter le meilleur compromis possible | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Distribution and kinetics of PBB residues in cattle.
Cows fed a constant amount of polybrominated biphenyl (PBB) reached a steady-state concentration in milk fat within 30 days. This concentration was approximately four times the concentration in the total diet. When feeding of PBB was stopped, the concentration in milk was adequately described as a sum of two first-order elimination rates. Biological half-life in environmentally contaminated cows, studied for 6 months about a year after contamination, was 60 days. The stage of lactation affected the rate of elimination, and in some concentrations increased shortly after calving. Residues were distributed in body tissues proportionally to concentration of fat in the tissues. Liver and brain were exceptions. Concentration in liver fat was generally higher than other tissues and possibly related to the treatment of some cows with phenobarbital. Residues in brain fat were significantly lower than all other tissues. The ratio of the concentrations in milk fat to concentration of residues in the blood of calves and fat of fetal tissues to the concentration in the corresponding tissue in the dams was 0.36:1. It was estimated that people consuming milk from the highly contaminated Michigan cows could have received PBB doses as great as 10 g from this source alone.
Introduction
The polybrominated biphenyls (PBB) are nonpolar and chemically unreactive. These two characteristics are the major determinants of the kinetics and distribution of PBB residues in the animal. Residues of PBB are distributed in close association with the fat of tissues, have a long biological half-life, and are mainly excreted in fat-containing products, i.e., milk and eggs. Thus, as expected, the kinetics and distribution of PBB residues are qualitatively similar to the kinetics and distribution of chemically related compounds such as polychlorinated biphenyls (PCB) and chlorinated hydrocarbon insecticides.
We studied the distribution and kinetics of PBB in controlled feeding studies with diary cattle (1) and in highly exposed dairy cattle involved in the Michigan feed contamination incident (2,3). Our studies are briefly summarized in this paper. All discussions are confined to the major hexabromobiphenyl component of a commercial PBB (FireMaster FF-1, Michigan Chemical Co., St
Distribution Tissue Levels
Distribution of PBB residues among tissues are determined in 41 dairy cattle (3). Of these, 32 were cows that had been exposed to feed contaminated with 0.4% PBB (4). The estimated PBB intake was about 400 g over 15 days. The other 9 animals were calves born to these exposed cows. The calves' PBB exposure was from both placental transfer and consumption of contaminated milk. The tissues examined were perirenal fat, omental fat, subcutaneous fat, skeletal muscle, cardiac muscle, kidney, liver, lung, and brain.
The animals were divided into four groups for summarization: group I contained 6 cows killed 9 months after exposure; group II contained 12 cows that became moribund 9 to 12 months after exposure; group III contained 14 cows killed 15 to 20 months after exposure and after they had been used in residue elimination studies (2); and group IV contained 9 calves killed at 24 months of age.
The geometric mean concentration of PBB in the fat of the tissues of the four groups are shown in Figure 1. All values are expressed as concentration in fat because the variation among tissues was greatly reduced when the values were expressed on (8,9). In those limited studies there was a much wider range of concentrations among tissues than in our study. In our study the cows were killed at least 9 months after exposure to PBB; in the other studies the cows were killed within a few days after exposure to PBB. The greater uniformity of the concentrations in our studies suggest that PBB is slow in establishing equilibrium concentrations in the fat of the various body tissues. this basis rather than as concentration in sue. In other words, the concentration tissue parallels the concentration of fat There were no significant differences; concentrations of PBB in perineal fat, o subcutaneous fat, skeletal muscle, cardi, and kidney when all of the animals are ( as a group. Some of the differences in cor among these tissues were statistically within individual groups (3). However, ences were small, inconsistent, and do no have biological or practical importance. Concentrations of PBB in liver were si higher than concentrations in other tissue III and IV but not in groups I and II. The the higher values in groups III and IV is but most of the higher values of group I sociated with 9 cows that received ph (2). Phenobarbital is a liver microsomal ( ducer, and the increase is possibly asso( enzyme induction.
The concentration of PBB in the fat significantly lower than the concentratio of all other tissues except brain; where t tration was significantly lower than in al sues. Phospholipids make up a large por lipid in brain and this may be one explanlower residue concentrations in brain.
Our findings on PBB distribution in thi consistent with the results or studies c tribution of chlorinated hydrocarbon con cattle (5)(6)(7). However, we are not in gi ment with some limited studies on PBB Placental transfer of PBB was evaluated with two independent sets of observations (3). Fetal samples .6 .8 1 were obtained from eight animals that were pregnant when killed, and blood samples were obtained from nine calves at birth and compared with blood samiyl in the fat of ples simultaneously obtained from their dams. The vs and calves, average ratio of PBB concentration in fetal or calf posure; group 11 thegndffent Subcutaneous body fat, blood, milk fat and feces were obtained from 12 cows every 4 weeks for 24 t appear to weeks (2). Some body fat biopsy samples had too ignificanl .little fat to provide reliable analytical information. Ignificantly Therefore, this discussion will involve the 6 cows ,s in groups from which complete sets of data were obtained. reason In in the fat where Y is the dependent variable, X is the indehe concen-pendent variable, and b is the regression coefficient. 11 other tis-This model differs from standard linear regression rtion of the because the regression line passes through the oriation of the gin. The model is considered theoretically more appropriate when the error is proportional to the magis study are nitude of the observed values (10). Our observations suggest the body burden of PBB can be reliably estimated by sampling the most accessible tissue or product. Blood or milk samples are usually much easier to obtain than body fat biopsies. Blood suffers a disadvantage at lower body burdens because the concentration is much lower than in milk or body fat. Thus, detectable levels could be present in milk or body fat but the levels in blood would be below the limit of analytical sensitivity.
The 0.42:1 ratio of concentration in milk fat to concentration in body fat (Fig. 5) provides the most reliable estimate of this useful measure. The average ratio was 0.37:1 in the six cows followed during lactation in spite of the greater variation with body fat biopsy samples. The average ratio found with four cows with body fat residue levels below 2 ppm was 0.43:1 (1). This suggests that a constant ratio holds over a wide range of concentrations and that milk fat can be used to reliably predict body fat concentrations. The constant ratios among the concentrations in the tissues and products were obtained from animals no longer consuming PBB. While functional relationships probably exist when animals are consuming PBB, the numerical values will probably be different.
Steady-State Levels
Residue kinetics were studied in four first-lactation cows (2). The cows were fed 10 mg PBB per day for 60 days. Residues in milk were determined during the 60-day feeding period and for 60 days after feeding stopped. The stage of lactation when PBB feeding started averaged 164 days and ranged for 121 to 191 days.
Average concentrations of the major hexabromobiphenyl and heptabromobiphenyl components in milk fat are given in Figure 6. Concentrations of both components reached stable levels after 20 days of feeding and declined rapidly for the first 10 to 15 days after feeding stopped. After 15 days the concentration of hexabromobiphenyl declined much less rapidly while the concentration of heptabromobiphenyl was below the limits of detection. Concentrations of the two components are normalized to equal intakes of each. Differences in the concentrations of hexabromobiphenyl and heptabromobiphenyls (Fig. 6) indicate that, relative to in-take, five times more hexabromobiphenyl than heptabromobiphenyl was transferred to milk. We found similar differences in the transfer of the two compounds into eggs of hens with a concomitant higher relative concentration of heptabromobiphenyl in the hen excreta (11). Similarly, the more highly chlorinated polychlorinated biphenyl components are, the less efficiently they are transferred to milk (12) and eggs (13). We have interpreted these observations to mean that less halogenated components of PBB and PCB more readily diffuse across biological membranes than the more halogenated components.
The contribution of heptabromobiphenyl to the total residue is small even when PBB is fed. Not only is there a fivefold difference in relative transfer to milk, but the PBB fed also contains five to six times as much hexabromobiphenyl than heptabromobiphenyl. Thus, the concentration of hexabromobiphenyl in milk was approximately 25 times the concentration of heptabromobiphenyl.
The steady-state concentration (30 to 60 days) of PBB in milk fat was about 3.1 ppm. This accounted for 18% of the daily intake, somewhat lower than is typical of chlorinated hydrocarbon compounds (14). The 3.1 ppm concentration in milk fat was about 4.5 times the concentration in the total diet. In order to provide a margin of safety, total diet concentrations should be less than 0.1 times any regulatory guideline in milk or body fat to prevent excessive residues.
Residue Elimination
We have previously proposed that a simple model, consisting of two open compartments with an irreversible exit to the exterior from the first compartment ( Fig. 7) that adequately describes the residue picture for halogenated hydrocarbon com- pounds (14). One may visualize compartment 1 as small with a rapid turnover; i.e. blood, and compartment 2 as large with a slower turnover, i.e., body fat. The third compartment is equivalent to milk and is a sink, not a true compartment. The model does not take into account elimination by metabolism or enterohepatic recirculation, but the net effect of these routes would be to reduce the value ascribed to input. We have discussed the major features of the model elsewhere (14). The concentration of PBB in milk fat when feeding stops is described by the equation where C is the concentration in milk fat, Cl is the initial concentration associated with the first compartment, C2 is the initial concentration associated with the second compartment, a and b are constants, and t is the time in days.
The shape of the concentration curve (Fig. 6) is consistent with the equation. The first term of the equation has a large rate constant and the value of the term approaches 0 within 10 to 15 days. Thereafter, the curve is described by the second term alone. Thus, the constants of the second term can be readily evaluated by least-squares fitting to the logarithmic form of the second term.
The constants of the first term cannot be accurately evaluated because of the too few points and the rapid change in concentration. In this study the concentration declined 70% within 10 days. The relative magnitude of the initial decline will be greater with a shorter feeding period and smaller with a longer feeding period (14).
The constants of the second component of the equation are of the greatest practical importance. The average value of b was 0.012/day. This is equivalent to a half-life of 58 days with a range of 49-85 days.
We have also determined the residue half-lives in 12 environmentally contaminated cows (group III, Fig. 1) over a 24-week period (2). The average halflife was 60 days but ranged from 36 to 301 days. Some of these cows did not fit the model particularly well. Some reasons for the poor fit will be discussed below.
Factors Affecting Elimination
The model (Fig. 7) and subsequent calculations of half-lives assume that the various physiological processes of the cow are at steady state. Although the conditions of this assumption can be approximated in some cases, a true physiological steady state never occurs in practice.
Three important factors could affect the half-life; the total amount of fat in the body, changes in the amount of body fat, and the level of milk fat production. Each unit of milk fat will clear the same fraction of the body burden when the amount of body fat and level of milk fat production are constant because of the constant ratio of the concentrations of PBB in milk and body fats (Fig. 5). Each unit of milk fat will clear a larger fraction of the body burden as the amount of body fat is reduced or as the level of milk fat production is increased. Conversely, each unit will clear a smaller fraction as the amount of body fat is increased or as the level of milk fat production is decreased.
Changes in amount of body fat can have a dramatic effect on the rate of excretion under some conditions. As body fat is lost, the concentration of PBB in the remaining body fat will be increased proportionally. Conversely, there will be a proportional dilution with body fat gain.
These effects were dramatically illustrated in the environmentally contaminated cows we studied (2). Those cows had suffered definite ill effects from PBB (4) and were in poor condition when studied.
Data from three representative cows studied immediately after calving are shown in Figure 8. Cows 935 and 745 showed dramatic increases in PBB concentration during the first 75 days of lactation when energy deficit and body fat loss are the greatest. However, the concentrating effect of fat loss was not universal as illustrated by cow 16. It should be noted that the concentrating phenomenon after calving has not been consistently observed in healthy well-fed cattle. Brown et al. (15) concluded that fresh cows did not have higher DDT residues than other cows in the same herd. We followed some cows from a PCB study (16) for as long as 200 days postpartum and the concentration in milk fat followed an exponential decline remarkably well (Fig. 9). Thus, the increased concentration of residue after calving is probably limited to cows that are in poor condition at calving.
The concentration of PBB in milk fat dropped rapidly for 5 to 10 days in all of the cows that we studied immediately after calving (Fig. 8). Willet and Irving (8) also noted this rapid drop after calving. We suggest that, during the dry period, PBB residues in mammary gland fat are in equilibrium with PBB in the remaining body fat. The PBB in the mammary gland is rapidly transferred to milk as lactation is initiated and after the gland is depleted, the lower transfer rate of PBB from body fat becomes the controlling factor in milk fat concentration. Compounds with larger transfer coefficients do not show this phenomenon after calving as exemplified by DDT (15) and PCB (Fig. 9). Caution should be exercised in applying our estimates of half-life despite the good agreement between our two studies. First, the ratio of PBB concentration in milk fat to body fat is wider than any other compound that we have studied and half-life should be directly related to the width of the ratio (14). We found longer half-lives with some compounds having narrower ratios which suggests that the special circumstances of our PBB studies favored shorter half-lives. Second, as lower residue levels are approached, there may be small slowturnover compartments that make a significant contribution to the milk residue. Third, in the field one may not have an entirely clean environment and the continuing low level intake would give a longer apparent half-life.
Human Exposure
The kinetic data presented here and other independent observations can be used to estimate possible exposure of farm families to PBB in the Michigan incident. The other observations include the time and level of animal exposure, observed residue levels in herds at the time the contamination was identified, and serum levels of exposed people.
Jackson and Halbert (4) provide the best information on the time and level of animal exposure. The cows were fed approximately 15 lb of a concentrate that could have contained as much as 0.4% PBB. Actual analysis of one sample from Halbert was 0.3% PBB (Fries, unpublished). The concentrate was fed for about 2 weeks in late September to early October, 1973. The intake of PBB by these cows could have been in the range of 20-28 g/day. Actual intake may have been somewhat lower because anorexia was experienced. The 0.4% level is assumed for our estimate.
If PBB was transferred from diet to milk fat as efficiently in Halbert's cows as the cows in our study (Fig. 6), milk fat concentration at the end of 15 days exposure could have been as high as 6000 ppm, with an average of 4500 ppm for the 15 days. While the intake of Halbert's cows was probably less than 28 g/day because of anorexia, milk production also dropped, and these two factors could have balanced each other. One would expect a decline to approximately 30% of the maximum exposure value (6000 ppm) within 15 days after exposure ended. This value would have been about 1800 ppm with an average of 2900 ppm for the second 15-day period.
Thereafter the concentration would be expected to decline with a first-order rate constant of 0.012/ day (half-life = 58 days). Approximately 230 days elapsed between the time of exposure (Sept. 20,1973) and the time that a significant number of herds were identified (May 10, 1974). The projected concentration at the time of identification would be 160 ppm.
Two observations support this projection. First, six randomly selected cows from the Halbert herd (group I, Fig. 1) averaged 159 ppm PBB in milk fat (range, 48-351 ppm) when killed in June 1974. Second, the Michigan Department of Agriculture (17) identified 22 herds with residue levels greater than 100 ppm. The discrepancies between the projected values and observed values are not great when one considers the possible introduction of uncontaminated replacements. Thus, the above projection provides a reasonable basis for estimating human exposure to PBB.
The following estimate of exposure of an individual in a farm family consuming its own milk assumed that 1 liter of milk containing 4% fat was consumed per day. The total PBB intake would have been 2.7 g over the 15-day period while the cows were being exposed, 1.7 g during the subsequent 15 days, and 5.4 g during the subsequent 200 days. The estimated total exposure was 9.8 g over the 230-day period. The cumulative intake over time is shown in Figure 10. Note that, if the problem had not been identified, the projected intake over infinite time would have been 10.4 g. The PBB levels in serum determined by the Michigan Department of Health provide a means of checking the reasonableness of our estimate of the higher exposures to farm families. It has been reported (18) that healthy men contain 16% fat (range 4 to 27%). Thus, a 70 kg man would contain about 11.2 kg fat. If there was no metabolism or excretion of a 9.8 g intake, the average fat level would be 875 ppm. In limited observations (19), the ratio of fat PBB to serum PBB was 232 to 1. Thus, one might predict serum PBB levels as high as 3.7 ppm. The highest serum value found by the Michigan Department of Health (20) was 3.8 ppm with a number of individuals in the range of 2 to 3 ppm. This is good agreement considering the assumptions that were made.
It is reasonable to conclude that the most highly exposed people consumed from 5 to 15 g PBB over a 230 day period. This estimate has only considered intake from milk. In a few cases it is known that individuals consumed contaminated meat and/or eggs. The number of cases where all factors coincided to provide maximum intake were probably small. However, if a cow as used in our example as slaughtered for home consumption within 45 days of initial exposure, the projected intake from meat would have exceeded the projected intake from milk (Fig. 10).
The exposure of an individual in the general population would have a pattern over time as projected for the farm family. However, the exposure level would have been many orders of magnitude less because of dilution in the normal marketing channels. The pattern of exposure over time (Fig. 10) does suggest that prevention of accidents is the only practical means of minimizing exposure. Even when efficient methods are available and used, identification will not be soon enough to prevent a major portion of the potential exposure from a "single-shot" accident. | v3-fos |
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} | s2 | Toxicity and residue studies in dairy animals with FireMaster FF-1 (polybrominated biphenyls)
Two studies (one in Holstein calves and one in Holstein cows) were conducted to determine potential toxicity and residue levels following oral ingestion of polybrominated biphenyls (PBB). The material was FireMaster FF-1. Administration was by gelatin capsules. Doses in calves were 0.1, 1.0, 10, or 100 mg/kg body weight, while doses in cows were equivalent to 0.01, 0.1, 1.0, or 10 ppm in the diet. The calves were sacrificed after 2, 4, 6, or 12 weeks. The cows were fed 158 or 228 days, and were then in a recovery period for 182 or 112 days. In the calf study, signs of toxicity were observed only in animals fed 100 mg/kg-day. Administration of 10 mg/kg-day or less for up to 12 weeks caused no overt signs of toxicity. Histologic studies were conducted upon selected organs and tissues taken at time of sacrifice. The only treatment-induced lesions among animals fed 0.1 mg/kg-day were minimal lesions in the kidney and skin in the one calf fed at this level for 12 weeks. Treatment-induced lesions were present in the kidneys, skin, and/or liver from some animals fed levels of 1.0 mg/kg-day and above. The relative severity of these lesions was related to the level and length of exposure. Treatment-associated changes were observed in the testes of all males in this study. The hypospermatogenesis observed was consistent with the age of the animals due to prepuberal development of the testes. No clinical signs of toxicity or histologic changes attributed to PBB were observed in the cows. Two cows were pregnant at the initiation of the study and give birth to normal, healthy calves during the study. These calves grew normally and appeared healthy when sacrificed at about 6 months of age. Residue levels were quantitated as the hexabromobiphenyl isomer (BP-6) which is the major isomer present in the PBB mixture. Tissue residue levels in calves increased with dose and duration of administration of PBB with highest levels being found in the fat. At 100 mg/kg the levels in fat were about 6000 to 6300 ppm after 6 to 12 weeks. Residue levels of BP-6 in milk and fat of the cows also increased with dose and duration of administration. Maximum levels in milk were about 1/3 the levels in the diet. In general, the levels plateaued after about 4 to 6 weeks and did not go appreciably higher even though administration of the FireMaster FF-1 was continued. Maximum levels in fat were on the order of approximately 4.5 times the levels in the diet, except at the lowest dose level. Residues decreased in milk after administration of PBB was discontinued, but detectable levels were still present 6 months later. Residues were found in the calves born of treated cows indicating passage of the PBBs through the placental barrier and/or ingestion through the milk.
Introduction
Toxicity attributed to polybrominated biphenyls which accidentally contaminated feed of dairy cattle was described by Jackson and Halbert (1). Therefore, the two studies reported here were initiated. The primary purpose of these studies was to study possible toxicity and residue levels following oral administration of known doses of the material. Since these studies were conducted, a study of toxicosis from polybrominated biphenyls in heifers has been reported by Moorhead et al. (2).
One of the studies was conducted to evaluate the potential toxic effects and to determine tissue residues following oral adminis;ration to Holstein calves. The other study was conducted to evaluate potential toxicity and to determine residue levels in milk, urine, feces and tissues following oral administration to mature Holstein cows.
Materials
The polybrominated biphenyls were fed as Fire-Master FF-1, which is a pulverized form of Fire-Master PB-6 with 2% calcium trisilicate added to prevent caking.
Calves
There were eight male and eight female Holstein calves, weighing 98 to 169 kg at the start of the study. Each calf was housed individually for 14 days prior to the start of the investigation.
The animals were divided into four groups of two males and two females each. Each group received daily oral doses of PBB in gelatin capsules, at dose levels of 0. 1, 1.0, 10, or 100 mg/kg body weight/day. These doses are equivalent to about 3.3, 33, 330, or 3300 ppm in the diet.
All calves were weighed each week, and the most recent weight was used for dose calculations. They were fed 1 kg of 16% grain ration daily. Hay and water were allowed ad libitum.
The calves were examined daily for signs of overt clinical toxicity.
The following determinations were conducted upon each calf just prior to the inception of the study and weekly thereafter: hematocrit, hemoglobin, erythrocyte count, total and differential leukocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, blood urea nitrogen (BUN), blood glucose, serum alkaline phosphatase (SAP), serum glutamic-pyruvic transaminase (SGPT), and serum glutamic-oxalacetic transaminase (SGOT).
One animal (male) from each group was sacrificed after 2 weeks of testing, one additional animal from each group (female) was sacrificed after 4 weeks, and after 6 weeks a third animal (male) from each group was sacrificed. The remaining female from each group was sacrificed after 12 weeks of testing. All major tissues and organs were examined grossly at time of sacrifice. The following tissues and organs were examined histologically: adrenal glands, brain, pituitary, gonads, heart, kidneys, liver, lungs, skin, spleen and thyroids.
Samples of liver, kidney, fat, and skeletal muscle taken from each animal at sacrifice were analyzed for BP-6 content.
Cows
Eight Holstein cows were used. Each cow was individually stanchioned for 14 days prior to the start of the investigation. All animals were in good health, mastitis-free, and tested TBand Brucellosis-negative.
They were divided into four groups of two cows each. Each group received daily oral doses of PBB in gelatin capsules, equivalent to 0.01, 0.1, 1, or 10 ppm in the diet (based on a daily food consumption of 17 kg per cow). The total daily food consumption was 4.5 kg of a 16%o grain ration and approximately 12.5 kg of clover-prairie hay daily; water was permitted ad libitum. The levels were equivalent to about 0.0003, 0.003, 0.03, or 0.3 m',/kg body weight per day.
The material was administered for 158 consecutive days to six of eight cows and for 228 consecutive days to the other two cows. The latter two cows (No. 2, 0.01 ppm and No. 6, 1 ppm) were pregnant. Therefore, administration was continued until their calves were about 2 months old. Administration of PBB was discontinued and the cows were placed on recovery for 182 days (6 cows) or 112 days (2 cows). The total duration of the experiment was 340 days.
Total daily milk production was recorded throughout the investigation. Initial, weekly and final body weights were recorded throughout the investigation. Observations for mortality, general behavorial reactions and pharmacotoxic signs were conducted daily.
The following determinations were conducted upon each cow just prior to the inception of the study and weekly (new calves, too) thereafter, hematocrit, hemoglobin, erythrocyte count, total and differential leukocyte counts, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, blood urea nitrogen (BUN), blood glucose, serum alkaline phasphatase (SAP), serum glutamic-pyruvic transaminase (SGPT), serum glutamic-oxalacetic transaminase (SGOT), albumin, glucose, pH, microscopic examinations leukocyte-erythrocyte. Thyroid function tests, T-3 or triiodothyronine by resin uptake, T4 or thyroxine by column and free thyroid index by calculation, were conducted 3 weeks before the end of the study in the cows.
At the end of the investigation the animals were sacrificed. All major tissues and organs were examined grossly during the necropsy. Specimens of the following tissues were preserved in 10% neutral buffered formalin for histological examination: adrenal gland, brain, bronchial lymph node, gall bladder, gonads, heart, kidneys, liver, lung, pituitary, spleen, thyroids, and bone with marrow.
Milk samples were collected weekly during the test period, with the collections for the first 90 days analyzed for BP-6. Weekly milk samples also were collected and analyzed during the recovery period. Blood, urine, and feces were collected weekly for the first 90 days and analyzed for BP-6. Samples of liver, kidney, fat, skeletal muscle, and bone were obtained at sacrifice for analysis of BP-6 content.
Extraction and clean-up techniques were developed and optimized for each type of sample to be analyzed. Gas-liquid chromatography (Tracor MT-220 gas-chromatograph with a 63Ni detector) was used to quantitate residues.
The analytical methods for milk, blood, fat, bone marrow, feces, and urine were validated by analyzing control tissues fortified at the limit of sensitivity of each method with BP-6 analytical standard. The recoveries obtained average 100% for milk at 1 ppb, 98% for blood at 5 ppb, 100% for fat and bone marrow at 50 ppb, 76% for feces at 5 ppb, and 84% for urine at 5 ppb. The highest fortification level made in fat was at 150 ppm, 93% recovery was obtained. The precision of analysis of selected tissues was determined by analyzing six aliquots of each tissue type after homogenization and fortification at the limit of sensitivity. Relative standard deviations of less than 9% were obtained.
Calves
Generally, poor weight gains or body weight losses were noted among animals at the 100.0 mg/kg level. Two calves (male 14 and female 15) lost 8 kg each, while a third calf (female 16) lost 22 kg. All calves in the other groups had normal weight gains.
Calves 14 and 16 (100 mg/kg) had decreased food consumption from test day 36 until sacrifice. There were no other differences in food consumption values among calves at any other level.
No deaths occurred. On test day 21, calf 15 (100 mg/kg) became lethargic and remained in this condition until it was sacrificed on day 28. Calves 14 and 16 (both 100 mg/kg) had signs of keratitis, and lacrimination beginning on day 35 which continued until time of sacrifice. In addition, on test day 50, calf 16 developed alopecia and hyperkeratosis involving the head, cervical, and dorsal thoracic region that progressed in severity until sacrificed.
Although slightly elevated from day 21, the elevated total leukocyte count was apparent on test days 77 and 84 (weeks 11 and 12) for female 16 at the 100 mg/kg level. Additionally, differential leukocyte counts revealed a reverse lymphocyte-neutrophil ratio from day 42 which was especially noticeable on test day 84, when it was sacrificed. Values for all other hematologic parameters were essentially the same among the other groups.
There were increases in SGOT and BUN values during the final 2 weeks (days 77 and 84) in blood from the animal (female 16) fed 100 mg/kg. In the same calf there were decreased SAP values after day 49. Male 14 (100 mg/kg) also had lower SAP values on days 35 and 42, at which time he was sacrificed. Values for all other animals were comparable and considered normal among groups for the clinical chemistry parameters investigated.
The histopathological evaluation of a series of tissues from the 16 calves revealed treatment-induced lesions in the kidney, liver, and skin. Lesions were also observed in the testes which were treatmentassociated and ascribed to several factors including the nutritional status, age of the animal (prepuberal development), and duration of exposure to PBB. One or more of the above tissues were affected in animals from all test groups. The relative severity of the treatment-induced lesions was related to dose level and length of exposure to the test material.
The treatment-related renal lesions comprised a wide spectrum of degenerative changes which represent various st-ages in the development of tubular nephrosis. The most severe lesions were present in female 16 which was exposed at the highest dose level (100 mg/kg) for 12 weeks. The renal lesions observed among animals exposed at the lower test dose levels for shorter periods of time were of lesser severity (minimal) and they consisted of focal dilatation of cortical tubules. Some affected tubules contained exfoliated epithelial lining cells within their lumen. Among animals at the higher dose levels or those exposed for a longer period of time, the tubular dilatation was slightly more severe, more extensive and was accompanied by regeneration of tubular epithelial cells. Some of the affected tubules also contained proteinaceous or granular casts mixed with cellular debris. Lesions of interstitial fibrosis and compression of glomeruli were also present in the kidney from calf 16.
Treatment-related hepatic lesions were confined to animals in groups fed 1, 10, or 100 mg/kg. Two animals (Nos. 6 and 8) at the I mg/kg level that were exposed to the test material for 6 to 12 weeks, respectively, had foci of slightly enlarged hepatocytes located in the centrilobular region of some lobules. The enlargement of hepatocytes was due to an increase in cytoplasmic mass with no nuclear changes. However, there was no significant increase in the liver weights of these animals. This lesion was not evident among animals at other dose levels. The liver of one female (No. 11) at the 10 mg/kg level and two females (Nos. 14 and 16) at the April 1978 100 mg/kg level revealed degeneration and/or necrosis of individual or small foci of hepatocytes with no lobular distribution. The necrosis of hepatocytes was accompanied by liver cell regeneration among those animals fed 100 mg/kg of PBB.
Treatment-related skin lesions were present among single female animals at 0.1 mg/kg (No. 4), 10 mg/kg (No. 12), and 100 mg/kg (No. 16) levels exposed for 12 weeks to PBB. These changes consisted of either acanthosis, hyperkeratosis and/or dermal infiltrates of mononuclear cells. The changes were similar in nature among all three animals but they were most severe in animal No. 16.
Treatment-associated changes were present in the testes and epididymides of all males in this study. The changes described as hypospermatogenesis of the testes and the absence of sperm in epididymal ducts are consistent with the age of these animals due to prepuberal development. Degeneration of the germinal epithelial cells lining seminiferous tubules was also present in many of these bull calves. The severity of the degenerative change was variable but may have resulted from impairment in the nutritional status of these animals rather than a direct effect of the test material.
Other lesions found were attributed to naturally occurring diseases or related to the method of sacrifice.
Samples of liver, kidney, fat, and muscle obtained from each calf at the time of sacrifice were analyzed for BP-6 residues. The results of the analysis are presented in Table 1.
The tissue residue concentrations of BP-6 were directly related to the dose level and the period of exposure. The highest levels of BP-6 were found in the fat. Lower levels were found in kidneys, liver, and muscle.
Cows
Milk production ranged from 7.6 to 31.4 lb/day prior to administration of the PBB. The production in two cows (Nos. 2 and 6) dropped considerably prior to parturition. After calving, even though administration of PBB continued, their production increased to about 30 lb/day for cow 2 and 50 lb/day for cow 6. Milk production was decreased in all cows during the latter part of the recovery period. This is consistent with the normal state and cycle of lactation.
The body weights of the cows ranged from 446 to 591 kg at the start of the study. All animals gained weight during the study. The two pregnant cows lost weight after delivery, as would be normally expected after parturition. The two calves weighed 46 and 50 kg, respectively, at birth. They had normal weight gains and at about 6 months of age they both weighed 229 kg.
There were no differences in food consumption values among cows at any levels. All were considered normal in this respect.
No deaths occurred during the investigation. The following reactions were observed. On day 71, cows 2 (0.01 ppm) and 5 and 6 (1 ppm) were stomping their fore and hindfeet and appeared uncomfortable. This lasted until test day 86 for cow 2 and day 75 for cows 5 and 6. On test day 240, a subcutaneous accumulation of fluid (about 200 ml) was observed over the right rib cage of cow 2. The fluid began to subside on test day 149 and was no longer evident Environmental Health Perspectives by day 157. On day 129, cow 5 had slight overgrowth of the hooves involving all feet which increased in severity for the remainder of the investigation. Hematologic data revealed no treatment-related changes among any of the parameter investigated. Cow 6 had an elevated total leukocyte count throughout the study with lymphocytes comprising a high percentage of the cells.
Clinical chemistry values were normal and reflected no changes related to treatment in any of the cows or in the two calves. The values obtained for thyroid function were considered to be normal for cattle of this age. These values compare favorably with those reported for Holstein cows in another study (3).
The results of the analyses for residues of BP-6 in milk are presented in Tables 2 and 3. The residues increased as the dose increased (0.01 ppm through 10 ppm in the diet). In general, the levels plateaued after about 4 to 6 weeks and did not go appreciably higher even though administration of the Fire-Master FF-l was continued.
Maximum levels of BP-6 found in milk in relation to levels in the diet were about 0.06, 0.07, 0.03, and 3 ppm at 0.01, 0.10, 1.0, and 10 ppm in the diet, respectively. After administration of FireMaster FF-I was stopped, the levels of BP-6 in milk were observed to decrease rather slowly. Some BP-6 was still detectable in milk from animals of all groups after the recovery period (approximately 6 months).
In most cases BP-6 was not detected in urine (limit of detection 0.005 ppm) after 90 days of administration, even at 10 ppm of PBB in the diet.
BP-6 was barely detectable in feces from some cows fed 0.01 ppm. Levels in feces in cows at other dose levels were about 0.02, 0.15 or 1.5 ppm at 0.1, 1.0 or 10 ppm in the diet, respectively. It appears that about 15% of the ingested dose is excreted in the feces.
Residues in blood were detected (lower limit 0.01 ppm) only in cows fed 10 ppm. The levels of BP-6 in their blood were in the order of 0.05 to 0.1 ppm.
The residues in bone marrow and fat from three sites (tail, stomach and brisket) for the cows and calves at sacrifice are presented in Table 4. The 0 3 7 14 21 28 35 42 49 56 63 70 77 84 91 166 229 1 residue levels, in general, increased as the level of oral intake increased. The two cows with shorter (112 days) recovery periods had higher residues then the other two at the same feeding level with longer (182 days) recovery periods. Levels in the fat were on the order of 50 times the levels in the 0.01 ppm diet and 4.4 times the levels in the 0. 1, 1.0, and 10 ppm diets. Gross and histopathological evaluations were conducted on a series of tissue from the eight cows and two calves. The lesions described were regarded as either those of naturally occurring diseases related to the age of the animal or ascribed to the method of sacrifice. With regard to the latter, there was focal hemorrhage in the lung and brain. The findings in the ovaries were indicative of a normal estrus cycle in the cows.
Discussion and Summary
One of these studies was conducted to evaluate the potential toxic effects and to determine tissue residues of BP-6 after oral administration to Holstein dairy calves. Dose levels of 0. 1, 1.0, 10.0, or 100.0 mg/kg were administered daily in gelatin capsules to groups of four calves (two males and two females) for 2 to 12 consecutive weeks. Calves were sacrificed at 2, 4, 6, and 12 weeks. Animals at the highest dose level lost weight and displayed alopecia, keratitis, and lacrimation from test day 35 until their sacrifice. There were increased BUN, SAP and SGPT values at the highest dose. Histopathologic examination of organs and tissues revealed treatment-induced lesions in the kidney, liver and skin which were dose related. Treatmentassociated changes were present in the testes among all males. Residue analyses of liver, kidney, muscle, and fat revealed treatment and time related increases in the concentrations of PB-6. Higher concentrations were found in fat than in the other tissues analyzed, with the lowest concentration found in muscle.
The second study was conducted to evaluate potential toxicity and to determine milk and tissue residue levels of BP-6 after oral administration to Holstein dairy cows. Doses were equivalent to 0.01, 0.1, 1.0, or 10.0 ppm in the diet and were also administered in gelatin capsules. The levels were based on a total daily food consumption of 17 kg per cow. Test material was administered daily for 158 consecutive days with the exception of two cows which were pregnant. Those cows received the test material for an additional 70 days. No effects upon milk production, body weights, amount of food consumed, and hematologic clinical chemistry, and urinalysis studies were noted. No deaths occurred. Analysis for BP-6 in milk, blood, feces, urine, and fat revealed a dosage related increase. However, after approximately 4 to 6 weeks of PBB administration the residue levels in milk plateaued and there appeared to be no further accumulation as the residue levels remained relatively constant. Gross and histopathologic studies conducted on all cows revealed no treatment-related lesions. Two cows were pregnant when the study was initiated. These cows were treated during pregnancy and allowed to calve. No problems occurred at parturition, and both calves were normal in appearance and healthy from birth to sacrifice. Though neither calf was treated, tissue residue analysis for BP-6 showed its presence, indicating the passage of the compound through the placental barrier and/or ingestion through the milk. | v3-fos |
2018-04-03T05:35:58.486Z | {
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} | s2 | Effect of low-protein diets on free amino acids in plasma of young men: effect of protein quality with maintenance or excess energy intake.
Hematological changes due to protein deprivation were studied in 34 young Japanese men who were given a standard diet with an N intake of 200mg/kg for one week and then low-protein diets with maintenance (45 +/- 2 kcal/kg) or excess (57 +/- 2 kcal/kg) energy and N intakes of about 44 to 99 mg/kg of whole egg or about 50 to 121 mg/kg of polished rice for three weeks. In the period of the low-protein diet the concentrations of most individual free plasma essential amino acids (EAA) decreased significantly in general, and thus the total EAA concentration decreased significantly. Lowering of the valine concentration and elevation of the alanine concentration were the highest changes. The total nonessential amino acid (NEAA) concentration increased significantly in men fed a low-rice-protein diet, but not in those fed a low-egg-protein diet. Consequently, in the former group the ratio of essential to nonessential amino acids fell significanly from 0.96 in the control period to 0.61--0.74 in the period of consuming the low-protein diet. The effect of protein deprivation on the plasma EAA concentration was also larger with egg protein than with rice protein, and the total EAA concentration of men fed egg protein changed significantly and in parallel with the N intake over the range of 44 to 99mg/kg. The decreases in serine and threonine and increase in alanine tended to be more when energy intake was over the maintenance level. The concentration of plasma proteins and especially albumin decreased significantly during the period of consuming the low-protein diet. The interrelation of plasma-free amino acids, the amino acid pool in tissures and dietary N and energy intakes is briefly discussed.
Hematological changes in protein deficiencies have been studied extensively in men and animals. YOSHIMURA (1) showed that measurements of the concentrations and total amounts of plasma protein and hemoglobin were useful methods for detecting protein deficiency in men. WHITEHEAD (2) found a characteristic change in 297 the pattern of plasma-free amino acids in infants with PEM and proposed a "nonessential to essential amino acid ratio" as a criterion for diagnosis of this condition. Abnormal amino acid ratios have also been observed by others in human infants (3,4) and adults (5) and in rats (6). There is also evidence that plasma-free amino acids are influenced by not only protein deficiency but also energy intake and other factors. GRIMBLE and WHITEHEAD (7) examined the effect of glucose adminis tration on plasma-free amino acids in kwashiorkor. LUNN et al. (8) showed that the plasma amino acid pattern was affected by the changes in hormone balance that resulted from decrease in food intake. This paper reports studies on the effect of low-protein diets of different qualities supplying maintenance or excess energy on the concentrations of plasma-free amino acids and other blood constituents in young men.
EXPERIMENTAL
The subjects tested were 34 healthy male university students of 20 to 27 years old. They were given a standard diet containing about 200mg N/kg of mixed protein for Hematological tests were carried out at the end of the preliminary period and once a week in the standard period. Blood was withdrawn from the antecubital vein at about 7:00a.m., before breakfast, for measurements of free amino acids, hemoglobin, plasma protein and albumin, the hematocrit and the circulating plasma volume. Plasma specimens for amino acid analysis were prepared by the procedure of STEIN and MOORE (10) with a slight modification (11), and analyzed in an automatic amino acid analyzer.2 The levels of Thr, Val, Cys, Met, Ile, Leu, Tyr, Phe, and Lys as essential amino acids (EAAs) and Asp, Ser, Glu, Gly, Ala, His, and Arg as nonessential amino acids (NEAAs) were measured. Plasma protein was calculated to be 6.55 times the difference between the total and non-protein nitrogen levels, determined by the Kjeldahl method. Plasma albumin was determined col orimetrically (12). Hemoglobin was measured by the cyanmethemoglobin meth od (13) and the hematocrit by high-speed centrifugation in a capillary (14). The circulating plasma volume was determined by the dilution method with Congo red (15) and the circulating blood volume was calculated using the hematocrit value. The total amounts of plasma protein and hemoglobin were also calculated.
RESULTS
Changes of the free amino acid concentrations in the plasma of subjects given low protein diets The mean plasma amino acid concentrations after 3 weeks on a low-egg-, or rice protein diet are compared with those in the preliminary period in Table 2. The mean values for all 34 subjects at the end of the preliminary and experimental periods are also shown graphically in Fig. 1.
The control aminogram obtained in the standard period is comparable with those reported by others (16)(17)(18). Most of the EAAs decreased significantly in the experimental period, the reductions in valine, leucine, and lysine being the greatest with both proteins. The total amount of EAAs was 16% less than the control with low-rice-protein and 23% less than the control with low-egg-protein. Among the NEAAs, alanine increased significantly with both diets and glycine increased significantly with rice protein but not with egg protein. Unlike in rats (19), in human subjects serine was not affected with a maintenance energy intake and tended to decrease with excess energy intake. Thus with the low-rice-protein diet the total 2 Yanagimoto Mfg . Co., Ltd., Yanaco, Model LC-5S. amount of NEAAs was significantly higher than with the control diet , whereas with the low-egg-protein diet it was not; moreover , the ratio of EAA to NEAA (E/N ratio) fell significantly from a mean of 0.96 in the control period to 0 .65 with low-rice protein and to 0.74 with low-egg-protein diet. As seen in Fig. 2, the total EAA concentration and E/N ratio decreased markedly at the first week and then remained at lower levels.
With both proteins, fall in threonine and rise in alanine were more marked with excess energy intake than with maintenance energy intake ( Table 2) . With egg protein, the total EAA concentration was significantly lower with excess energy than with maintenance energy. With each protein , there was no significant difference between the total NEAA concentrations in the groups with excess and maintenance energy intakes because increase in alanine occurred concomitantly with decrease in serine concentration in the groups with excess energy intake . Y. YOSHIMURA, and G. INOUE Correlation between the free amino acid concentration in the plasma and protein intakes in the submaintenance range The correlations between the plasma levels of free amino acids and dietary N intake in the range of about 44 to 121mg/kg were tested statistically ( (Table 3, last column). In general, with egg protein the correlation coefficients for individual amino acids were estimated to be comparably high, while with rice protein they were considerably lower and in many cases were inverse. Alanine and histidine in rice and valine in egg were relatively sensitive to protein deficiency, and thus there was a significant correlation between total EAA and N intake with egg protein. This correlation is shown graphically in Fig. 3. No significant correlation was observed with rice protein.
Changes in hemoglobin, plasma protein and other characters
Other hematological data are summarized in Table 4. A low-protein diet for 3 weeks did not decrease the concentration or total amount of hemoglobin signi fi cantly, but decreased the plasma protein, affecting the concentration more than the total amount. The mean albumin concentration in 13 subjects decreased significantly, being 4.54g/100ml in the control period and 4.09g/100ml after one week of the experimental period (Fig. 4). No change was observed in the hematocrit or circulating plasma volume and supply of excess energy did not affect the values either.
DISCUSSION
As shown here and by others (2)(3)(4)(5)(6), the fall in the E/N ratio in the plasma of subjects given low-protein diets was mainly caused by decrease in the concentration of total EAA, and especially branched-chain amino acids, and partly by increase in the concentration of alanine. The decrease in concentration of plasma EAA was primarily due to reduced supply of these amino acids but it was also related to energy supply, because addition of extra energy to the low-egg-protein diet resulted in reduction of plasma EAAs. Further evidence that decrease in the levels of plasma EAAs in protein deficiencies may be intensified by supply of excess energy is as MA FREE AMINO ACIDS IN MEN 305 Table 4. follows: (i) LUNN et al. (8) observed hyperinsulinemia resulting from ingestion of high-carbohydrate meals during the early stage of kwashiorkor and suggested that utilization of amino acids in tissues was enhanced in this condition; (ii) We found previously (9) that the nitrogen balance and NPU observed with submaintenance N intakes were greatly affected by the protein sparing effect of energy intake; (iii) MUNRO et al. (20) showed that the lowered levels of plasma amino acids observed in subjects loaded with glucose were due to enhanced utilization of the amino acids in the muscle through the action of insulin; and (iv) recently, GRIMBLE and WHITEHEAD (7) also reported similar findings to (iii), in patients with kwashiorkor after administration of glucose.
In this work the levels of branched-chain amino acids decreased most during protein deficiency, and this is consistent with findings in cases of kwashiorkor (21). The threonine concentration was significantly lowered in our subjects receiving excess energy; however, it was reported to increase in obese subjects during starvation (22). This discrepancy needs further study . LUNN et al. (8) observed that the plasma alanine concentration in infants with kwashiorkor was increased by high-carbohydrate food . Moreover, ADIBI (17) observed a high concentration of plasma alanine in adults fed a protein-free diet , but a low concentration in starved subjects. Furthermore , GRIMBLE and WHITEHEAD (23) reported high alanine levels in children fed diets low in protein but adequate in energy. We found that elevation of plasma alanine was intensified by supply of excess energy ( Table 2). Supply of energy probably increases the alanine con centration because it reduces gluconeogenesis, for which alanine is a major substrate (24).
In contrast to plasma alanine, plasma serine was normal or slightly reduced in protein deficiency. These findings are consistent with those of YOUNG et al. (25). On the other hand, FALLON et al. (26,27) observed that in rats on low-protein diet , serine degrading enzyme activity decreased and serine synthetic activity in the liver increased, suggesting a decrease of plasma serine. We have also observed marked increases of plasma and liver serine in adult rats on a low-protein diet (unpublished data3). Species differences in serine metabolism in protein deficiency must be examined.
We found a significant correlation between the total EAA concentration in the plasma and N intake over the range of 44 to 99mg/kg in subjects given a low-egg -protein diet, but not in those given a low-rice-protein diet. YOUNG et al. (25) observed a similar correlation in young men receiving 0.2 to 0 .5g/kg of a low-egg-protein diet. However, in our subjects the E/N ratio was not significantly correlated with the protein intake, indicating that individual amino acids in the plasma were affected in complex ways by not only the protein level but also the protein source and energy content of the diet.
Our previous work showed that the protein sparing action of energy intake had more effect than the protein intake itself on N utilization in men with a submaintenance intake of N (unpublished data by WOVE et al. 4). SINGH et al. (28) observed increased levels of plasma EAAs in marasmic children and similar findings were obtained by SALEM et al. (6) in protein-energy-deficient rats. These observations suggest that increase in plasma EAAs may be due to protein catabolism in skeletal muscle during energy deprivation. To elucidate the nutritional significance of change in the level of plasma amino acids in protein deficiency, the dynamic states of plasma amino acids in relation to amino acid pools in the tissues must be clarified (29,30). The relationship between the effects of nitrogen and energy intakes on amino acid utilization must be studied.
Many years ago, in comprehensive long-term studies YOSHIMURA'S group (1, 31-33) observed significant decreases in the concentrations and amounts of plasma proteins and hemoglobin in young men receiving a low-protein diet. In the present study, the concentration of plasma protein, and especially albumin, was also found to be a sensitive parameter of protein intake. This finding is also consistent with results in malnourished children by DUZEN et al. (34) and PADILLA et al. (35) and in protein-deprived dog by HEARD et al. (36). As reported previously (9), the mainte nance N requirements for subjects with a maintenance energy intake were about 90 mg/kg for egg protein and about 120mg/kg for rice protein. This maintenance level of N intake corresponds to an E/N ratio of 0.71 and plasma protein concentration of 6.91g/100ml and these values are significantly lower than those of 0.95 and 7.30g/100ml, respectively, for the same subjects in the control period. This implies that an N intake sufficient to maintain a zero balance may not be sufficient to maintain hematological values at physiologically normal levels. 4 Inoue , G., Kishi, K., Miyatani, S., Fujita, Y. and Yamamoto, S.. Interrelationship between the effects of energy and nitrogen intakes at near maintenance level on egg protein utilization in young men. | v3-fos |
2016-05-04T20:20:58.661Z | {
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} | s2 | Ten years experience with cow indexing
HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. TEN YEARS EXPERIENCE WITH COW INDEXING (*) S. Trodahl, O. Syrstal
tion for breeding bulls. There we have the problem that the average genetic values of the bulls used, and the applied selection intensities on the female side are very different from one herd to the next. It will be shown, how far these difficulties could, at least in theory, be successfully dealt with. Cow indexing has been utilized in the Norwegian breeding scheme since 19 6 7 . The indexes are estimated relative breeding values for milk production, based on individual production, herd average, .and indexes of sire and dam. The overall average of indexes computed is slightly above ioo. -The standard deviation of the index is estimated at 4 . 5 units. Cows with indexes above I IO (i.e. > -f-z, st. dev.) are recommended for planned matings with selected bull sires. The final selection of bull dams is carried out when bull calves are born from these matings. Besides the cow index, other traits like ease of milking, fertility and conformation are considered.
The average index of bull dams have in recent years been II4 , about 2 .7 standard deviations above the overall average, corresponding to the top o. 9 p. 100 from a normal distribution.
In intra herd selection a partial regression of daughters on dams of 17 kg FCM per index unit has been found. This in only about 70 p. i o 0 of what should theoretically be expected. This may be due to a number of causes.
The accuracy of cow indexes as a basis for bull dam selection can not be studied satisfactorily since only sons of cows with the highest indexes are,entering A.I. service and subsequently progeny tested.. However, positive correlations have been found, but these have not proved to be significant. All parameters relevant for the estimation of cow's breeding value int he German Fries,ian population of lower Saxony were analysed on a sample of I3 , 0 6 3 .cows with 3 complete la!tations. Special attention was paid to, systematic errors. , '. The investigation was based on a mixed model analysis of variance and least-squares techniques were applied. The following results were obtained: i. All effects, obtainable through official milk recording, as region, calving season, age at calving, herd level and calving interval turned out to be significant and should be adjusted for when estimating cow's breeding value. Special attention should be paid to the fact that the effect of calving interval is more distinct on the current lactation (effect of pregnancy) than on the subsequent lactation. The influence of age at calving, however, might be neglected in the case of subsequent lactations.
2 . Interactions are obvious between calving season and specific conditions in different regions and there are some indications of an interaction between calving season on production level of the year. However, due to the large variation in production conditions within regions ;, only. !LIimited advantage in adjusting for such interactions seems to be obtainable by applying corr g etionffa q tors within grassland and fodder cropping areas. 3 . Regardiiig the, differences in production potential, production systems and farm structures between regions and countries no general procedures for adjusting for systematic effects can be put forward; The main problem to be solved in each individual situation is to optimize the accuracy of environmental description and the validity of the contemporary mean. ' | v3-fos |
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} | s2 | Freeze-fracture observations of the lactating rat mammary gland. Membrane events during milk fat secretion.
Membrane events during milk fat secretion were analyzed by freeze- fracture of the rat mammary gland. Two modes of milk fat secretion were observed: extrusion of fat droplets surrounded by a portion of the apical plasma membrane of the alveolar epithelial cells and, less frequently, release into the alveolar lumen of fat droplets contained in intracytoplasmic vacuoles. The extrusion process consists of two asynchronous events: clearing of membrane particles (probably including integral membrane proteins) and bulging of the apical plasma membrane. Most fat droplets are extruded with a bilayer membrane envelope (milk fat globule membrane) partially devoid of particles. The segregation of membrane particles may represent the onset of a process of structural degradation of the milk fat globule membrane.
Under the complex hormonal environment and other stimuli of pregnancy and onset of lactation, the resting mammary gland is transformed into an actively secreting organ (8,40). This process of growth and differentiation leads to the extensive development in the alveolar epithelial cells of rough endoplasmic reticulum, Golgi apparatus, and other endomembranes which are involved with the plasma membrane in the synthesis and secretion of milk (2-4, 9, 15, 16, 20, 28, 35, 37, 42).
The ultrastructural, physiological, and biochemical aspects of lactation have been investigated (4,9,16,23,35,42). Early ultrastructural observations demonstrated that, during milk fat secretion, most lipid droplets are extruded into the alveolar lumen with a bilayer membrane envelope derived from the cell plasma membrane (2,3). This secretion mechanism, as well as biochemical (10,21,28,30) and morphological (10) evidences, led to the deduction that in freshly drawn milk the fat globule is still surrounded by a membrane (milk fat globule membrane, MFGM; for review see references 1,29). Other studies provided additional aspects of milk fat secretion, namely on the origin, composition, and mechanism of formation and stabilization of the MFGM: (a) Golgi vesicles have been reported to contribute directly to the formation of the MFGM (45); (b) lipid droplets included in intracellular secretory vacuoles can be secreted without participation of the plasmalemma (47); (c) after secretion, the membrane of the milk fat globule may undergo a process of vesiculation and fragmentation and is gradually lost (14,38,44,46); (d) some fat droplets are extruded with a crescent of cytoplasm TxE JouaNAL OF CELL BIOLOGY 9 VOLUME 76, 1978 " pages 767-778 (12,13,22,36,37,39,48); and (e) enzymic and chemical analysis of the MFGM detected "marker enzymes" of Golgi apparatus (24,25,34) and endoplasmic reticulum (17,25).
We have studied the freeze-fracture morphology of the apical membrane of the rat mammary gland during lactation. Our results show that the process of extrusion of fat droplets generally coexists with regional segregation of integral components of the apical plasmalemma.
MATERIALS AND METHODS
The inguinal mammary glands of Spragne-Dawley rats, aged 8-10 wk during the first pregnancy and 20-22 wk during the second pregnancy, were used 2 days before and 1, 4, 6, and 14 days after parturition. The animals were anesthetized with 0.2 g of sodium pentobarbital, intraperitoneally, and the tissue samples were fixed by immersion in 3% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, for 2 h at 4~ After fixation the specimens were washed in buffer, gradually impregnated in 28% glycerol in 0.1 M sodium cacodylate, pH 7.4, placed on gold specimen holders, and rapidly frozen in partially solidified Freon 22 (26). The specimens were freeze-fractured at -ll0~ in a Baizers 300 apparatus (Balzers AG, Balzers, Lichtenstein) equipped with a turbomolecular pump, shadowed at 45 ~ with a platinumcarbon electron gun, and reinforced by carbon at 90*. The replicas were cleaned with a 5% sodium hypochlorite solution containing 2.5% sodium deoxycholate and 2.5% Triton X-100 for 24 h, followed by a 2:1 chloroform:methanol mixture, rinsed in distilled water, mounted on uncoated grids, and observed with a Jeol 100C electron microscope at 80 kV. The micrographs are mounted with shadow direction from bottom to top; shadows are white. Interpretation and nomenclature of fracture faces are given elsewhere (32).
RESULTS
The lactating rat mammary alveoli are formed by actively secreting cuboidal epithelial cells, arranged in a monolayer around a lumen where casein micelles, fat droplets, and other secretory products are collected (Fig. 1). The apical plasma membranes of these cells delimitate the lumen and are directly involved in the secretory process. The lateral membranes between neighboring cells present junctional complexes for intercellular contact and sealing of the alveolar lumen ( Fig. 1 ; see reference 33 for illustrations). The basal membranes, in close relation with myoepithelial cells, capillaries, and extracellular connective structures that surround the alveoli, are involved in the uptake of blood-borne substances essential for milk biosynthesis.
Before the onset of fat secretion, a large amount of fat droplets is accumulated in the apex of the mammary epithelial cells. The lumina of the alveoli are already filled with numerous milk protein components. The casein granules are easily identiffed by their typical profiles consisting of fiat, circular clusters of rugosities without enveloping membrane. The fracture-face A of the apical plasma membrane presents an apparently random distribution of numerous membrane particles, and views of microvilli. The particles are also present in the fracture faces of the microvilli.
The observation of the apical plasmalemma during lactation revealed structural alterations related to milk fat secretion (Figs. 2-11, and 13). A mild or pronounced bulging of microvilli-free regions of the membrane was observed. These areas were either smooth, i.e. devoid of particles ( Fig. 2) or, less frequently, showed no alteration of particle distribution in relation to the surrounding apical plasmalemma. Fractures which allowed for simultaneous view of the plasma membrane and cytoplasm (Figs. 3, 5, and 7-9) showed that the bulges were occupied by fat droplets, sometimes associated with small portions of intracellular components. The events of fat droplet extrusion could be reconstructed from views of A and B faces of the apical plasma membrane and crossfracture profiles. These included the gradual enveloping of the fat droplets by the apical plasmalemma. Frequently, the fracture-face A of the fat droplet membrane envelope displayed a smooth, particle-free surface in continuity with apical plasma membrane regions which presented an unaltered pattern of particle distribution (Figs. [3][4][5][6]. Equivalent aspects of membrane particle clearing on the fracture-face B were also observed ( Fig. 8). Less frequently, the fat droplets were enveloped by a membrane presenting a fracture face with particles ( Fig. 9). Intermediate aspects consisting of emerging droplets surrounded by a membrane with some particle-free areas and the consecutive extrusion of more than one fat droplet were also observed. Other observations suggested that the secretion of larger fat droplets could also occur by extrusion of its components enveloped by a particle-free membrane as if the fat was being squeezed through a "locus minoris resistentiae" of the cell membrane (Figs. 10 and 11).
In the cytoplasm of the lactating cells most fat droplets showed smooth fracture faces. By cross fracture they often displayed a lamellar or paralamellar organization. Intracellular vacuoles con- lipid droplets surrounded by numerous, small (fat?) globules with a smooth fracture face were occasionally observed (Fig. 12). The following observation suggested that the secretion of these fat droplets followed a mechanism different from that of fat droplets which have no membrane: intracytoplasmic vacuoles were observed in close contact with the apical plasma membrane, and the region of the alveolar lumen directly opposite to these vacuoles presented emerging particle-free globules suggesting that fat was being released through a small opening in the plasmalemma (Fig. 13).
In the alveolar lumen, milk fat appeared as globules, heterogeneous in size and structure (Figs. 1, 14, and 15). Fracture usually followed a plane close to the surface; most fracture faces were smooth (Fig. 1). 1 Less frequently, a membrane with particles was seen around one or more globules (Fig. 14). In this case, the pattern of distribution of particles on the membranes enveloping fat globules ranged from uniform to segregated (Fig. 14) or reticulated. Cross fracture of the fat in the lumen revealed aspects similar to those observed in the cytoplasm (Figs. 14 and 15). Fat globules surrounded by cytoplasmic crescents were also observed. Other observations suggested the incorporation of small lipid droplets in larger globules, as well as the blebbing off of The smooth surface of most fat globules observed in the alveolar lumen probably corresponds to the fracture face of the fat globule membrane in view of the clearing of membrane particles during extrusion, and the concept of membrane splitting in freeze-etching (31). In some instances it might also correspond to the fracture through an outer lipid lamella of the fat globule. In this case, the cross-fracture profile of the fat globule membrane should often be evident (see Figs. 5 and 14).
segments of the membrane envelope of the fat globules (Fig. 15).
DISCUSSION
Analysis of freeze-fracture results indicates two possible modes of milk fat secretion: (a) extrusion of the fat globule surrounded by a portion of the apical plasma membrane, the prevalent mechanism; and (b) release into the alveolar lumen of free fat globules, i.e. without enveloping membrane, a process observed much less frequently and restricted to the secretion of small fat droplets contained within intracellular vacuoles.
The fracture faces of the apical plasmalemma during the extrusion of fat droplets illustrate two membrane events: clearing of membrane particles and bulging of the plasma membrane. In some instances, as the droplets are pushed out of the cell there is a progressive clearing of particles in the areas of membrane bulging so that, at the moment of separation from the cell, the globule is surrounded by a bilayer membrane without particles. In others, the extrusion process is not preceded nor accompanied by clearing of membrane particles, and so, the extruded fat globules are enveloped by a membrane with particles. Intermediate aspects, i.e. fat droplets extruded with a variable degree of membrane particle clearing, are also found. In consequence, our results demonstrate an asynchronism between bulging of the plasma membrane and redistribution of integral membrane components, as revealed by the progressive clearing of particles. The extrusion of fat droplets with a bilayer membrane envelope supplied by the apical plasmalemma of mammary epithelial cells has been described in thin-section studies in several species (2-4, 11-13, 16, 22, 35, 36, 39, 41-43). Our freeze-fracture results I~OURE 2 Initial events of fat droplet extrusion. The fracture-face A (aa) of the apical plasma membrane shows bulging of particle-free areas (arrows). Microvilli are observed on cross fracture, x 40,000. FI~UaE 4 High magnification of the transition from the area with particles to the smooth area of the apical plasmalemma of Fig. 3 shows the continuity of these membrane segments, x 120,000.
FIGu~ 5 Fracture-face A (an) of an area of the apical plasmalemma with particle clearing (*). The outer lipid lameUa of a fat droplet is exposed, and the cross-fracture profile of the surrounding apical membrane is evident (arrowheads)? L, alveolar lumen, x 40,000.
show that this process is often accompanied by segregation of membrane integral components. 2 It is also possible that, during extrusion, peripheral membrane components at the inner surface are segregated. This could facilitate the extensive bulging of the apical cell membrane during the extrusion process and account for the absence of actin and spectrin in MFGM preparations (19)) Less frequently, fat droplets seemed also to be released from intracytoplasmic vacuoles into the alveolar lumen. Our findings agree with previous thin-section observations (47). This exocytotic mechanism presumably involves the fusion of the secretory vacuoles, containing lipid droplets and other secretory products (47), with the plasmalemma, and results in the release of fat globules without an enveloping membrane.
Morphological and biochemical studies of secretion in different cells, including mammary epithelial cells during lactation, provide evidence for a continuous turnover between endomembranes and the plasma membrane segments related to the secretory processes (20). These events could explain the presence of Golgi (24,25,34) and endoplasmic reticulum enzymes (17,25) in MFGM preparations and their phospholipid composition intermediate between that of the Golgi and the plasma membranes (34), even if contami-2 Although clearing of membrane particles was more frequently observed, it may eventually not correspond to the prevalent mechanism if the extrusion without particle segregation is accomplished faster. a The segregation of actin during extrusion is, in view of the particle rearrangements which we observed, a preferable alternative to account for the absence of actin in MFGM preparations. Mammary epithelial cell surface antigens (6) and concanavalin A binding sites (18) have been detected in MFGM. It is not established, however, whether these sites correspond to integral or peripheral membrane proteins.
nations from cytoplasmic components extruded with some fat droplets could be avoided (29) or if the Golgi vesicles did not contribute directly to the formation of the MFGM (45). In consequence, the enzymic or chemical analysis alone cannot provide definitive evidence for the origin of the membrane of the fat globule, but must be seen in perspective with cell membrane dynamics. After fat secretion, electron microscope observations suggest that, in some globules, the membrane is gradually lost by a process of fragmentation and vesiculation (5,14,38,44,46). It is possible that during and after structural degradation of the membrane envelope of the fat globules, lipid and protein components of the initial (44) fat globule membrane may be adsorbed to the surface of the fat globules (14). Thus, the unit membrane profiles observed on thin sections of MFGM preparations, obtained from cream by freezing, sonication, and other procedures (20,21), may represent not only initial MFGM but also reflect bilayered structures reconstituted be, fore and during the isolation procedures. The structural rearrangement of the MFGM during or after secretion has been postulated to account for some biochemical and ultrastructural results (14,21,27). However, it has been recently assumed that the overall composition of the MFGM relative to the plasma membrane of the alveolar epithelial cells is not affected (19). Unfortunately, analysis of the apical plasmalemma of the lactating mammary cell is not accessible (34).
Our freeze-fracture observations suggest that the events which occur within the apical membrane during milk fat secretion may account for some compositional (19), physicochemical (7), and ultrastructural (14,21) differences between MFGM and plasma membrane preparations. The mechanism of segregation of membrane particles is, at present, unclear. It is possible that the Fig. 6. The fracture process exposed the lameilar appearance of the fat droplet (.fd). Membrane particles are observed on the area of the plasma membrane which surrounds but is not in direct contact with the fat droplet (arrowheads). aA, fracture-face A of the apical plasma membrane. • 40,000. reorganization of the membrane integral components results from the interaction between the fat droplet core and the apical plasma membrane, during extrusion. It might also involve the concomitant displacement of peripheral membrane proteins at the inner surface.a In summary, the process of segregation of integral components from areas of plasmalemma which envelop the fat droplets may differentially restrict the membrane components which form the milk fat globule membrane. This could correspond to an initial event of membrane structural degradation, which progresses after extrusion. Although most of the components found in purified preparations of M F G M were at one time associated with the apical plasmalemma, the fat globule membrane cannot be considered as structurally identical to the apical membrane of the mammary epithelial cells.
We wish to thank Dr. Pietro M. Gullino for helpful discussions, and Messrs. Clifford Parkison and Bela Berghoffer for technical assistance.
A. Peixoto de Menezes was supported in part by Instituto Nacional de Investiga~o Cientifica, Portugal.
Received for publication 13 July 1977, and in revised form 24 October 1977. FiGt.ri~ 10 and 11. Extrusion of fat droplets (fd) suggesting the sqee~dng through a "locus minoris resistentiae" of the apical plasma membrane. Fig. 10, the fat material is surrounded by the apical plasma membrane, without particles (an). L, alveolar lumen. • 35,000. Fig. 11, dumbbell profile as a result of a constriction at the plasma membrane level. • 17,000. Fig. 14, fracture-face A of a fat globule membrane with a smooth particle-free area. The arrowheads point to the cross-fracture profile of the fat globule membrane? x 38,000. Fig. 15, vesiculation process of the fat globule membrane. The segment of blebbing (arrowhead) presents a fracture face with an irregular distribution of particles, x 38,000. | v3-fos |
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} | s2 | A genetic study of partial egg production records in a randombred Fayoumi flock
Summary A genetic study of the annual egg production and 23 different partial records was done on 950 Fayoumi pullets, daughters of 40 sires and 3dams, belonging to the Faculty of Agriculture Cairo University. The partial records include the egg production till 4044452 and 70 weeks of age using full trapnesting and partial trapnesting ( days every week), together with the annual partial trapnesting record, beside the production during six fixed periods all in the first year of the bird’s life (also using full and partial trapnesting). Sire, dam and combined heritability estimates were computed for the 24 traits beside the genetic correlations between the annual full record and each of the 23 partial records. Relative efficiencies of the genetic progress expected in the annual record using the different part records were studied. It was shown that the highest annual relative efficiency could be obtained by using for selection the production of the pullet during the 4 week period between 40 and 44 weeks of her age (trait Di2). A selection index using the production from first egg to the age of 40 weeks (trait Ai) beside trait Di2 was shown to excel that of D
Introduction
Partial recording, during specific periods or till fixed ages early in the production life of the fowl, beside reducing the generation interval, may help increase the annual progress when breeding for egg production. The partial trapnest records may also be effected by trapnesting only a few days every week to decrease further the amount of labour needed to evaluate the experimental birds. The significance of using partial records depends of course on the size of correlated response gained in the annual egg production record.
A considerable amount of work has been done on partial recording and trapnesting (e.g. : OLIV!R Et al., 1957 ;WHEAT and EMS!,!Y, 1973 ). Many of these studies showed that more genetic gain could be obtained in the full year egg production record when using partial records for the evaluation of animals. The objective of this work is to evaluate the necessary parameters of partial records in a randombred Fayoumi flock, in an attempt to seek the most economic method of recording that leads to the highest expected genetic gain in the annual egg production.
Material and methods
This work was carried out on the randombred Fayoumi flock belonging to the Faculty of Agriculture, Cairo University. The flock was not previously subjected to any kind of genetic selection. Fourty males and 400 females were randomly chosen and divided into 40 groups each of one male and ten females.
Nine weekly hatches were obtained during the period from mid January to mid March, 1974 . The number of females actually contributing to the progeny used in this study was only 311 . Individual daily egg production records were kept for each of 950 pullets of the progeny of these parents for a full year starting from sexual maturity. The following 24 traits were recorded for each individual (designations between brackets): i. The total number of eggs laid till the ages of: beside (A6); the complete annual egg production record (that is, the total number of eggs laid during a 3 6 5 day period from date of first egg).
2 . The total number of eggs produced during the following periods of the first year of the bird's life: 3 . The number of eggs trapped in the first three consecutive days every week till the ages, or during the periods of the 12 traits described above. These traits will be given the same designations of the corresponding complete trapnesting traits using the lower case letters (thus, for instance, ai stands for the 3 -days-aweek record of the number of eggs laid by the pullet till 40 weeks of her age).
Data were all corrected for hatch effect (which proved to be highly significant in all traits except D 12 and diz) before performing the hierarchical analyses of variances and covariances. Sire, dam and combined heritabilities were estimated for all the traits beside the combined genetic correlations between A6 and the rest of the characters. The following table i shows the degrees of freedom and the k values of the analyses. The standard errors of the heritability estimates were calculated according to the method suggested by W OOLF (ig6i), and those of the genetic correlations were estimated by Ros!RTSOrr's ( 1959 ) formula.
Results and discussion I. -The means The least squares means of the number of eggs laid by the fowl from first egg till the different ages used in this study are presented in table 2 . The egg production of this Fayoumi base population is indeed low. As mentioned above, this flock has not been previously subjected to any kind of genetic selection. Starting from the age of 40 weeks it can be seen that the rate of egg production increased with the advancement of age till it reached its maximum during the period from 5 2 to 7 o weeks of age (when the rate was 39 p. 100 ). It may be mentioned that the least squares mean of age at sexual maturity was 3 8 weeks ( 2 66. 14 ::J:: 1 .5 day), and thus the average age at which the bird completes its annual record may be taken as go weeks.
II. -The heritabilities 1 . Egg production till different ages : .-Sire, dam and combined heritability estimates (h2!, h 2 d and h 2 c resp.) of the number of eggs laid till the ages described above (the &dquo; A &dquo; and &dquo; a &dquo; traits) together with the amount of genetic and phenotypic variances are shown in table 3 . The sire heritabilities of A I and ai (egg production till 40 weeks of age) are a little bit higher than the dam heritabilities. This was not the case in the other &dquo; A &dquo; or &dquo; a &dquo; traits in which the dam heritabilities were higher than the sire ones. The combined heritability estimates and the genetic variances increased in magnitude with the length of the recorded laying period till the age of 52 weeks. The combined heritability value of A5 (the number of eggs laid till 70 weeks of age) is a little bit lower than that of A 4 . Table 3 shows that the amount of genetic variance of A 5 is almost double that of A 4 ( 143 . 2 vs 7 q.5), and the lower heritability of A 5 is due to the very high increase of the phenotypic variance. The same picture could be seen also with respect to a 4 and a5. The combined heritability value of A6 (the complete annual record) is indeed low ( 0 . 09 ) and table 3 makes it clear that the amount of genetic variance of this character is almost the same as that of A 4 , and that the amount of phenotypic variance is about three times the corresponding amount of A 4 . Again an exact picture could be observed with respect to a6 and a 4 . The combined heritability of A6 is lower than most of the published comparable heritabilities (e.g., KING It might be of interest to note that the combined heritability values of the 3 -days-a-week trapnesting records are all lower than the comparable values ofthe complete trapnesting records. All the &dquo; a &dquo; traits have less genetic variance (except ai) and more phenotypic variance compared to the corresponding &dquo; A &dquo; traits (that is; after multiplying the a 2 9 and a 2 p of the &dquo; a &dquo; traits by 49/9 ).
2 . -Egg production o different periods o f pullet's production li f e: Table 4 shows the sire, dam and combined heritability values together with the amount of genetic and phenotypic variances of the number of eggs laid during the different periods of the first year of the pullet's life (the &dquo; D &dquo; and &dquo; d &dquo; traits). It could be seen in all these traits that the dam heritabilities are higher than the sire estimates, and that the heritability estimates of the &dquo; D &dquo; traits are higher than the corresponding estimates of the &dquo; d &dquo; characters. The amounts of phenotypic variance of the number of eggs laid during the 4 week periods of Di2, D 23 and D 34 are almost the same, but the genetic variance of the first period is markedly higher than the other two periods ( 4 . 2 , 2 . 4 and o.8 resp.). In the 8 week periods (D 13 and D 24 ) it could be seen that the amount of genetic variance of the number of eggs laid in the first period (Di 3 ) is more than double that of the number laid in the second period (D 2 ¢). The same holds true with respect to dig and d 24 . It could also be observed that the records which include the egg production during the period from qo to 44 weeks of age (D 12 ,Di 3 and D 14 ) showed all the highest sire, dam and combined heritability estimates, compared to the other three part records D 23 , D 3 q. and D 24 . The same holds true also with respect to the &dquo; d &dquo; traits. The period of production that showed the lowest heritability values is that between q.8 and 52 weeks of age.
III. -Genetic correlations and index efficiencies I . -Genetic correlation.s with the annual record: Table 5 shows the genetic correlations and genetic covariances between A6 and all the part records used in this study. The genetic correlation between A6 and a6 is complete. Among the other 22 part records, D 23 showed the highest correlation with A6 (r 9 = 0 . 935 ), and the following characters showed genetic correlations of more than o.8 (in descending order): d 12 , D I2 , D 13 , A 5 , A 3 and A 2 . The lowest y 9 values were those between A6 and d 23 , d 34 and d 24 ; the partial trapnesting records collected during the first year of the bird's life, during the periods that do not include the production of the period from 40 to 44 weeks of age.
2 . -Relative efficiency of selection on part records in improving the annual egg !roduction: The significance of using part records depends upon the expected correlated response gained in the annual record (A6) by selection on the part records(FAr,-CONER , ig6i). The amount of correlated response in the annual record is equal to (i sgr 9 h x ) ; where i is the selection intensity, 6 g is the genetic standard deviation of A6, rg is the genetic correlation between the A6 and the record x and h x is the square root of the heritability of trait x. The response to direct selection on A6 will then be ia,h A6 . The ratio of the response obtained by indirect selection on trait x to that gained by direct selection (that is; rgh x /h AS ) would show the relative efficiency (R. E.) per generation of the use of any part record in selection for A6. The part records, usually completed early in the production life of the pullet, is supposed to decrease the generation interval, and the relative efficiency per year would then be the best yardstick for comparison. To compute the value of the relative efficiency per year, 8 weeks will be allowed after the age at which the part record is completed (for the collection and hatching of eggs to produce the new generation), bearing in mind that A6 is completed at an average age of go weeks. Table 6 shows the relative efficiency per generation and per year for the part records used in this study. D I 2 , Dr 3 , A 3 and A 2 give the highest expected yearly genetic gain in A6, followed by d 12 , A 4 and Ai. The first six of these seven part records involve the production of the 4 week period between 40 and 44 weeks of age. The egg production of this period, with complete or partial trapnesting (Di2 and di2) would be very convenient part records for the breeder since he can produce one generation every year ( 52 weeks). These two traits, D 12 and d 12 , were also the only ones, among all the traits studied, which slowed no significant hatch effect. Selection indexes involving two or more of the disussed part records could be used to attain higher efficiency. The following index is the one adopted now for selection in the Fayoumi flock of the Faculty of Agriculture, Cairo University. It involves the A I and D 12 traits; that is, the A 2 record divided in two pieces. The index equation to be solved was a follows: (23.96 ! the phenotypic covariance between Ai and D 12 , other figures are shown in tables 3 , 4 and 5 ). The two regression coefficients are then The accuracy (R) of the index (or the squared efficiency) is where !6.39 is the genetic variance of A6. Thus the efficiency will be equal to 0.39! and the genetic standard deviation of the index will be 3 . 474 (the square root of 12 . 0 6 5 ). The relative selection efficiency of this index per generation is 134 p. 100 and per year is 252 p. 100 . If we select 20 p. 100 of the pullets (i = z. 4 ) yearly using this index, the expected genetic superiority in A6 of the selected individuals will be: or about 4 . 1 p. 100 of the mean of A6. Half of this superiority is expected to be recovered in the progeny every year. | v3-fos |
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} | s2 | Halothane-test in pig breeding
HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HALOTHANE-TEST IN PIG BREEDING W.A. G. Cop, D. Minkema, G. Eikelenboom
Index selection combining scores for backfat and daily gain was practiced in a selection experiment with upwards (HP-line) and downwards (LP-line) selection on the index. A control line was maintained from the 3 . year. Results from 8 generations are presented: in this study. The LP-line (low growth, high backfat) showed higher standardized selection differentials, selec-+ tion responses and realized heritability than the HP-line (high growth, low backfat) (h' L p = . 5 z h l lp = . 34 ). Calculations of actual weights on backfat and daily gain in retrospect showed a shift towards backfat in the LP-line and towards daily gain in the HP-line. Joint estimates o, realized he and r G of the index traits gave h a = .8 0 for backfat and . 3 6 in daily gain. Geneticf correlation between the index traits was calculated to i6.
Correlated respcnses in slaughter traits showed higher values in the I,P-line than in the HPline. Joint estimates of r G to the index showed favourable values for all slaughter traits except number of inverted teats. Correspondance to the f o values estimated from half sib analyses in the control line were very good. Daily food consumption increased in both lines. Feed conversion ratio showed higher response in the HP-line than in the LP-line.
Regressions on generaticn number for litter size traits were all negative. Correlated responses to the index were unfavourable but genetic correlations between fertility traits and the index would have fallen in the range of 0 During a one year survey a total of 1 , 304 Dutch Yorkshire and 1 ,6 40 Llutch Landrace pigs were subjected to the halothane-test after their arrival at one'of the national pig testing stations.
The average percentage reactors was 3 . 07 p. 100 in the Dutch Yorkshire breed and 22 . 2 p. 100 in the Dutch Landrace breed.
Death losses during the fattening period and during the transport of the sows to the slaughterhouse were almost ten times higher in reacting as in non-reacting Dutch Landrace pigs ( 5 , 27 p. 100 vs 0 . 5 6 p. 100 ).
In the Dutch Landrace breed significant differences were found between reactors and nonreactors in the growth traits of the boars and in all carcass and meat quality characteristics of the sows, which confirm previous observations. Hovewer, in the Yorkshire breed no significant differences were found in these traits between reacting and non-reacting animals. The conflicting results obtained in this breed are discussed.
It is suggested that the halothane-test will be most effective for elimination of stress-susceptibility and abnormal meat quality when used as a selection criterion in commercial pig breeding and selection of Dutch Landrace pigs. Zeist, The Netherlands A simulation study describing the influence of direct and indirect selection on the gene frequency for hypersensitivity to halothane is presented in this paper. The factors studied are fitness, culling level on index, initial gene frequency and type of inheritance of the trait.
The results show that indirect selection (effect of fitness and positive effect on the selection index) will rise the gene frequency for hypersensitivity to halothane, as long as no direct selection is applied against the trait.
It is estimated that the gene frequency without direct selection in the Dutch Landrace will increase within 5 generations from 0 . 47 to about o.6 5 and within 10 generations to 0 .8 3 .
In order to select against hypersensitivity to halothane, 3 alternatives are discussed: building in the trait in a selection index, direct selection by excluding all positive boars and possibly also gilts from performance testing, and the set-up of lines of homozygous negative pigs. Genetic correlations were estimated between hatchability traits of purebreds and egg number and egg weight of crossbreds. While hatchability and egg number were positivly correlated genetically in one strain, essentially no correlation existed in the other. Correlations pertaining to egg weight were unfavorable in both strains but more so in one. Correlations with number of saleable chicks were positive for egg number and negative for egg weight. Selection according to a restricted selection index egg number and egg weight (this to be held constant) should impair hatchability in one strain. The investigation described in this paper is based on 2 , 33 o veal calves and young bulls after 13 6 sires of the breeds Red Danish, Black Pied Danish and Danish Red and White. For these animals information on gestation length, birth weight, daily gain, feed intake and carcass compo- | v3-fos |
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} | s2 | Bioaccumulation and detection of trace levels of cadmium in aquatic systems by Eichhornia crassipes
The water hyacinth (Eichhornia crassipes) may be used as a sensitive biological indicator for continuously monitoring trace quantities of toxic heavy metals in aquatic systems. A river water system polluted with cadmium was simulated while other factors of temperature, day-night cycle, water quality, and light intensity remained constant. When the water hyacinth is maintained in river water containing 0.001 mg/l. of cadmium chloride, the plant's root system will concentrate this element at an average rate of 0.9, 1.4, and 3.0 μg Cd/g root dry weight after 24, 48, and 72 hr exposure periods, respectively. At a higher cadmium concentration of 0.01 mg/l., cadmium was concentrated in the roots much faster to levels of 6.8, 13.6, and 39.1 μg/g root after 4, 8, and 24 hr exposure periods, respectively. At initial concentrations of 0.05 mg/l. cadmium, the roots contained 29.5, 48.8, and 156 μg/g root following 4, 8, and 24 hr exposure periods, respectively. During these same time intervals, the water hyacinth sorbed 56.7, 153, and 281 μg/g root when the initial cadmium concentration was increased to 0.10 mg/l. The water hyacinth tops can also assist in the monitoring process when cadmium contamination levels are 0.10 mg/l. and greater. At this initial cadmium concentration, cadmium is translocated into the tops. After 8 hr, the tops averaged 1.1 μg/g top. After 24 hr, this concentration was increased to 6.1 μg/g top.
Introduction
Cadmium is a toxic, heavy metal which can present a serious threat to human health. Toxic effects from this heavy metal are well documented. Excess levels of cadmium can cause kidney and liver damage, pulmonary disease, and cancer in experimental animals (1)(2)(3)(4). Chronic low levels of cadmium may contribute to hypertension, decreased growth, and alterations in blood cholesterol and trace element metabolism (5).
Heavy metals present a serious form of pollution in aquatic systems since they do not degrade as do most organics. Even trace quantities of toxic metals in water systems are serious potential health problems because of the ability of certain aquatic plants to concentrate heavy metals which are then consumed by fish that form a part of man's diet (6,7).
Using biological indicators such as plants for monitoring both air and water pollution has been --ecognized and used to a limited extent over the * National Aeronautics and Space Administration, National Space Technology Laboratories, NSTL Station, Mississippi 39529.
years. Mosses and lichens strongly sorb metal ions from the air and water and are useful for detecting atmospheric and aquatic lead and other metal contamination (8)(9)(10). Leaves and twigs of woody plants have also been used to indicate atmospheric pollution (11).
One of the most promising candidates for biological indicators of trace levels of heavy metals in aquatic systems is the water hyacinth (Eichhornia crassipes). In static laboratory experiments, this plant demonstrated an amazing ability to sorb and concentrate cadmium, as well as other metals such as mercury, lead, and nickel (12,13). Water hyacinths have been used successfully by NASA at the National Space Technology Laboratories to remove organics and heavy metals from its chemical waste prior to discharge (14). In the course of this system's evaluation, water hyacinths were found to contain detectable levels of heavy metals, especially in the roots, although these same heavy metals in the waters were below normal detection limits by atomic absorption-flame spectrometry. The study presented in this paper is an outgrowth of this observed phenomenon. Water hyacinths are used to develop a rapid, biological monitoring system for December 1978 establishing cadmium pollution in the aquatic environment.
Procedure
For each different cadmium concentration, twelve glass aquariums were filed with 15 liters of river water. Nine of the aquariums were polluted with sufficient 1000 mg Cd/l. standard solution to produce an approximate initial cadmium concentration of 0.1 mg/I. for run 1, 0.05 mg/I. for run 2, and 0.01 mg/I., for run 3. Three aquariums were left unpolluted. Four groups of nine water hyacinths were thoroughly washed and placed in three of the polluted containers and one of the unpolluted containers. After 4 hr, three plants from each aquarium were removed for analysis, and the remainder of the plants were transferred to four fresh aquariums. This procedure was repeated again after 8 hr. Each experiment was terminated after 24 hr.
For run 4, twelve glass aquariums were filled with 30 liters of river water. Nine containers were polluted to an approximate cadmium concentration of 0.001 mg/I. The experiment was conducted in the same manner as outlined above, except the plants were removed and transferred at 24, 48, and 72 hr intervals.
During each experiment, the plants were maintained with growth lights supplying approximately 500 FC to the plants during a 16 hr photoperiod and 8 hr nyctoperiod. The air temperature was 23 + 5oC. The initial unpolluted river water samples were analyzed according to Standard Methods (15) and found to contain the following average concentration: 0.25 mg/I. total Kjeldahl nitrogen; < 0.13 mg/I. phosphorus; 52 mg/I. dissolved solids; 9 mg/I. total organic carbon; pH 6.9. The initial cadmium concentration of 0.1 ,ug/l. was determined with an IL 555 flameless atomizer and an IL 351 AA/AE spectrophotometer.
The roots and tops of the plants to be analyzed were separated, washed, dried at 60°C to a constant weight, ground, and homogenized in a Waring Blendor. A 0.500 g of each plant sample was weighed and transferred to a 75 ml volumetric digestion tube. The plant samples were charred at 400°C with 10 ml concentrated H2SO4 for 2 min and then digested for 20 min at 400°C with an additional 10 ml concentrated HNO3. The samples were allowed to cool, and then 2 ml 30%o H202 was added.
The tubes were again heated to 400°C for 10 min. Following the digestion process, the samples were diluted to volume with deionized, distilled water, and the cadmium content determined by flame spectrometry by using an IL 351 AA/AE spectrophotometer. A reagent blank was also digested in the same manner, and any cadmium introduced into the plant samples from the reagents was subtracted from cadmium concentrations in the plants.
Discussion
The experiments for assessing the potential of using water hyacinths as biological indicators for estimating the level of cadmium pollution in aquatic systems were designed to simulate real conditions. A fresh volume of polluted river water was supplied to the plants at regular intervals. The cadmium concentrations were varied while other factors of temperature, day-night cycle, water quality, and light intensity remained constant. The data in Tables 1 and 2 are the results of this series of four experiments.
The first experiment conducted with 0.1 mg Cd/l. was a relatively high cadmium concentration for potable or recreational water systems. The water hyacinths were found to average 56.7 ,ug Cd/g root (dry weight) after only 4 hr of exposure. The concentration in the roots continued to increase to an average of 153 ,tg/g root after 8 hr and 281 ,ug/g root after 24 hr. At this high cadmium level, cadmium was first detected in the leaves after only 8 hr of exposure.
The concentration in the second experiment was decreased to 0.05 mg Cd/l. The quantity of cadmium sorbed per gram dry root weight over the same time intervals was almost exactly half of the concentrations found at the 0.1 mg Cd/l. level. The cadmium was concentrated to average levels of 29.5, 48.8, and 156 ,g/g root after 4, 8, and 24 hr, respectively.
No cadmium was detected in the leaves of these plants, nor was any cadmium detected in the leaves of any of the later experiments.
This same trend was also observed in the third experiment when the cadmium concentration was decreased to 0.01 mg/l. The cadmium concentrations in the roots averaged 6.8, 13.6, and 39.1 ,g/g root after 4, 8, and 24 hr, respectively.
The exposure time in the fourth experiment was increased in order for the water hyacinths to accumulate sufficient cadmium at the 0.001 mg/I. level to be detected by the procedure outlined above. This very low concentration of cadmium in the water had to be determined by atomic absorption using a flameless atomizer. The cadmium in the water could not be detected without concentrating it if the normal method of atomic absorption-flame spectrometry had been used. The first root samples analyzed after 24 hr exposure contained an average of 0.9 ,ug/g root. This concentration was far less than the expected value of one-tenth of the 39.1
Conclusions
The data from Table 1 were plotted in Figure 1, which demonstrates how a family of curves can be used to estimate low levels of cadmium in river water utilizing water hyacinths. At very low levels of cadmium this graph must be expanded as in Figure 2.
The leaves were found to be useful for estimating high cadmium concentrations. At the highest level of cadmium in this study, the leaves contained detectable levels of cadmium even after 8 hr of exposure.
The sorption rates of cadmium as well as other toxic heavy metals will vary from one system to another depending on environmental factors. However, the data necessary to obtain a family of curves such as Figure 1 for a particular aquatic system can be obtained without much difficulty. | v3-fos |
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} | s2 | Pesticide use in agriculture.
During the last three decades, the use of modern organic synthetic pesticides has increased about 40-fold. Total U.S. production, for domestic and expert use, in 1976 was about 1.4 million pounds. Crops receiving the most intensive application of various pesticides were cotton for insecticides, corn for herbicides, and fruits and vegetables for fungicides. Examination of use trends of pesticides indicates that the volume in pounds of herbicides used on crops is increasing, whereas the quantities of insecticides and fungicides remain stable. New chemical classes of compounds such as the synthetic pyrethroid insecticides are being introduced, but are not yet significant in terms of their share of the market. The increased usage of pesticides, together with knowledge of some of their adverse effects, has alerted the public to the need for regulation. To assist in the regulatory decision-making process, emphasis is being placed on benefit-cost analyses. Additional and improved biological inputs and methodologies are needed to provide accurate analyses.
Introduction
Chemicals are continually becoming a more intricate part of modern society. Pesticides used in agriculture constitute less than 3% (1,500 out of over 63,000) of the commonly used commercial chemicals in the United States (I) but are often highlighted as being of special concern because of their relatively high intrinsic toxicity and direct application to food crops. This presentation reviews pesticide use in agriculture, with emphasis on crop use in the U. S., and includes discussions of mutagenicity of pesticides and of benefit-cost analysis. Pesticide use on crops in the U. S. is discussed within the framework of land use and crop production, though significant quantities of pesticides are used for control of pests of livestock, man, stored products, structures, home gardens, lawns and ornamental plants, and in industrial processes.
nematodes are probably in excess of 30o of total production. Information is even more limited on the extent to which actual pest losses have been reduced as a result of evolution of pest control technology. Nevertheless, the development of pesticide technology, together with the increased use of improved varieties, fertilizers, irrigation and other new technologies have resulted in enormous increases in crop yields since 1900 (3) ( Table 4).
Pesticides and Farm Management
Although benefits associated with pesticide use are most frequently identified as a reduction in /acre 1900 187 28 13 56 1910 191 27 15 58 1920 160 27 12 63 1930 179 26 14 65 1938 236 28 13 73 1941 251 32 17 80 1944 267 32 17 86 1947 266 34 18 121 1950 275 40 16 145 1953 310 39 18 152 1956 405 42 20 175 1959 459 49 22 185 1961 445 60 25 192 1964 502 67 26 202 1967 490 75 27 211 1970 436 80 32 225 1973 485 86 31 236 1975 453 86 31 losses due to pests, other less apparent factors contribute to the dependence of modem agriculture on the use of pesticides. An example is the use of pesticides in lieu of costly and frequently unavailable labor. For instance, the alternatives to the use of herbicides are either mechanical or manual methods of weed control. Mechanical methods may require more energy, lead to increased soil erosion and sedimentation in water, and are often not as effective. Manual methods require an able and willing labor force. Due in part to pesticide use, the labor required to produce agricultural products has been markedly reduced over the past 40 years (8) ( Table 5). Notable examples are the increased use of herbicides for weed control on corn to replace manual Environmental Health Perspectives and mechanical methods and the use of harvest-aid chemicals (desiccants and defoliants) on cotton followed by mechanical harvesting to replace manual harvesting. Also, it has been suggested that increased pesticide use in reduced tillage systems may reduce the total energy requirements in the production of crops such as corn (9). Others question this conclusion as it relates to energy requirements, but support the increased use of pesticides to reduce soil erosion (10). Pesticides are therefore an essential part of current agricultural production technology; however, we should also be cognizant of the increasing interest in the development of an alternative agriculture which is labor intensive and emphasizes use of biological methods of pest control (11).
Pesticide Production and Use
In the present discussion of pesticides, major emphasis is placed on insecticides, herbicides, and fungicides, which constitute about 90%o of all pesticide use in agriculture. Fumigants including most nematicides, growth regulators, desiccants and defoliants, miticides, rodenticides, and repellents, are discussed when relevant data are available.
Production and Value
During the period 1945 to 1976, production of synthetic organic pesticides in the U. S. increased from less than 35 million pounds per year to over 1.4 billion pounds with a high of 1.6 billion pounds produced in 1975 (3, 12, 13) ( Table 6). In recent years, about one-third of this production has been exported, 596 million pounds in 1975 and 574 million pounds in 1976; while quantities of pesticides equal to less than 5% of U. S. production were imported, 53 million pounds in 1975 and 65 million pounds in 1976 (13). World production of pesticides in 1975 was an estimated 3.7 billion pounds; thus in 1975, the U. S. produced nearly one-half of the world's pesticides (14). Also, pesticide sales by manufacturers in the U. S. (valued in 1970 dollars) were reported to have increased from $0.9 billion in 1970 to $1.7 billion in 1975 with herbicides accounting for 62%, insecticides for 32%, and fungicides 6% of the total sales (13) ( Table 7). In another report, U. S. domestic sales (valued in 1976 dollars) have been estimated at $2.7 billion in 1976 with world sales estimated at $7 billion (14).
It is important to emphasize that the increase in both production and value of pesticides sold is probably associated with increased production of existing pesticides, since the number of new pesticides being introduced annually has declined from nearly 30 per year to less than 10 per year since 1965 (15).
Crop Use
In 1964, the U. S. Department of Agriculture initiated a survey of farmer usage of pesticides; subsequent studies have been conducted for 1966,1971 and 1976 (16)(17)(18). These surveys indicate that pesticide use on farms has about doubled since 1964 and in 1976 was an estimated 663 million pounds of active ingredients (Table 8). When this quantity is compared with net U. S. production (less exports and plus imports), agricultural uses account for only about 70%o of the pesticides produced in the U. S. (13,(16)(17)(18).
Estimates of the crops receiving the most poundage of pesticides in the U. S. in 1976 show that 41% of total 1976 farm use of insecticides was on cotton; 54% of herbicide use was on corn; and 84% of fungicide use was on fruits, nuts, and vegetables (16)(17)(18) (Tables 9-11). Likewise, total pesticide use based on expenditures by crops throughout the world indicates that pesticide use on cotton, corn, and fruits and vegetables accounts for over 70% of all pesticide use (Table 12). U. S. crop-use trends of pesticide types expressed in pounds of active ingredients for insecticides, herbicides, and fungicides show the volume of herbicides used on agricultural crops has increased five times since 1964 while insecticide and fungicide usage remained relatively stable (16)(17)(18) (Tables 9-11).
When farm use in the U. S. of the major pesticides is categorized by chemical classes, some major changes are evident during the period 1964-1976. There has been a significant decline in chlorinated hydrocarbon insecticide use and corresponding increase in the use of organophosphate and carbamate insecticides (Table 13). Among the herbicides, there have been major increases in the use of triazines and amides, but use of phenoxy Cook (14). compounds has remained about the same (Table 14). Only fungicides have not experienced any sigificant changes; dithiocarbamate and the phthalimide compounds are still the principal chemical classes (Table 15).
Noncrop Use
The most important noncrop farm use of pesticides is on livestock. Insecticides, used both externally and systematically for the control of livestock parasites, remained relatively stable over the 1964-1976 period (16)(17)(18) (Table 16).
Substantial quantities of pesticides also are used for other noncrop activities including stored products, structures, (termites, cockroaches, etc.), home gardens, forestry, ornamental (lawns, trees, December 1978 shrubs), industrial processes, and mosquito and fly control. Information on quantities used for these purposes is limited. However, it is obvious from data on farm use and from information on total sales, that these uses are substantial. For example, in one study conducted in 1972 on the use of pesticides in suburban homes and gardens in three representative cities, a population of 5.5 million people was reported to have used 759,000 pounds with the average per acre of application between 5.3 and 10.6 pounds. One must exercise caution extrapolating the results of this one study into broader suburban home and garden use of pesticides, but anything near such a rate would represent 30 or 40 million pounds of use in the U. S. for this noncrop activity (19).
Since available data apparently are not adequate to provide a good understanding of total pesticide use in the U. S., there is a need to gather more information particularly on noncrop pesticide use.
Mutagenicity of Pesticides
The use of pesticides in agriculture generates considerable benefits. At the same time, the potential of adverse effects of pesticides must be recognized. While the benefits of pesticides are usually quantified from an analysis of direct costs associated with pesticide use and of relative crop production, a quantitative evaluation of risks is often much more difficult to obtain. The difficulty of obtaining reliable quantitative risk estimates is particularly formidable in the case of mutagenic and carcinogenic risks for a variety of technical reasons which have been discussed elsewhere (20)(21)(22).
The quantitation of mutagenic and carcinogenic risks is of particular importance because of recent reports that representatives of the major classes of pesticides are suspect due to mutagenicity and/or carcinogenicity (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Included in these studies are triazines, organochlorines, carbamates, dithiocarbamates, organophosphates, phthalimides, amides, phenyl ureas, phenoxyacetic acids, and benzimidazoles. These classes of pesticides represent well over 80%o of all synthetic organic pesticides now used in the U. S. (Tables 8, 13-15). It is not unlikely that further experimental scrutiny of structually related pesticides within these classes will reveal additional compounds which exert an adverse genetic effect under some test conditions. This does not imply that these compounds necessarily pose a significant genetic hazard to humans under the conditions of their use, but rather emphasizes the extent to which regulatory decisions related to possible genetic effects of these compounds will influence pesticide technology during the next decade; it also illustrates the need for translation of laboratory findings into quantitative measures of the actual risk to humans under use conditions.
Regulatory decisions related to the use of the above groups of pesticides will be a major determinant of the pesticide technology available during the decade ahead. The importance of basing these decisions on meaningful, quantitative, risk analyses is obvious. It is not our intention to discuss in detail the complex issue of genetic risk assessment. Suffice it to say that the cellular mechanisms for DNA replication and repair, like all major physiological systems, involve a complex set of enzymatically and genetically controlled biological reactions which may be altered in many ways. Not only are several types of genetic damage possible, but each may arise via a variety of mechanisms. Evaluation of findings in terms of risk is often impossible if these mechanisms are unknown. The interpretation of the finding that certain herbicides cause structural abnormalities in plant chromosomes, for example, would be quite different if the effect resulted from direct alkylation of the DNA rather than, as has been suggested, as a secondary effect of severe physiological disturbances in the plant (37,38). Likewise, a reversible increase in repair synthesis in bacteria from a near lethal dose of a compound which exhibited no other adverse genetic effect would not be of the same concern as would a significant increase in the frequency of heritable translocations in mice at a dose near that expected from environmental exposure. Unfortunately, distinctions are seldom so clear-cut in practice. Interpretation of findings with agents that are mutagenic in microorganisms but which do not readily produce observable mutations in mammals are particularly difficult to interpret quantitatively. This occurs because alternative explanations for the results generally lead to markedly different conclusions regarding potential health hazards (27,39). Indeed, the problem of reliably extrapolating into dose ranges approximately environmental exposures is still formidable even if a health hazard of undisputed significance is established in man, due to the wide range of minimal risk levels predicted by the alternative models which might be assumed (21,22).
The major purpose of this workshop is the evaluation of potential uses of higher plants as monitors of mutagens in the environment. Data obtained from such monitoring with higher plant systems can play a valuable role in determining if a pesticide, in the environmental milieu in which it is employed, poses a mutagenic hazard. One example of the value of such data is provided by consideration of Environmental Health Perspectives the environmental data obtained on the triazine herbicides. This group of herbicides is used in the U. S. in quantities greater than any other group of organic pesticides (Table 14). Atrazine does not itself induce mitotic gene conversion in yeast, but extracts from maize kernels or seedlings treated with atrazine do induce this genetic effect (23,24). A mutagenic effect is also observed in pollen grains from maize plants treated with atrazine in the field (25). This information indicates that laboratory data based on the exposure to test organisms to atrazine per se will not necessarily be an appropriate base for a risk assessment of atrazine residues in maize or in field runoff after atrazine use. Monitoring the mutation frequency in field plants thus revealed a mutagenic effect due to environmental conversion products which might not have been appreciated from conventional laboratory tests of the pesticide per se, or from chemical determinations of atrazine residues in the field.
Hopefully, future developments will permit the integration of such monitoring data with laboratory and epidemiological data to permit translation into a quantitative measure of health risk. Undoubtedly, the development of substantive risk evaluation methods is one of the most important challenges to environmental health scientists. Fortunately, research in genetic toxicology is moving rapidly toward a better understanding of the fundamental mechanisms of genetic damage and the foundation provided by this work will, hopefully, resolve the major difficulties associated with quantitative risk assessment.
Benefit-Cost Analysis
To aid in the formulation of regulatory policy action a specific decision-making approach is evolving that assesses the benefits and costs (including human health risks) associated with pesticide use. This approach, known as benefit-cost analysis (or benefit-risk analysis), is concerned with the economy as a measure of societal welfare, which refers to the state of well-being of the individuals in the society (40).
Benefit-cost analysis requires that many questions be examined for tradeoffs associated with the effects of regulating pesticide use. The most obvious question addressed by such an analysis is, "do the benefits or gains of the given pesticide use pattern exceed the costs or losses incurred?" This then lends itself to simulating posited changes in the given pesticide use pattern and measuring the potential impacts. In this way, analysts can determine optimal resource use through the allocation of productive inputs, the substitutability between productive inputs or control practices, and the substitutability between commodities produced. Other effects resulting from a policy change are the possible redistribution of social benefits such as impacts to the producers' and consumers' incomes.
Although complete benefit-cost analyses are yet to be conducted, the recent Environmental Protection Agency decision not to suspend trifluralin registration for weed control immediately used such an approach to balance relative cancer risk against the economic benefit of continuing registration pending further evaluation (41,42).
A recent study designed to identify or specify inputs needed for benefit-cost modeling of pesticide use represents perhaps the most complete effort to develop a conceptual framework (43). The impact areas identified in this study are summarized in Table 17. The principal areas considered are: agricultural, material and property damage, human health, environmental and aesthetic values, distributional effects, and regulatory control costs. The first component, agricultural impact, is the focus of most benefit-cost analyses of pesticides. The crop yield, quality of the crop, and cost of production are areas of market-valued benefits resulting from the use of pesticides. On the other hand, the final item within this agricultural component, quality of land, deals with such effects of pesticides as soil erosion, soil compaction, and soil microorganisms. This variable is not intuitively apparent nor easily quantified and evaluated, but integral to the cropping decisions of the farmer. These characteristics are considerations when modeling the agricultural sector to simulate the effects of a change in pesticide use resulting from regulatory action. The biological data necessary to complete the agricultural impact equation involves collaboration between economics and various biological disciplines.
The second component, protection of material and property from damage by pests, is another quantifiable benefit of pesticide usage. The measurement of such an impact is made from the prices or defined values of such goods. Right-of-way maintenance includes the use of herbicides along roadsides, a labor-saving use. The use of pesticides to insure the structural integrity of buildings from pests such as termites extends the life of the buildings, a tangible benefit. Control of pests damaging commodities during storage and personal belongings is, of course, of real value.
The third component, human health, is the major issue in risk identification and quantification. The difficulties of quantifying the risk associated with possible mutagenicity and carcinogenicity discussed in the preceding section will probably constitute the greatest barrier to the practical application of such an analysis. Nevertheless, even at our present state of knowledge, assumption of conservative extrapolation criteria will permit assessment of a maximum feasible risk for many types of agents. The calculated permissible levels may then be adjusted to higher values as more precise risk information becomes available. Such maximum risk estimates may then be balanced against the benefits of pesticide use. If there are measurable health hazards and the total value of these hazards are a function of the size of the population affected, then an estimate of the value of adverse health effects can be made. Conversely, of the various items and groups affected under the human health component, the disease vector control item is a definite benefit. The control of disease carrying insects is often overlooked, but has saved innumerable lives. Biological estimates of human health benefits must be made to insure the safety of the public and, at the same time, allow adequate productive resources to maintain prosperity.
The environmental and aesthetic impacts also are difficult to measure. Pesticides are formulated to kill or control a wide array of species considered harmful to man. The basic problem with these chemical formulations is that their effects often are not limited to the intended or target organism. Through the use of pesticides, nontarget organisms can be killed either directly or indirectly via the consumption of contaminated foodstuffs. Pesticides can be transferred from the original application sites to other locations by erosion, drift, runoff, and biological transfer.
The final components, distributional effects and regulatory control costs, are treated as secondary effects in the original model specifications (43). We feel, however, that these effects, to the extent possible, should be quantified and included in the benefit-cost analysis conducted for pesticide use policy. An example of one of the distributional impacts that has rather significant impact on both the agricultural community and the consumer is the U. S. balance of payments accounts. Over the past four years, agriculture has consistently provided a net contribution to the balance of payments of over $10 billion per year (3). Thus, at a time when the U. S. deficit in balance of payments has reached a negative $27 billion per year and has caused the value of the dollar to decline, agriculture's role in the balance of payments is more important than ever. Judicious, objective investigations of pesticide use is warranted since the role of agriculture in the nation's economic welfare is more far reaching than some people realize. Regulatory costs at Federal, State, and local levels are very real costs than can be integrated into models designed to provide a total benefit analysis.
There is perhaps an area of consideration that has been essentially ignored in previous discussions of benefit-cost analysis of pesticides. This is the inclusion of methods of pest control practices other than pesticides. The inclusion in pesticide regulation analyses of nonpesticide control methods such as biological control agents, genetical methods, host plant resistance, cultural and physical methods and attractants and repellents, as alternatives to the use of pesticides also should be considered.
Future Outlook
During the past 20 years, quantities of pesticides used have increased about 10%o per year. Pesticide use will likely continue to increase but at a somewhat slower rate. The future use of pesticides can be speculated to be largely a function of changing social mores and subsequent institutional regulation Environmental Health Perspectives rather than solely a function of demand for increased production and labor substitution. But, of course, the latter mentioned variables will remain in the pesticide use equation.
Insecticide use in terms of pounds is not likely to increase substantially. However, shifts in compounds used will continue to occur. Continued decreases in chlorinated hydrocarbons and changes among the organophosphates, with emphasis on greater specificity, can be expected. Also, some important new classes of compounds will play a major role in the years ahead. For example, among the insecticides a wide range of highly effective synthetic pyrethroids have been recently introduced (44). Also, a group of benzoyl phenylureas that act as insect growth regulators by inhibiting chitin synthesis look quite promising (45). These latter compounds are also active against nematodes.
Herbicide use will continue to expand but perhaps at a reduced rate. The amide and triazines will continue to represent the major chemical classes with some significant increases in other classes. Also, regulatory actions will likely have more impact on herbicide use in the future than they have in the past.
Nonsystemic, inorganic fungicides have been in use for over 100 years and continue to be of importance in select instances, especially the copperbased compounds; however, regulatory restrictions have had a major impact on these materials. Of the organic fungicides, dithiocarbamates will continue to be the most important group. Most current research and development into the fungicide area is focused on systemic products, so more fungicides of this type can be expected in the future.
Lastly, new compounds such as plant growth regulators and harvest aids are becoming more important with the advent of the energy shortages. However, there are regulatory issues to be resolved in obtaining approval for at least some of the materials which modify tissue growth (14).
Pest Control Policy
Although the use of pesticides in agriculture is the subject here, it should be emphasized that the U. S. Department of Agriculture is not only committed to the safe and effective use of pesticides, but to the development and use of alternatives to pesticides. Perhaps the Department's policy on pest control might be most accurately expressed by quoting from the Secretary's Memorandum No. 1929 dated December 12, 1977 (46): "It is the policy of the U. S. Department of Agriculture to develop, practice, and encourage the use of integrated pest management methods, sys-tems, and strategies that are practical, effective, and energy-efficient. The policy is to seek adequate protection against significant pests with the least hazard to man, his possessions, wildlife and the natural environment. Additional natural controls and selective measures to achieve these goals will be developed and adopted as rapidly as possible." The Department's new pest management policy should provide a framework for the U. S. Department of Agriculture to work more effectively with all segments of society interested in improving pest control. Certainly, a better understanding of the role of chemical mutagens in our environment and the way their impact might be measured is a critical part of hazard assessment. The exchange of information and the innovation stimulated by this workshop should help all of us carry out our responsibilities more effectively. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Effects of PBBs on cattle. I. Clinical evaluations and clinical chemistry.
Toxicosis was induced in pregnant heifers by feeding 25,000 mg/head/day of FireMaster BP-6, a commercial blend of polybrominated biphenyls (PBB). The PBB feeding decreased dry matter intake approximately 50% by 4 days exposure. Emaciated animals became anorexic a few days prior to death at 33 to 66 days. Weight losses of heifers average 80 kg. Other clinical signs observed were dehydration, diarrhea, excessive salivation and lacrimation, fetal death, abortion, and general depression as evidenced by depressed heart and respiratory rates. Clinical signs were apparent after 10 days exposure and progressively intensified along with loss of condition until death. Clinicopathologic changes included significantly increased serum glutamic-oxaloacetic transaminase and decreased serum calcium by 30 days exposure. Lactate dehydrogenase, urea nitrogen, and bilirubin were elevated, and serum albumin decreased by 36 to 40 days. Principal urine changes were decreased specific gravity and moderate proteinuria. Pregnant heifers fed 0.25 or 250 mg/head/day for 60 days and nonpregnant heifers fed 250 mg/head/day for 180 days displayed neither clinical signs nor clinicopathologic changes indicating adverse effects from PBB exposure. Post-exposure, all heifers exposed to PBB for 60 days calved normally with zero calf mortality and were successfully rebred. Milk production was not different from control animals. Birth weights of calves from dams exposed to 250 mg PBB/head/day were significantly greater than calves of dams exposed to 0 mg or 0.25 mg/head/day. PBB exposure of dams produced no detrimental effects on calves as indicated by clinical signs, clinicopathologic changes, or performance.
Introduction
In 1974 the industrial fire retardant FireMaster FF-l (PBB), a pulverized form of FireMaster BP-6 with 2% calcium trisilicate added to prevent caking, was advertently mixed with livestock ration and fed to dairy cattle (1). Widespread attention has been focused on the incident, a major concern being the hazard present to the exposed livestock and consumer health. Several field studies have indicated increased cow and calf mortality, decreased milk production, and a wide variety of nonspecific clinical signs attributed to PBB exposure (2,3). However, a field study of contaminated and noncontaminated herds failed to associate PBB exposure with reported signs of toxicosis (4). The discrepancies between these field study reports possibly reflected the great variations of levels and durations of exposure, farm management practices, and lack of adequate farm records. The immediate need arose for controlled studies to evaluate the acute and latent health hazard present to cows and their offspring at varying levels and duration of exposure to FireMaster.
Materials and Methods
Sixty dairy animals were involved in seven studies to determine distribution and clearance of FireMaster BP-6 and to evaluate the health hazard present from this residue. Table 1 lists the animals involved in the various experiments, the daily and total amount of FireMaster received, and the duration of the experiment. Specific details of procedures and animals for experiments 1, 2, 3, (5); 4 (6); and 7 (7) have been reported previously. Experiments 1, 2, and 3 were designed to carefully monitor the short-term distribution and clearance of FireMaster. Changes in health of animals were recorded by attending technicians, but no quantitative procedure was used to evaluate health. All but one of the animals involved were necropsied. The surviving animal is currently under observation with health, breeding, and milk production records being maintained. Experiment 4 was designed to systematically and quantitatively evaluate the health, performance, and residue dynamics of pregnant heifers given 0 to 25 g of FireMaster for 60 days. Feed intake and body weight measurements were recorded, along with frequent clinical evaluations performed by a veterinarian. Clinical evaluations included the grading of general health and eight reported clinical signs of PBB toxicosis (2) on a 1-10 scale, plus measurement of heart rate, respiration rate, and body temperature (6). In addition, blood and urine were collected for clinicopathologic determinations.
Following parturition, milk production alnd breeding records were maintained. Three animals from each group were allotted to necropsy for histological examination of tissues. Of these, two were necropsied on day 61 and the other and her calf 10 datys postpartum. All moribund animals wer-e also necropsied.
In experiment 5, feed intake and growth of the calves of the heifers from experiment 4 were recorded, along with clinical evaluations and clinicopathologic determinations. In addition, the attending technician recorded daily ratings of health and evaluated the appetite, breathing, eyes. and stool of each calf daily for 180 days. Blood and urine sampling for clinicopathologic determinations is being continued. Experiments 6 and 7 were toxicity studies designed to monitor specific target organ effects from PBB exposure. However, clinical observations and blood and urine were collected to give additional information on the overall health hazard present to dairy animals from PBB exposure.
In all experiments the animal that were dosed were given the FireMaster in gelatin capsules with a balling gun to insure that the total dose was received and to reduce cross-contamination. Management practices were followed that minimized housing effects, reduced cross-contamination, and assured that all animals had a similar environment.
The clinical evaluations of animals were performed at frequent regular intervals throughout the experiments. Previously recorded evaluations were not available to the veterinarian at the time of each clinical evaluation to minimize bias. Blood for clinicopathologic determinations was collected by venous puncture in untreated (serum) and sodium fluoride treated (whole blood) vacuum tubes. Urine was collected by induced micturition, filtered, and stored with benzoate preservative. The following serum and plasma parameters were determined: glutamic-oxaloacetic transaminase (SGOT), alkaline phosphatase (AP), lactate dehydrogenase (LDH), total protein, albumin, glucose (total reducing sugars), bilirubin, urea nitrogen (BUN), uric acid, creatinine, cholesterol, calcium, and inorganic phosphorus. Globulin and the albumin: globulin ratio were calculated. Urinary reducing sugars, calcium, potassium, and specific gravity were measured. Urine was screened for the presence of glucose, protein, ketones and blood. Color and appearance were evaluated and the urine sediment was examined for the presence of epithelial cells, erythrocytes, leukocytes, crystals, casts, and mucus. Detailed descriptions of each method and typical coefficients of variation for the chemical determinations have been reported (8).
Results and Discussion
These results are intended to provide an overview of our clinicopathologic findings from studies of the effects of FireMaster in dairy cattle. Seven separate experiments were conducted over a period of three years under var-ying conditions and with differing objectives. The various doses of FireMaster given were 0.25 mg, 250 mg, 3 g, and 25 g daily for periods of I to 202 days. In addition, calves were exposed in l/t(o and thr-ough consumption of milk from contaminated dams. The results obtained are reported in two primary categories: exposures which elicited a toxic response and exposures which did not.
Exposures Which Did Not Elicit a Toxic Response
The first study with FireMaster was designed to obtain information on short-term residue dynamics. Toxicosis was not expected, so the protocol did not include measurements of clinicopathologic responses. No clinical signs or lesions in necropsied animals that could be attributed to PBB exposure were observed in mature cows given single 3 g doses or in calves which received the residue in utero or through milk from contaminated dams (5).
Studies to quantitate, clinicopathologic responses precisely were designed and conducted because of the increasing evidence of toxicity reported in field studies of contaminated animals. Pregnant heifers in experimental groups that were exposed to 0.0, 0.25, and 250 mg FireMaster/daily for 60 days did not exhibit signs of toxicosis (6). Intake of dry matter and body weight significantly increased for the three groups. Feed efficiencies were not different among the three groups. Heart rate, respiration rate, and body temperature were unaffected and mean general condition scores from clinical evaluations improved ( Table 2). The change in the condition of the animals was attributed to improved management and nutrition. Clinicopathologic values of blood and urine samples remained within reported normal ranges for cattle (Table 3) giving no indication of a toxic response attributable to PBB. No gross pathologic changes or changes in tissue sections were observed in animals necropsied (17).
Animals that were not necropsied have remained under study throughout their lactation. A relationship between exposure to PBB and milk production, incidence of mastitis, and the number of insemina- tions required per conception was not evident in these studies (Table 4). Milk production was considered adequate for these grade heifers as superior animals with good milk production potentials had not been selected for toxicity studies.
Calves from the above dams were exposed to FireMaster through placental transmission and their dams' milk. Mean birth weights of calves were 35, 38, and 44 kg from dams fed 0.0, 0.25, and 250 mg/ day for 60 days. Weight gains of calves by day 42, weaning, were 18, 15, and 13 kg and after 180 days Table 4. Summary of mean daily milk production, incidence of mastitis, and insemination of heifers exposed to PBB. a Mastitis related to an injury to the teat. b Mastitis related to a prepartum injury and resulted in the loss of production of one quarter for the entire lactation. ' Persistent mastitis necessitated discontinued milking of one quarter on day 127 of lactation. 107, 121, and 113 kg. Clinicopathologic data revealed no significant changes suggesting a toxic response associated with FireMaster exposure.
In order to determine if prolonged exposure would elicit a toxic response, one pregnant and four nonpregnant heifers were given 250 mg FireMaster daily for 180 or 202 days. Clinicopathologic determinations indicated no toxic effects that could be attributed to PBB exposure. The pregnant heifer had dystocia at parturition, which resulted in the stillbirth of a 56*Xkg calf. Necropsy of the calf revealed no abnormalities. The reproductive tract of the cow regressed normally, and she was rebred.
In summary, analysis of clinicopathologic data showed no toxic response in animals given either a single 3 g dose of FireMaster or continuous doses of up to 250 mg/day to give an accumulative dose of up to 50.5 g. Actual mean concentrations of PBB residue in fat tissue exceeded 30 parts per million in animals fed 250 mg/day, exceeding the FDA interum guideline by 100 times. The toxic syndromes observed in field studies of contaminated animals were not evident in any of these experimental animals. Although at present there is no suggestion of toxicity, the animals will remain under study throughout their productive lifetimes.
Exposures Which Elicited a Toxic Response
Toxicity was induced in six pregnant heifers fed 25 g doses of FireMaster daily for 32 to 60 days. These animals consumed between 0.8 and 1.5 kg of Environmental Health Perspectives FireMaster. Intake of dry matter was reduced 50% by 4 days exposure and declined to approximately 1 kg by 30 days (Table 5). Subsequently, body weight was significantly reduced with losses of 80 kg among heifers surviving 40 days (Table 5). Heart and respiratory rates progressively decreased (Table 5). Excessive lacrimation and salivation were observed as early as day 11 and increased progressively in severity. Dehydration and emaciation developed and persisted throughout the trial. In four of the heifers, diarrhea developed and persisted from 3 to 9 days with occasional tenesmus. Three heifers aborted on days 30, 33, and 38, and three had retained dead fetuses when necropsied. All heifers were moribund within 33 to 66 days.
Changes in some clinicopathologic values occurred in these heifers. SGOT and serum calcium had a significant response by day 15. After 30 days exposure SGOT, LDH, BUN, and bilirubin were significantly (p < 0.01) increased and serum calcium and albumin decreased from pretreatment normals ( Table 5). Although significantly different from pretreatment, albumin and bilirubin did not deviate from the normal range reported for cattle (Table 3). Changes in the urine were not marked. Urine calcium of heifers in group II was lower on days -1 and 15. Concentrations in heifers of Group IV, showed a marked decrease by day 15 and remained lower until necropsy. Urine calcium concentrations of animals in groups I, II, and III were lower on days 150 and 190 following a ration change to haylage on day 110. Interpretation is difficult because calcium concentrations are based on single samples and not 24-hr collections. Samples taken immediately prior to necropsy revealed a trace to moderate proteinuria, a slight decrease in pH, and decreased specific gravity. The specific gravity decreased from a pretreatment mean of 1.038 to a mean of 1.017 in terminal samples. All animals were maintained as long as possible. When death was impending heifers were necropsied on days 33, 36, 39, 40, 41, and 66. Principle changes were observed in the gall bladder and renal tubules. These and other changes in tissues of the terminally toxic heifers have been reported in detail (17).
In an experiment designed to measure renal function in PBB exposed animals, two mature cows were given 25-g doses of FireMaster daily for 25 days and necropsied 35 days post dose (7). It was clinically apparent that one cow was more severely affected. The other was beginning to show clinical signs when necropsied. Primarily a decreased appetite and weight loss were observed in both with a 30% decrease in body weight of one and a 12% decrease in the other at necropsy. Clinicopathologic responses for the more toxic animal were similar to those observed in the six heifers exposed to 25 g/day (18).
The changes in routine clinicopathologic parameters examined in these eight animals have indicated a definite toxic response in dairy animals fed 25 g/day of FireMaster, but were not specific or sensitive so as to provide a clear indication of the exact mechanisms of toxicity. However, target organ effects were indicated. Several parameters suggested that liver was affected by PBB, but necrosis was not evident (17). The mild rise in bilirubin values, a good indicator of biliary obstruction and hepatocellular damage, was considered a reflection of gall bladder changes and minimum hepatocellular damage (17). Serum values of SGOT and LDH peaked at approximately the time of fetal death and/or absorption and were at least partially attributed to uterine cell necrosis.
The increase in BUN, a good indicator of renal damage, and the decrease in urine specific gravity suggested FireMaster was primarily a renal toxin. This was supported by microscopic examination of renal tissue (17). The elevated BUN possibly reflected decreased flow of urine through the tubules, and decreased specific gravity indicated an alteration in the ability of the tubules to concentrate urine (19). The primary sites of the renal lesion were distal tubules and collecting ducts which are primarily responsible for the forming of a concentrated urine (19). Alterations of intrarenal blood flow or a washout of the corticomedullary concentration gradient are two mechanisms which could affect the flow through and the reabsorption abilities of the tubules. However, recent studies in our laboratory have suggested these mechanisms are not affected in PBB exposed dairy animals (7). An effect of PBB on production or action of the antidiuretic hormone (ADH) cannot be entirely ruled out; however, there is no evidence from current work to implicate the role of ADH in this syndrome.
The mild hypocalcemia of toxic animals was possibly due to decreased uptake of calcium from the gastrointestinal tract because of the marked decrease in feed intake of the animals and/or some disruption of the calcium metabolism. Synthesis of the active metabolite of vitamin D (1,25-dihydroxycholecalciferol) which acts directly upon its target organs, the small intestine and the bone to mobilize calcium stores, occurs in the renal tubules (20,21). This suggests that the renal tubular damage may have interfered with 1,25-dihydroxycholecalciferol production or action and possibly affected calcium availability. Decreased serum calcium concentrations were reported in a survey of 16 low-level exposure herds (4). In our studies, effects on serum calcium were observed only at extremely high levels of exposure.
The clinicopathologic parameters examined indicated the kidney and possibly the liver as target organs, but were not specific enough to relate an exact mechanism of action of PBB.
In summary, we have gained several insights into the clinicopathological effects of PBBs in dairy cattle. They are categorized as: dose response to toxicity, and physiological and clinical manifestation of toxicity. Firstly, PBB toxicity as determined from clinicopathologic parameters measured in these studies, was not dose responsive. Instead, PBB appeared to be a threshold toxin since toxicity was not evident during exposure and in the two years following exposure in our experimental animals given 250 mg/day or less. Although placental transport of PBB has been demonstrated in these animals (22) teratogenic effects have not been observed.
In contrast, the higher dose of 25 g/day elicited an unequivocal toxic response. At this level the animals could no longer tolerate or detoxify the compound(s), and toxicosis was induced. The toxic syndrome was evidenced by a variety of clinical signs, changes in blood and urine chemistry, and a reproducible and specific pathological lesion in the kidney: the mechanisms of PBB toxicity in the kidney or other affected tissues (i.e., liver, gall bladder, etc.) could not be determined. Further research is being conducted in our laboratory to explain the toxic syndrome through understanding of the primary toxicity to the target organs, the mechanisms by which the clinical signs are elicited, and by distinguishing target organ effects from secondary effects. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Genetic formulas for the colour in the Texel, the Dutch and the Zwartbles sheep in the Netherlands
The Texel sheep of the Netherlands, as well as the common sheep we have called Dutch, are usually white. Their most frequent formula for the three loci of coloration Agouti (A), Extension (E) and Irregular Spotting (S) is !t ’""! ’"’’!+!+S+5+, where Awh is a gene for light tan or white.The blacks segregating in Texel are given by the recessive a (black) in Agouti. The black sheep found in Dutch sheep, on the contrary, are usually A -hA wnEaE+ (or EdEd) S+S+. The black pigmentation being due to the dominant Ed allele in E which is epistatic on A gouti genotypes. The Zwartbles which is black with white stripe in head, white socks and white tip of tail (HST) has a formula which is probably AwhAwhEaEa (or EdE+)SbSb where Sb is the allele for HST or Bizet design. The frequency of a in Texel sheep is around .oi8 that of Ed in Dutch sheep lies between .015 5 and .030.
Introduction
According to H A G >;DOORN and H A G>; DOORN ( 1914 ) the black coloration segregating in white flocks in the Netherlands was due to a gene recessive to a gene for white.
Nevertheless a note by KAr,ss>;!x ( 19 6 3 ) seemed to indicate that a dominant type of inheritance for the black was also present in the sheep of the Netherlands and this was confirmed by HOOGSCHAGEN (1963). Later on data were collected among breeders in the Netherlands (H OOGSCHA . GEN , 19 6 4 ) and presented in two articles: one of which was devoted to hereditary transmission of white markings on black background (HOOGSCHAG!N,19 66) and the other had to do with the two types of black (recessive and dominant), HOOGSC H AGE N (1967).
Since that time additional information has been presented concerning the transmission of coat color in Sheep. In this article a consideration will be given to a more accurate interpretation of the inheritance of coat colour in the Netherland sheep. According to the May-census there were in 1977 about 8 00 ooo heads of sheep in the Netherlands, thereof about 330 ooo mature ewes and q 5 o 000 lambs.
We have considered three breeds of the Netherlands: the Texel, the Dutch and the Zwartbles sheep. The Texel is a registered white breed originating from the Texel island, with about 6 0 00 o ewes registered.
The Dutch sheep is the name we propose in this article to the non-registered common sheep. It presents many similarities with the Texel as many Texel rams are among its progenitors. The number of Dutch sheep can be estimated on 250 00 o ewes, most of them are white, about 10 to 15 ooo are black.
The so-called Zwa y tbles breed gathers flocks of black sheep deriving from Dutch populations and wears the white design sometimes called HST (white head, spotted socks, white tail tip). The number can be estimated between i ooo and 2 000 .
It is in extension with the need for black wool for handicraft. The data are those already presented by KA>,ss!!K ( 19 6 3 ) and HooGSC H nGrrr (1963,1966,1 9 6 7 ) plus those resulting from a survey in pure Texel Sheep and others recently gathered by one of us (P. H.).
In order to measure the economical importance of breeding black sheep in the Netherlands we have checked the observations of the Netherlands Wool Board (Co 6 fieralieve Nedeylandse Wolfederatie G .A. * ) which commercializes more than 50 p. 100 of domestic wool.
II. -Results
The results of crossings are presented in table I and 2 .
White
The existence of two black factors: one acting as a dominant, the other as a recessive towards white, has been described for years in the Sheep (see R AE , 195 6 for a review). For a geneticist in mammalian coloration it is rather puzzling as, usually, black may not be obtained by alleles of loci giving white designs as these genes act only as suppressors of any kind of pigmentation (SEAR!,E, 19 68). Moreover it is hardly to admit a dominant black as well as a recessive one being at the same locus.
Nevertheless it has been shown that an allele in Agouti may play the role of a white gene. This is the case for the allele A 1 of A DALS TE IN S SON ( 1970 ) further on (rg 74 ) named A -h by the same author. This factor induces a tan pigmentation (black or brown eumelanin being suppressed) which at its turn may turned offset, giving pure white. This offseting is apparently due to modificater genes which may lead to pure white breeds.
Later on L AUV E R G N E ( 197 6) studying in France various crosses between deep red Solognot, white Berrichon, black with HST Bizet and a black and tan HTS Finish ram has shown that, in this situation, a modificator gene for obtaining pure white from a red allele in Agouti was the piebald HST gene called S h (ADAI,ST!INSSON'S 1974 s). Whose behaviour was very different according the pigmentary background. This red allele was nemed A!'h but one can wonder if it is the same as ADn!,sTW NssoN's one.
) Recessive black
White being explained by an allele in A gout i the recessive black is, as in may Mammals, the recessive term of this allelic series.
) Dominant black
Reviewing previous works, specially those by R OB E R T S (i 9 2 4 ), R OB E R TS and WHITE ( 193 0) and ZO PH O N IASSO N ( 1934 ) and taking account of the homology between colour loci in Mammals R!ND!r, ( 1957 ) was the first author to consider the dominant black gene in the Sheep to be an allele at the Extension (E) locus. This interpretation supposes that E d is not only dominant upon E + but also epistatic on any kind of allelic combination in Agouti locus. Recently one of us (I,nuv!xG:!!, 197 6) has analysed data with the two type genetic factors for the black, with that scope in mind.
B. -Inter!yetation of our data I ) The white colour and the recessive black According the above considerations the white of Texel could result of the action of an allele in Agouti dominant towards the recessive black a. As the blacks segregating are solid black the gene for HST S b is apparently absent from the breed.
One can admit that the A g'OM!' allele for white is !4'&dquo;'', as in Iceland, provide one considers that, in some cases this factor for tan may allow some black pigmentation on the muzzle and around the eyes as in the white Gotland sheep described by L6F V E NB ERG and JOHANSSON (1952).
The formula of white Texel becomes A w h A '&dquo;!5'+5'+ or A &dquo;'!a5'+5'+, as some recessive blacks aaS + S + are sometimes produced. The formula of white Dutch is probably identical to that of white Texel.
2 ) The dominant black The crosses no 3 , 4 , 5 , 6, and m demonstrate the existence of a dominant black, the black parents being homozygotes as in 3 and 4 or heterozygotes as in 5 , 6 , 7 and m ( resp . AwhAwh E a E a and AwhAwhD.a!.+).
The crosses 8, 9 and io, show that the two genes for black, dominant and recessive, are definitely non allelic.
) The white marks on black background
The hereditary transmission of HST among pigmented animals appears monofactorial recessive as tested in table 2 : s b < S + . Some very thin dominancy (white hair on the forehead) may be detected in some heterozygotes.
These interpretations concerning heredity of pigmentation and of white markings are summarized in table q..
It may seem strange that the black is due in two closely related breeds in the same country to different genetical formulas. This is apparently due to the fact that only white Texel rams are raised and sailed out, which prevents the spreading out of the recessive black a. On the other hand in Dutch breed it is easier to obtain black offspring with a ram wearing a dominant black.
C. -Com!arison with previous interpretations A.n hypothesis with two independant factors for the black was already brought by one of us (H OO G S C HA GE N , 19 6 3 ) : W fw (w = recessive black) and Z /z (Z = dominant black). For statistical tests there is no difference between this hypothesis and the present interpretation. But, if the alternative W lw fits well with A /a, the dominant black Z upon a white z does not correspond exactly to the new interpretation. The relationship of E d towards white, as a matter of fact, is not of dominancy but of epistacy. The allele E d gives a black coloration whichever the formula in Agouti may be and is dominant on E + whose behaviour is simply to allow the normal expressivity of genotypes in Agouti locus.
For the piebald gene giving HST there is a close corresponding between I IO OGSC HA GE N ' S n ( 19 66) With the data of the survey in pure Texel breed ( 4 in 13 000 ) one can estimate the frequency of the gene a: (q a = 0 . 01 8 in that breed).
The data of table 3 show that the frequency of pigmented wool is about 2 p. 100 (established on seven years). According the experts this value must be majored of at least I point, as a greater part of black wool is not checked by the Wool Board, as it is used for handicraft.
If all the black sheep on Dutch breed are homozygote the frequency of the gene E d is 0 . 03 . In case all are heterozygotes the frequency is only 0 . 015 . The frequency lies probably somewhere between these two figures.
Conclusion
Two genetical types of black do exist in the sheep in the Netherlands. One is given by the recessive a allele in Agouti; the other by a dominant allele E d in Extension, which is epistatic on Agouti genotypes.
Pays-Bas
Le mouton Texel des Pays-Bas, au même titre que le mouton commun de ce pays que nous avons appelé Dutch, est généralement blanc. La fosmule la plus fréquente aux trois loci de coloration Agouti (A), Extension (E) et Panachure Irrégulière (S) est A w h A w h E + E + S+S+ où A wh est le gène pour le fauve ou le blanc. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Self-incompatibility systems as bioassays for mutagens.
Many flowering plants are unable to set seeds with their own pollen because a system known as gametophytic self-incompatibility is operating. The basis of this system is a single multiallelic locus S, and if the S allele carried by a pollen grain matches one of the two S alleles carried in the style, as it is certain to do upon self-pollination, then pollen tube growth is inhibited. Should one of the self-pollen grains carry a mutated S allele, however, it would not match either of those carried in the style and would therefore, not be inhibited. Gametophytic self-incompatibility thus provides a mechanism for discriminating between such mutant and nonmutant pollen grains. Knowing the numbers of pollen grains available to the stigma, and also the numbers of seeds produced, it becomes possible to estimate the frequency with which mutations occur at the S locus. Assay systems of mutagenesis which employ gametophytic self incompatibility will allow very large numbers of pollen grains to be screened for S allele mutants, which should indicate the mutagenicity of the environment. These systems have the added benefit that screening is done by the stylar tissues, rather than technicians. Finally, they may be used to construct largely autonomous assay systems which would provide continuous monitoring of the environment.
bility will allow very large numbers of pollen grains to be screened for S allele mutants, which should indicate the mutagenicity of the environment. These systems have the added benefit that screening is done by the stylar tissues, rather than technicians. Finally, they may be used to construct largely autonomous assay systems which would provide continuous monitoring of the environment.
Flowering plants have evolved a variety of mechanisms which insure that accidental selfpollination is not followed by self-fertilization. Such mechanisms are known as self-incompatibility systems and generally function by preventing either the germination or the tube growth of self pollen. Gametophytic forms of self-incompatibility, certainly the most widespread type in existence, are so named because their function basis is the haploid, or gametophytic, genotype that is carried within the individual pollen grains.
In the simplest case of gametophytic selfincompatibility, a single multiallelic locus, S, controls the reaction between pollen and style. If the S allele in the pollen matches either of the two S alleles in the stylar cells, pollen tube growth is inhibited. It is important to note that, with this form of self-incompatibility, pollen grains from a single plant differ in their incompatibility type; this fact provides the basis of a system for monitoring environmental mutagenesis. If a plant possesses a rigorous form of gametophytic self-incompatibility, numerous self-pollinations, involving many millions of pollen grains, will not result in a single seed, unless, however, the S allele carried by a particular * Department of Botany, University of Massachusetts, Amherst, Massachusetts 01003.
December 1978 pollen grain has mutated. In that case, the pollen S allele will not match those of the style, and pollen tube growth will proceed normally, resulting in fertilization and the formation of a seed. Determining the numbers of seeds resulting from pollination by a known number of pollen grains provides a good measure of mutation rates at the S locus.
Advantages of the S System as a Bioassay for Mutagenesis Devreux and de Nettancourt (1) have pointed to two major advantages demonstrated by the self incompatibility system. (1) Since pollen is a haploid system, the difficulties of detecting recessive alleles do not arise as they do in diploid systems. Thus, most mutations in the S allele will be expressed. (2) If mutant pollen tubes are allowed to effect fertilization, the mutant genotype can be recovered and subjected to further study and verification. This is particularly important since, to this point, no S allele has ever been changed by a mutagenic agent from one specificity to another. That is, S, mutates to S2. Instead, all mutations have represented deactivation of some component in the S allele, thus S, mutates to S,'. This deactivation may be limited to the pollen, to the style, or exhibited by both (2). Detection of a new specificity among presumptive 85 mutants would thus certainly indicate contamination rather than mutation.
To these important advantages, we may add the following. Very large numbers of pollen grains can be screened with little effort. The stigma of Oenothera organensis will accommodate nearly 5,000 pollen grains, a fact which allowed Lewis (3) to screen 65 x 106 pollen grains for mutants in the S allele. Taking advantage of the small pollen grains and large stigmatic surfaces of Nicotiana alata, Pandey (4) found that over 17,000 pollen grains could be screened in each pollination. These large numbers allow rare mutational events to be detected and their frequencies to be measured with great accuracy.
Another major advantage of the self-incompatibility system is that the actual screening of mutant versus nonmutant pollen grains is accomplished by the stylar tissues themselves. This eliminates the need for great skills on the part of technicians.
Environmental mutagenesis is very likely to vary greatly over both time and space. Thus, it becomes imperative that in situ assay systems be developed. Several of the species carrying the S allele may provide this capability and, as outlined in a following section, it now seems likely that a largely autonomous assay system, employing the self-incompatibility system, may be feasible. This would allow a significant increase in our capacity to measure environmental mutagenesis.
Disadvantages of the S System
As is discussed below, previous studies of the self-incompatibility systems have indicated that they are presently less sensitive to mutagenesis than is the Tradescantia stamen hair system, for example. Until now, however, no efforts have been made to maximize sensitivity of the system and many opportunities exist for doing so. It thus seems likely that this disadvantage can be overcome.
Although the self-incompatibility system is often extremely effective in discriminating between self and "other" pollen, this is not so in some species. Cheiranthus cheiri (Cruciferae) exhibits what is termed, "cryptic self incompatibility." That is, it rigorously excludes self pollen tubes when pollinated with a mixture of self and other pollen. However, it is fully self fertile when pollinated with only its own pollen (5). In other cases, a wide spectrum of environmental stresses as well as aging and, possibly, infection, may significantly weaken the selfincompatibility system (6). Susceptibility to such influences is apparently a clonal characteristic (7) and thus careful selection of plant material will greatly diminish this possible source of error (2).
Nature of S Allele Mutations
Two general models of S allele mutations have been suggested. The first of these is based on Lewis's (3,8) now widely accepted suggestion that the S allele is actually tripartite, one component of which codes for the S allele specificity (S, or S2, etc.), another which activates the incompatibility system in the style, and a third for activation in the pollen. Lewis (3,8) was the first to suggest that simple point mutations could destroy the specificity code or the pollen activator of self incompatibility, either of which would result in a failure to reject "self" pollen. Plants bearing such mutations are thus capable of self pollinations.
A second model for mutations to self compatibility was proposed by Brewbaker and Natarajan (9). It is based on the finding by Lewis (10) that in some systems of gametophytic self-incompatibility, the presence of two, rather than one S allele, may severely limit or even eliminate the self incompatibility reaction. The presumed explanation for this fact is that competitive interactions between the two S alleles prevent either one from functioning normally. Brewbaker and Natarajan (9) employed this hypothesis to explain why, after irradiating plants of Petunia to induce self-compatibility, they found that each resultant mutant carried an extra centromere-bearing fragment of chromosome. They postulated that these fragments carried an S allele which interfered with the functioning of the normal S allele, thus inducing the self-fertile condition. Another view is that these fragments may serve also (or instead) to complement a genome which contains an otherwise lethal mutation and it is this mutation which causes the self-compatibility (4). More recently, de Nettancourt et al. (11) presented the very interesting hypothesis that centric fragments in Nicotiana alata may contain a nucleolar organizing region and, while the normal n.o.r. of the pollen genome is blocked by the self-incompatibility reaction, the n.o.r. of the fragment functions, thereby circumventing inhibition of pollen tube growth. In other studies, however, 11 out of 24 self-compatible mutations in Nicotiana alata were found to be free of centric fragments (12), although Pandey (4) has argued that chromosomal fragments may be inserted into the normal karyotype and carried there rather than on centric fragments. This may indeed be the case, but since centric fragments have never been associated with induced self compatibility in any species outside the Solanaceae (12), and Lewis (13) has presented evidence that they do not occur in Oenothera organensis (Onagraceae), we must conclude that they are not inevitable associates of S allele mutations.
The possibility that extra chromosomal segments Environmental Health Perspectives may be required to induce self compatibility in some taxa is an important consideration because such fragments will not result from a single chromosomal break. Instead they require two breaks and, because the probability of accomplishing this is reduced, low levels of radiation or low concentrations of mutagens will not be very effective in inducing multiple breaks. Indeed, van Gastel and de Nettancourt (14) found that chronic y-irradiation was almost totally ineffective in the induction of mutations to self compatibility. They suggested however, that this may be due to the fact that only very small doses of irradiation could be absorbed during the brief period of maximum sensitivity, the meiotic metaphase I. Thus, although the total dose accumulated may be large, most of this occurred during periods of low sensitivity to mutagenesis.
Sensitivity of the Self-incompatibility System to
Induced Mutation
Certainly one of the most successful higher plant assays for mutagenesis is that of determining the frequency with which stamen hairs of Tradescantia change from blue to pink (15). Accordingly, it may be useful to compare the sensitivity of the Tradescantia stamen hair system with the selfincompatibility system. Figure 1 shows a dose response curve for Tradescantia clone 02 (15). The mutagenic agent was x-ray, and the frequency of mutations to pink cells are indicated. The spontaneous mutation rate (control) has been subtracted from the values shown. Also shown in Figure 1 are data obtained by Lewis (3) with self-incompatibility systems of Prunus avium and of Oenothera organensis. For these studies, the mutagenic agent was also x-ray, although given in roentgens. The spontaneous mutation rate has been subtracted from the values shown. (To allow comparisons between rads and roentgens of x-rays, roentgens should be multiplied by 0.96, although the gross differences between these studies largely obviate the utility of this correction.) Among several studies of mutability at the S locus, that on Prunus avium provides the most nearly complete dose response curve, and clearly, there is a significant difference between the Tradescantia stamen hair curve and the Prunus self incompatibility curve. Several factors may function to produce this difference.
Sparrow et al. (16) have demonstrated that radiosensitivity may be considered in terms of target theory, and the genetic basis for pigment production in Tradescantia stamen hairs may perhaps present a larger total target area than does the S locus of Prunus or Oenothera. In that case, the stamen hair system would be inherently more sensitive than is the self incompatibility system. Another factor that must be considered is the difference between numbers of pollen grains and numbers of viable pollen grains. Mutagenic agents invariably reduce the porportion of viable pollen grains contained in any treated pollen sample. Thus, the number of viable grains used in pollinations decreases, often quite drastically, with higher doses of mutagen. Figure 2 was originally published by Evans (17) and shows the decline in pollen viability which accompanies increasing exposure to acute x-ray irradiation. In other studies, the decline has not been so dramatic: van Gastel and de Nettancourt (12) found that 300, 600, and 825 rads of x-rays reduced pollen viability in Nicotiana alata, as indicated by staining with iodine, to 81.9%o, 63.2% and 51.6% respectively. Exposure to 825 rads of x-rays reduced the germinability of Tradescantia paludosa, in comparison, to approximately 5.0% (17). Part of this difference between the two studies may be due to differential radiosensitivity (18). Another important factor is that germinability (used as a criterion by Evans), is frequently a much more accurate indicator of viability than is stainability, used by van Gastel and de Nettancourt. Exceptions to this differential are those cases where staining requires active metabolism. Fluorescein diacetate is one such exception (19,20). It is thus very likely that doses of 600-1000 rads, frequently employed by investigators of the incompatibility system, may be reducing pollen viability to a small fraction of control values and many of the pollen grains placed on the stigma are incapable of germination. Although some investigators have allowed for this factor (12,14), the frequency of S allele mutants may sometimes be grossly underestimated. Consequently, it is not presently possible to determine how sensitive the self incompatibility system is as an assay for mutagenesis. With few exceptions, for example, van Gastel and de Nettancourt (12,14) investigations of mutations to self-compatibility have been concerned with the nature, rather than the quantity, of mutational events. Thus, no attempts have been made to develop systems which will be sensitive to mutagenesis. The following section presents evidence which suggests that such attempts would be very fruitful.
Future Development of Self-incompatibility Systems as Assays for Environmental Mutagenesis To maximize the utility of self-incompatibility systems as assays for mutagenesis, the following tasks must be accomplished: (1) species which exhibit a high frequency of induced self compatibility mutations should be identified; (2 within these species, clones which exhibit a high degree of self incompatibility should be selected; (3) techniques for employing the selected clones as in situ assay systems should be developed.
The first goal, that of identifying species which exhibit a high frequency of induced mutations has perhaps been brought within reach by investigations of complex systems of self incompatibility. Lundquist (21,22) has shown that in several species of grasses (Secale cereale, Festuca pratense, Horn deum bulbosum, etc.) self incompatibility is gametophytically controlled and involves two distinct loci, termed S and Z. Inhibition of pollen tube growth requires that alleles found at both of these loci in the pollen must be matched by alleles carried in the style. A pollen grain of the genotype S,Z3 would be inhibited in a style of the genotype S,S2 Z,Z3. S1Z3 pollen, would not, however, be inhibited in an S2S3 Z1Z3 style. Thus, if either the S or the Z allele in the pollen is inactivated by mutation, pollen tube growth proceeds without inhibition. Presuming that the mutation rates at the S and Z loci are comparable to those of other taxa, we should thus expect the frequency with which pollen grains carry a mutation to self-compatibility in the grasses should be twice the frequency of that in taxa which carry only one self-incompatible allele. Furthermore, Ranunculus acris pollen grains each carry three loci (Sa, Sb, Se), all of which must be matched in the style to cause inhibition of pollen tubes (23) and in Beta vulgaris pollen must be matched in no fewer than four loci (Sa, Sb, Se, Sd) (24). These last two taxa should thus exhibit three and four times the probability of mutating to self compatibility than do single locus taxa. Presently, as was the case with most other examples of self incompatibility (6), it is necessary to identify clones of both Ranunculus acris and Beta vulgaris which exhibit a rigorous degree of self-incompatibility. The product of such selections may be new and highly sensitive assays for mutagenesis. In addition to increasing the sensitivity of the incompatibility system to mutation and also selecting for increased rigor in the rejection of self pollen, the development of largely autonomous, in situ assay Environmental Health Perspectives systems would be of great utility.
Requirements for such a system are surprisingly modest and met by many self incompatible species, including all of the above mentioned multilocus systems (the Gramineae, Ranunculus acris, and Beta vulgaris). The species should be capable of vegetative reproduction, in order that useful clones can be produced and maintained. Fruits produced by the flowers should be uniovulate so that it is not necessary to induce fruit development artificially. In each of the currently operative selfincompatibility assay systems (Oenothera, Nicotiana, and Petunia) the number of seeds borne in the ovary is quite large. This quality is generally considered advantageous in that it allows recovery of several mutants from one pollination. With it, however, comes the requirement that each ovary be treated with lanolin containing 8-naphthoxyacetic acid in order to prevent abscission of fruits which contain only a fraction of the normal complement of developing seeds.
Members of the Gramineae, Ranunculus acris, and Beta vulgaris all produce achenes; that is, oneseeded fruit. This eliminates the necessity of inducing parthenocarpy. A final requirement for the proposed autonomous assay is that the self incompatibility system of the test species may be circumvented in order to allow self-fertilization during establishment of the test material. This, too, is easily accomplished, since bud pollination is known to be frequently effective in this respect (25).
To construct an autonomous assay system in a species showing the prerequisites of sensitivity, rigor, and vegetative propagation, it is necessary to first self-fertilize one clone. If the original clone was heterozygous at one locus, e.g., S1S2, the resultant progeny will contain the genotypes S,S,, S1S2, and S2S2. Of these, the heterozygote S1S2 will be able to fertilize both its S,S, and S2S2 siblings. The homozygotes will be interfertile but neither one will be able to fertilize the heterozygotes. If rametes of an S,S, clone are interplanted with those of an S,S2 clone, a very interesting situation is created (see Fig. 3). A flower of gentotype S1S1 will receive some pollen which is produced by itself or by other flowers on the same plant, but I shall ignore this quantity, in part because in a large population it is probably very small in comparison to the amount of pollen received from other plants, and in part because only specific field tests will allow any estimation of what this quantity is. That same S, S, flower will receive pollen also from other plants of genotype S,Sl, all of which will normally be rejected as incompatible. Furthermore, an equal quantity of pollen will be received from plants of genotype S, S2, half of which will be rejected and half of which SI S2 S1 S S1 S, Sl S2 FIGURE 3. An autonomous, in situ, assay for environmental mutagenesis. SI S2 indicates a plant which is self incompatible and heterozygous at the S locus. One half of its pollen, that carrying the S2 allele, will be compatible on S1S1 stigmata. This compatible pollen flow is represented here by arrows and it will serve as a control, indicating how many seeds would be produced when compatible pollen is available. For SlS2 plants, the only compatible pollen grains will be those which carry a mutated S allele. Seed production by S,S2 plants, as a fraction of that in S,S, plants, will thus measure the frequency of mutation.
will not. Thus, one-quarter of pollen received by an S,S flower is compatible (S2) and three-quarters is incompatible (Si). For every 1000 seeds produced on clone S, SI we may assume that 3000 seeds were prevented from forming by the incompatibility system. Rametes of clone 52S2 should produce no seeds, but, for every 1000 seeds borne on clone S, SI, clone S S2 should have received a quantity of pollen which, if compatible, would have resulted in the production of 4000 seeds. Any seeds produced on clone S S2 will presumably carry mutants in the S locus. Harvest and planting of these seeds will allow rigorous testing of this presumption. The mutation rate may then be stated in terms of x mutants retrieved from clone Sj S2 per 1000 seeds produced in clone S1S1. This has the great advantage of allowing for changing quantity and quality of pollen and ovules produced by the plants throughout the flowering season. The result would be a true in situ assay system, monitoring the environment for long periods of time and perhaps detecting transient but biologically significant mutagenic events. The numbers of pollinations made would be many times those which could be made by experimentalists. Furthermore, such a system could be composed of individuals which carry easily identifiable electrophoretic markers. This would enable determining if presumptive mutants were derived from a single pollen source and thus perhaps represented a large mutant sector within one plant or if many plants contributed mutated pollen, indicating a high frequency of mutation.
On the basis of these and other considerations, such a system would enable self-incompatibility to provide useful monitors of environmental mutagenesis. | v3-fos |
2017-08-16T02:22:40.295Z | {
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} | s2 | Interrelationships among zinc, copper, lead, and cadmium in food, feces, and organs of humans.
Concentrations of zinc, copper, lead, and cadmium were determined in 20 samples of food collected over a period of 20 days, 221 samples of feces collected over a period of 5 days from 19 males, 17 females, and 11 children and 85 samples each of renal cortex and liver from autopsied human cadavers in order to investigate the relationships among the four metals and among the various martices. In food the highest correlation was observed between copper and zinc (0.34). In feces the highest correlation was also between copper and zinc (0.45). In the highest correlation between cadmium and zinc (0.33), but that in the renal cortex was between copper and cadmium (0.52). These findings suggest that the relationships among the concentrations of the four metals in food and feces are almost equal to each other, but differ greatly from the concentrations in human organs due to the differing metabolic actions of the metals once they are absorbed into the body. In addition, it was observed that zinc and cadmium concentrations in the renal cortex increase with age, but copper and lead concentrations do not show much variation with age.
Introduction
Concentrations of heavy metals such as zinc, copper, lead, and cadmium in human tissues and organs as well as in foods have been studied by many authors (1)(2)(3)(4). Interactions between two metals in animal experiments, e.g., cadmium and zinc or methylmercury and selenium, have also been well documented (5,6). The findings from these studies are very important for a better understanding of the toxicokinetics of metals. However, these animal studies used unusually high doses of metals, much higher than any exposure (including occupational exposure) to which humans would be subjected. In addition, rather few studies have been carried out on the concentrations of zinc, copper, lead, and cadmium in human feces. The present study is designed to determine and evaluate the dynamics of these four trace elements, two of which are essential and two nonessential, in the organs, food, and feces of humans who have not been subjected to unusually high exposures of these metals. The working hypotheses were (1) whether accumulation and magnification in a particular organ or tis-sue occurred because of the different metabolism of each metal or possible interactions between metals, and (2) whether the ratios of these four metals in food changed during ingestion, resulting in different compositions in feces. The study aims to provide some background information on the ratios of the four metals in these media even though the samples of food, feces, and organs were not collected from the same individuals. There is no doubt that much work needs to be done on these hypotheses in the future.
Organ Study
The concentrations of the four metals in the renal cortex and liver were determined in a total of 85 cases (male and female) autopsied by coroners of the Keio University Department of Legal Medicine. These cases were sudden death victims without chronic diseases or chemical poisoning. The organ samples were digested by wet ashing and the concentrations of the four metals were determined by atomic absorption spectrophotometry after DDTC-MIBK extraction. Since the methods of pretreatment for feces and organs were different, ten samples each of feces and organs were measured using each pretreatment method in order to cross-check the methods. No differences were found.
Food Study
For the food study, samples were provided by the administrative office of Keio University Hospital. The foods submitted for testing were from a regular hospital diet, and included beverages such as tea and milk. The three meals from each day were mixed and freeze dried, and then stored in plastic containers. The samples were collected for 20 days (20 samples, 60 meals). Ten grams dry weight were used for analysis and the analytical methods were the same as those used for feces described below. The weight of the food items for each day, total daily calories, proteins, fat, and carbohydrates were recorded.
Feces Study
The feces study was carried out on 19 male medical students at the Keio University School of Medicine (21-24 years old), 17 female nursing students at the same school (20-22 years old), and 11 children (3 months-5 years) of the staff of the Keio University School of Public Health and Preventive Medicine. Feces were collected daily for five days. Information concerning the diet on the day previous to feces collection, smoking and drinking habits, history of residence, and regular intake of medicinal drugs was obtained for reference purposes. Feces samples were collected in plastic bags, weighed, and immediately stored in a freezer. Frozen samples were then treated by a vacuum freeze dryer and weighed. Then 2-g portions were analyzed for zinc, copper, lead, and cadmium content. The freezedried samples were then ashed in an electric furnace at 400°C (36 hr) and diluted with 0.5N acid. Zinc was measured directly by atomic absorption spectrophotometry. Copper, cadmium, and lead were measured by atomic absorption spectrophotometry after extraction with DDTC-MIBK. Figure 1 shows the concentrations of zinc, copper, lead, and cadmium in the renal cortex according to age for both sexes. Cadmium increases sharply from 0 to 19 years of age, continuing with a gradual increase up to about 50 years of age, and leveling off thereafter. The zinc concentrations for the 0-9 year age group are rather high, whereas the cadmium concentrations for this same age group are much lower. Both copper and lead show almost the same concentrations throughout all age groups. The lead level is the lowest among these four metals for all age groups. Figure 2 shows approximately the same pattern of metal concentration levels in the liver as was found in the renal cortex, with the exception of copper. Copper shows the highest concentration in the liver in the 0-9-year age group, after which there is a slight decrease. The concentrations of copper in the liver are several times higher than in the renal cortex. There is not much difference in the zinc concentrations in the liver and renal cortex, but the cadmium concentrations in the renal cortex are al- Table 1.
Results
The Cd/Zn ratio in the renal cortex and the liver by sex and age is shown in Figure 3. The ratio in the renal cortex increases according to age up to the 40-49-year age group, after which it decreases. This pattern is very similar to the cadmium concentrations in the renal cortex by age group. The Cd/Zn ratio in the liver, on the other hand, seems to show no leveling off, but rather a sharp increase from the 0-9-year age group to the 10-19-year age group, with a very gradual increase thereafter according to age. The Cd/Cu ratio in two organs showed very similar patterns to the Cd/Zn ratio. However, the Cd/Cu ratio is ten times higher than the Cd/Zn ratio.
The Cd/Cu ratio in food, feces, and organs according to sex is shown in Figure 4. When this figure and Figure 5 are studied, it must be remembered that the food, feces, and organ samples were taken from different individuals. It is very interesting to note that the Cd/Cu ratio in food is almost equal to that in feces. However, the ratio in the organs is much higher than that in food or feces, being ten times higher in the liver and almost 100 times higher in the renal cortex. Figure 5 shows the Cd/Zn ratio in food, feces, and organs. Again, the ratio in food and feces is the same, with a much higher ratio in the renal cortex and liver: almost 100 times higher in the kidney and 10 times higher in the liver. But the Cd/Zn ratio in food, feces, and organs is about 10 times lower than the Cd/Cu ratio in these three media. Tables 2 through 5 show the correlations of concentrations of these four metals in food, feces, liver, and renal cortex respectively. As shown in Table 2, no significant correlation is observed between any two metals in food, most probably because of the small number of samples examined, although a rather high correlation is observed between copper and zinc, copper and cadmium, as well as copper and lead. four metals in feces, indicating significant correlations for all paired combinations of the metals. The highest correlation (r = 0.45) was seen between copper and zinc, as is observed in food. It should be remembered that the correlation between cadmium and zinc is very low in both food and feces. It is not surprising that correlations are seen among these metals in food and feces, because these are expressed by concentrations. But the purpose of this study was to discover which metals show the highest and lowest concentrations in food and feces and to study the relationships of these correlations in food and feces with those in the organs. When Tables 4 and 5 showing the correlations of these metals in the two organs are studied, a significantly high correlation between zinc and cadmium in the liver is seen but a reverse correlation appears between copper and cadmium, as well as a significant correlation between zinc and lead in the liver. There is also a significant correlation (r = 0.37) between cadmium and zinc in the renal cortex, as shown in cant correlations between copper and cadmium as well as copper and zinc in the renal cortex. However, no correlation is seen between lead and other metals in the renal cortex.
It was discovered from analyses of the data in these four tables that although correlations in food and feces were seen in almost all pairs of any two metals as expected, the correlations in the liver and renal cortex were quite different from those in food and feces. In particular, the correlation between zinc and cadmium in food and feces is quite low, whereas it was significantly high in the liver and renal cortex. Table 6 shows the daily excretion in feces of the four metals in inhabitants of an area designated by the Japanese Ministry of Health and Welfare as requiring observation because of environmental pollution by cadmium, as well as in-inhabitants of a control area and Tokyo. The daily excretion of cadmium in the polluted area is about 200 ,ug/day, even though the persons concerned were no longer consuming rice contaminated by cadmium. The cadmium excretion in the control area is 50 ,ug/day and in Tokyo is 40 ,ug/day. The daily excretion of lead is also very high in the polluted area, almost 230 ,ug, but in both the control area and in Tokyo, the daily excretion of lead is much lower than expected. It should also be noted that copper and zinc excretions are much higher in the polluted area than in the control area or in Tokyo.
Discussion and Conclusions
With regard to the concentrations of cadmium in the renal cortex and liver according to age, the present study indicates almost the same patterns as reported by Friberg et al. (7) and Tsuchiya,Seki,and Sugita (8), as well as by many other investigators. There are data on simultaneous determinations of zinc and cadmium (II) and lead and cadmium (12), but simultaneous determinations of copper and cadmium in the renal cortex and liver are not well documented. It is especially interesting to note that copper (an essential element) showed a higher level in the liver than cadmium and lead among all age groups. It is also important to note that the copper concentrations throughout all age groups are much higher in the liver than in the renal cortex and that lead concentrations in both the liver and renal cortex were lowest of all metals.
The Cd/Cu and Cd/Zn ratios in the renal cortex and liver increased sharply until about 20 years of age. In the renal cortex, the ratios level off by the age of about 50, and then decrease slightly in the very elderly (after 70 years). However, the ratios in the liver do not seem to level off, but continue to increase slightly by age. The Cd/Cu ratio was about ten times higher in the renal cortex and liver than the Cd/Zn ratio. The Cd/Zn ratio observed in this study was much higher than those reported by Schroeder et al. (9) from the U. S. and by Piscator and Lind (10) from Sweden. Both reported ratios of 0.6-0.7 and 0.5-0.6, respectively, for ages 35-55, while the present study indicated 0.9-1.1 for the same age group. This difference seems to depend not only on the fact that concentrations of cadmium are generally higher among Japanese than among Americans or Swedes, but also on the fact that zinc is present at a higher level in the renal cortex of the Japanese.
It was also noted in this study that despite the fact that the same pattern of Cd/Cu and Cd/Zn ratios was noted in food and feces, both the ratios in the liver and renal cortex were greatly amplified, being about 10 times higher in the liver and 100 times higher in the renal cortex than in food and feces.
There were some correlations between all pairs of the metals in feces (221 samples) but no significant correlation in food, probably because there were only 20 samples. The correlation between zinc and cadmium in feces was rather low. However, there was a significant correlation (r = 0.37) between cadmium and zinc in the renal cortex, and a significant correlation (r = 0.33) between these same metals in the liver. It is very important to note that there was a significant correlation between copper and cadmium in the renal cortex. This may imply very close relationships between zinc and cadmium as well as copper and cadmium in the renal cortex, whereas no such relationship was noted between copper and cadmium in the liver.
The differences and interrelationships among metals in food, feces, and organs observed in this study show the need for further investigations, since it is difficult to explain the findings in this study from the viewpoint of the toxicokinetics of these four metals. | v3-fos |
2019-01-02T22:51:46.755Z | {
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} | s2 | RESPONSE OF DRILLED EARLY CORN TO SEVERAL PLANT POPULATIONS
Dryland corn acreage has been increasing in Kansas because of the use of early-maturing hybrids that are planted earlier than full-season hybrids. Earlymaturing hybrids planted early (early corn) reach their reproductive stages before the mid-summer heat and drought occur. Also, farmers have been interested in reducing corn row spacings from the traditional 30 inches. Equidistant plant spacing, which occurs with narrower row spacings, reduces within-row competition. Narrow row spacings for grain sorghum and soybeans planted with a grain drill have increased yields, while reducing erosion. intercepting more light, and increasing water uptake. This study was designed to determine the influence of environment on various plant populations of drilled early corn.
Dryland corn acreage has been increasing in Kansas because of the use of early-maturing hybrids that are planted earlier than full-season hybrids. Earlymaturing hybrids planted early (early corn) reach their reproductive stages before the mid-summer heat and drought occur. Also, farmers have been interested in reducing corn row spacings from the traditional 30 inches. Equidistant plant spacing, which occurs with narrower row spacings, reduces within-row competition. Narrow row spacings for grain sorghum and soybeans planted with a grain drill have increased yields, while reducing erosion. intercepting more light, and increasing water uptake. This study was designed to determine the influence of environment on various plant populations of drilled early corn.
Procedures
Six fields studies were conducted over a 2-year period from 1991 to 1992. The two locations in 1991 were in Labette and Marion counties, and the four locations in 1992 were in Labette, Marion, Riley, and Republic counties. The site characteristics and management practices for all study sites are listed in Table 1. Conventional tillage operations were employed for seedbed preparation at all locations. Four commercial hybrids representing different corn maturities were Cargill 1077 (75-day) 1991, 1992Cargill 2927Cargill (85-day) 1991Cargill , 1992Cargill 3627 (95-day) 1991, 1992: Pioneer 3737 (95-day) 1991, replaced by Golden Harvest 2404(105-day) in 1992 Four plant populations of 20,000; 40,000; 60,000; and 80,000 plants/a were used at each location. In 1991, the Marion County site was overseeded and thinned to desired populations. The remaining locations for both years were planted at 15% above the desired populations. The experimental design was a randomized complete block with three replications. Grain yield was determined at maturity.
Results
In 1991, the average monthly rainfall during the growing season was below the 30-yr average, and temperatures were above the 30-year average ( Table 2). Conversely, in 1992, the average monthly rainfall was above the 30-year average, and temperatures were near or below it. Consequently. corn yields were considerably lower in 1991 than in 1992. In 1991, grain yields ranged from 4 to 50 bu/a. whereas in 1992, yields ranged from 51 to 195 bu/a.
1991. The analysis of variance indicated that the hybrid x population interaction was significant at both locations in 1991 (Table 3). At Labette County, yields for the 85-day and both 95-day hybrids were greatest at 20,000 plants/a and fell dramatically as populations increased ( Fig. 1). However, for the 75-day hybrid (Cargill 1077), yields at 20,000 and 80,000 plants/a were similar, and yields at 40,000 and 60,000 plants/a were significantly lower but similar. Yields for the 85day (Cargill 2927) and both 95-day hybrids were lower than yields of the 75-day hybrid, thus causing the interaction. Heat and moisture stress adversely affected the three later-maturing hybrids, whereas the 75-day hybrid avoided the brunt of the stress because of its early maturity.
Similar results occurred at the Marion County site (Fig. 2). All hybrids obtained their greatest yields at 20,000 plants/a; however, the 85-day hybrid (Cargill 2927) had significantly lower yields (28 bu/a) than the other hybrids. The 75-day hybrid produced similar yields at all plant populations, whereas yields for the 85-day hybrid fell dramatically. Interestingly, yields of Pioneer 3737 (95-day maturity) did not decline as sharply when populations increased as did yields for the other two hybrids. These factors accounted for the significant hybrid x populations interaction. Again, the 75-day hybrid avoided much of the stress. The 85-day hybrid, which had long, broad leaves, took advantage of the early-season moisture and grew excessively compared to the other hybrids. When available moisture was depleted, it was hurt during the reproductive stages.
1992. The analysis of variance for all locations indicated no hybrid x population interaction, but significant differences occurred in hybrids and populations (Table 3). This is indicative of the excellent growing conditions, because all hybrids responded similarly to the various populations. At the Labette County site, the 75-day hybrid produced 60 bu/a, which was significantly lower than yields of the other three hybrids (Table 4). Grain yields significantly increased with each incremental increase in population (Table 5). The lowest population produced 89 bu/a, and the highest population produced 165 bu/a.
At the Marion County site, the 75-day hybrid again produced the lowest yield (65 bu/a), which was sig- nificantly lower than yields of the other three hybrids ( Table 4). The yield of the 95-day hybrid (Cargill 3627) was significantly lower than the yield of the 85-day hybrid but was not significantly different from the yield of Golden Harvest 2404 (105-day maturity). Yields increased significantly from 106 to 145 bu/a as population increased from 20,000 to 60,000 plants/a (Table 5). Yield at 80,000 plants/a (151 bu/a) was not significantly different than yield at 60,000 plants/a (145 bu/a). Results at the locations in Riley and Republic counties were similar to those at the Labette County site, in that the 75-day hybrid had the lowest yields, but no differences occurred among the other three hybrids at each location (Table 4). Yields at the Riley County site significantly increased up to 190 bu/a with each incremental increase in population (Table 5). At the Republic County site, the lowest population produced 150 bu/a, which was significantly lower than yields of the other plant populations. No differences occurred among the three highest populations. 1991 1992 1991 1992 1991 1992 1991 1992 Hybrid *** *** *** *** *** *** --Population *** *** *** *** -*** -*** Hybrid x *** NS ** NS -NS -NS Population **,***= Significant at the 0.01 and 0.001 probability levels, respectively NS = not significant Yields of four corn hybrids of different maturities at four plant populations in Marion Co., KS, 1991 (LSD 0.05 = 11 bu/a) This publication from the Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information is available from http://www.ksre.ksu.edu. Kansas, 1992. | v3-fos |
2018-04-03T01:54:55.019Z | {
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} | s2 | Effects of polybrominated biphenyl on milk production, reproduction, and health problems in Holstein cows.
PBB found in relatively low levels among animals present on a cross-section of Michigan farms during the time PBB was inadvertantly added to dairy feeds had no effect upon these animals' milk production, body weight, weight gain, breeding and reproduction performance, incidence of commonly experienced health problems, calving rate, and the health of their calves. No significant differences in these vital areas could be seen between Michigan animals exposed to PBB and equivalent Wisconsin animals which had not been exposed to PBB when both groups were subjected to equivalent management practices. No pattern of gross of histopathological lesions was seen upon necropsy between test animals and control animals.
In May 1973, Farm Bureau Services received 80 50-lb bags of a dairy feed additive invoiced as magnesium oxide at its feed plant in Battle Creek, Michigan. Subsequent events have indicated that an unknown number of 50-lb bags of a flame retardant known as polybrominated biphenyl (PBB) were accidentally commingled with the May, 1973 magnesium oxide shipment. As a result, some dairy feeds made after May 1973 and into which bagged magnesium oxide was added directly contained small quantities of PBB. Other feeds which were mixed after the feeds into which PBB was mistakenly added, or which were stored in bins where feeds containing PBB were stored also contained minute quantities of the chemical.
The chemical was identified in late April of 1974. Since then many Michigan dairy producers whose animals consumed various quantities of PBB have claimed that PBB caused a variety of clinical symptoms, including: decreased milk production, loss of body weight, decreased appetite followed by an increased appetite, increased incidence of mastitis, increased breeding problems, abnormal hoof growth, hyperkeratosis and other skin problems, abnormal tooth wear, stunted or deformed calves, increased calf mortality, and decreased resistance to normal infections. Other farmers whose animals consumed quantities of PBB equivalent to, or in * Farm Bureau Services, Inc., Lansing, Michigan 48909. some cases higher than, the amounts consumed by animals claimed to have been adversely affected by PBB have reported that their animals did not exhibit any of the clinical symptoms commonly attributed to PBB.
Controlled PBB feeding studies conducted at Ohio State University, USDA-Beltsville, Maryland, and Industrial Bio-Test Laboratories in Northbrook, Illinois, have shown that dairy animals did not exhibit the clinical symptoms commonly attributed to PBB until they consumed comparatively massive doses of PBB.
The precise amount of PBB fed in controlled feeding studies may not duplicate the time and dose parameters of exposures to PBB encountered at the farm level. Therefore, Farm Bureau Services and its supplier of magnesium oxide, the Michigan Chemical Corporation (now Velsicol Chemical Corporation), commissioned a 2-year study to evaluate the effects of PBB on milk production, reproduction, offspring performance, and overall health problems under dairy farm conditions.
Procedures
Beginning in July 1976, 46 adult Holstein cows from six herds across Michigan which had been exposed to PBB were purchased as test animals. Each Michigan herd had been and was on a Dairy Herd Improvement Association, Inc. (DHIA) testing program. The test animals were selected on the basis of April 1978 28.7 a Cows received and lost neck chain magnets when their milk production exceeded or dropped below 55 lb of milk per day. b Dry matter. c The grain consisted of a 14% and 16%o commercial pellet for producing cows and a 34% pellet for dry cows containing grain products, plant protein products, processed grain by-products, forage products, cane molasses, pellet binders, animal fat, vitamins, and minerals. PBB tissue levels, previous milk production, health problems and reproductive problems.
Beginning in August 1976, 40 adult Holstein cows from five unexposed herds in different areas of Wisconsin were purchased as control animals and placed with the Michigan animals to form one herd. The control animals were selected in an effort to match the animals selected from Michigan in milk production, stage of lactation, housing, and management conditions. All animals, test and control, were examined by a licensed veterinarian for any permanent anatomical and physiological defects, leptospirosis, brucellosis, and tuberculosis. Blood samples were collected from the animals at or before the time(s) they joined the experimental herd. Blood samples were collected from 41 animals selected at random in April, 1977. Fat tissue samples were collected from each animal at or before the time she entered the experimental herd and were analyzed for the presence of PBB. The average PBB tissue level of the test animals was 0.31 ppm, with a maximum of 1.8 ppm. There was no PBB detected in the control animals.
Once on the experimental farm, all animals were separated into three groups according to their milk production level and stage of pregnancy. The groups consisted of: (1) high producers (40 lb milk per cow per day and above), (2) low producers (less than 40 lb milk per cow per day) and (3) dry animals.
Health and breeding records were maintained on each animal. Grain and forages were balanced nutritionally and fed in accordance with the level of production and stage of lactation. The composition and amounts of feeds fed to each group of animals was recorded and appears in Table 1. Daily milk records were maintained for each cow. The herd was also maintained on a DHIA testing program.
The animals were housed in barns containing free stalls and a loafing area. Forages were fed in outside, covered bunks. Grain was fed in one or more areas-the milking parlor, out of forage bunks, and from a magnetic feeder-depending upon the animals' level of milk production.
All calves were identified and tagged at birth. They were fed their dam's colostrum for the first three days after birth and housed in individual stalls in an insulated and ventilated calf barn. Calves were grouped in pens within the calf barn at one month of age. At three months the calves were moved to a noninsulated, ventilated barn. The calves' feeding program consisted of milk replacer, grain and forages at levels recommended for acceptable growth (Table 2).
Results
The cows' milk production for the first year of the experiment is summarized in Table 3. Two months after initiation of the experiment, the test cows were producing 8.0 lb more milk per cow per day than the Environmental Health Perspectives a Total milking days for all cows within each group divided by number days in respective month. control cows. At the end of 12 months, the control cows were producing 2.2 lb more milk per cow per day than the test cows. The accumulative milk production for the test group and the control group was virtually identical at the end of 12 months. The mature equivalent milk production data prior to and during the experiment is summarized in Table 4.
8 a Mature equivalent record is defined as the amount of milk or fat that the same cow would have produced during the same lactation under the same environmental conditions had she been a mature cow.
The control cows' mature equivalent milk production was 15,757 lb at the initiation of the experiment, and test cows' mature equivalent was 14,819 lb. At 12 months, the control cows' mature equivalent had an increase of 625 lb to 16,383 lb. During the same 12-month period, the test cows' mature equivalent increased 1,427 lb, to 16,246 lb.
The body weight data for the test and control animals are summarized in Table 5. The weight gain for the two groups of animals was nearly identical-155 lb per control cow and 166 lb per test cow. April 1978 The reproduction and calf mortality data summarized in Table 6 indicate no significant (p < 0.05) differences between the two groups, although the number of services per conception was slightly higher for the test cows. None of the calves born alive have died. All are in good health and growing at a satisfactory rate. The incidence of common dairy animal health problems summarized in Table 7 indicates no differences in the incidence of mastitis, milk fever, ketosis or metritis in the two groups of cows. Blood data are summarized in Tables 8 and 9. Blood collected from test animals prior to the initiation of the study showed significantly higher levels (p < 0.05) of calcium, phosphorus, total protein, albumin, lactic dehydrogenase, and creatinine and lower levels of thyroxine than blood taken from control animals at the initiation of the experiment. However, all mean values were within normal limits (David A. Morrow, Michigan State University; Jerry Stevens, University of Minnesota). Data from the blood samples collected at 8 months of the experiment indicated no significant (p < 0.05) difference in any parameter between the test and control animals. The 8-month blood means agreed with the initial means and were within normal ranges.
The necropsy data are summarized in Tables 10 and 11. Nine mature cows (five test and four control) and seven fetuses (two test and five control) were submitted to Michigan State University. Six of the adult cows (four test and two control) were alive when submitted. Three (one test and two control) had died on the farm.
Of the six live cows submitted for necropsy, three had been purchased for the express purpose of monitoring their health and thereafter submitting them to necropsy in order to observe the results of histopathological lesions observed. The remaining three live animals (one test and two control) were culled because of fertility problems.
One of the three adult cows (a control animal) submitted for necropsy died after an IV injection of a calcium solution was administered to treat a case of milk fever. The other two adult animals (one test and one control) died from acute mastitis during the heat of the summer.
The gross and microscopic lesions seen in the live adult animals which were posted were variable and without consistency, including pyelonephritis, pyometra and chronic mastitis, obesity and endometritis (one test and one control), chronic abomasal ulcer and endometritis, chronic mastitis and degenerative arthritis, and chronic displaced abomasum. The dead adult cows submitted for necropsy had suffered variable amounts of post-mortem decom- position. Diagnoses included acute mastitis and salmonellosis, acute mastitis, enteritis and pneumonia, and one animal that likely died as a consequence of calcium administered for milk fever. No pattern of gross histopathological lesions was seen in the test animals and their fetuses as compared with the control animals and their fetuses. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Polybrominated biphenyl (PBB) in the growing pig diet.
Twelve pigs which averaged 13.7 kg were randomly allotted from litters to a corn-soybean meal grower diet containing 0, 20, or 200 ppm of polybrominated biphenyls (PPB). During a 16-week growth trial, average daily gain (kg), average daily feed (kg) and feed/gain for pigs on diets containing 0, 20, or 200 ppm of PBB, respectively, were 0.82, 2.45, 2.99; 0.67, 1.88, 2.79; 0.45, 1.23, 2.70. Mean daily gain differences between all lots were highly significant (p < 0.01). Blood from each pig was withdrawn biweekly through the first 8 weeks of the trial and at 4 week intervals thereafter. Hemoglobin and hematocrit differed significantly only at the 6 weeks bleeding, being reduced in pigs receiving 200 ppm of PBB. Erythrocyte reduced glutathione concentration and glutathione peroxidase activity were not significantly influenced by level of dietary PBB. Serum lactic dehydrogenase activity was significantly higher in control pigs than in either PBB supplemented lots at 16 weeks. There was no significant influence of PBB upon serum glutamic oxaloacetic transaminase, serum alkaline phosphatase or serum creatine phosphokinase. Based on these enzyme assays, PBB produced no evidence of significant necrosis of liver, myocardium, or skeletal muscle. There was no consistent effect of dietary PBB upon total serum protein concentration or electrophoretic profile. Pigs on either level of PBB did not have overt clinical signs of toxicity during the 16-week test period with the exception of a dermatosis on the ventral surface of two of the pigs receiving 200 ppm of PBB. There was a marked increase in liver weight of pigs receiving either level of dietary PBB. Heart, kidney, and adrenals of pigs receiving either level of dietary PBB were heavier as a percent of body weight than that of control pigs. Fat retention of PBB and urinary and fecal PBB excretion were significantly affected by dietary PBB level. Grossly, the glandular portion of the stomach appeared somewhat hyperplastic in pigs on 200 ppm of PBB. Two pigs which had received 200 ppm of PBB were placed on the control diet and over the next 14 weeks normal growth rate occurred. One of these pigs was killed and organ weights were normal. The other pig, a gilt, came into estrus. She was bred and conceived. At the end of gestation, four pigs were born. Three survived and grew normally; the one death at birth examined at gross necropsy did not reveal changes in organ size or other tissue alterations. ImagesFIGURE 2.FIGURE 3.
Introduction
Polybrominated biphenyls (PBB) are organic chemical compounds that are widely used for fireresistant applications in both industrial and consumer products. In 1973, these chemical compounds were accidentally introduced into Michigan cattle feed. Since then many researchers have indicated a concern, not only about the effects of PBB on livestock performance, but also about PBB's potentially hazardous effects on human health.
Studies of PBB's effects have been reported on other species (1)(2)(3)(4)(5). However, information on the effect of PBB on swine is limited and further research is needed. The purpose of the present study was to evaluate the effect of different levels of PBB on performance of growing pigs and to determine if a high dietary PBB level during the growing phase of gilts precludes subsequent reproduction.
Materials and Methods
Twelve pigs averaging 13.7 kg were randomly allotted from litters into three groups of four pigs each fed a corn-soybean meal grower diet (Table 1) containing 0, 20, or 200 ppm of PBB. These diets, plus water, were offered ad libitum. The feeding trial lasted for 16 weeks. All pigs were housed in a slotted floor pen about 1.5 m above ground level, and total excreta were collected and incinerated to destroy the PBB. Pig weights and feed consumption were recorded biweekly. Blood samples were collected from the anterior vena cava biweekly through the first 8 weeks and at 4-week intervals thereafter for hemoglobin (Hb) (6), hematocrit (Hct) (7), red blood cell reduced glutathione (RBC GSH) (8) concentration, and red blood cell glutathione peroxidase (RBC GPx) (9) activity determination. Serum samples were collected for alkaline phosphatase (AP) (1O), glutamic-oxaloacetic transaminase (SGOT) (11), lactic dehydrogenase (LDH) (12), and creatine phosphokinase (CPK) (13) activity assay. Total serum protein concentration was determined by the Lowry method as modified by Miller (14), and the serum protein fraction profile was analyzed by agar gel electrophoresis (15). At week 4, urinary and fecal samples were collected and PBB concentrations were determined. Subcutaneous adipose tissue was also obtained by biopsy from the back immediately over the shoulder for PBB analyses by the GLC method (16). At termination, all pigs but two at the 200 ppm level were killed for gross necropsy examination. Selected organs were weighed, and samples of muscle, liver, kidneys, and subcutaneous and visceral adipose tissue were taken for PBB determination to characterize the pattern of PBB retention in growing pigs.
Results and Discussion
The effects of dietary treatment in pigs during a 16-week feeding trial on weight gain and feed consumption are shown in Table 2. Average daily gain and feed intake were significantly (p < 0.01) decreased by dietary PBB levels. The differences between pigs on 20 ppm and 200 ppm of PBB diets were also significant (p < 0.01). It would appear that the higher the dietary PBB level, the greater the effect on feed intake and weight gain. It is also interesting to note that efficiency of feed conversion to gain was better for pigs on PBB treatments than for the controls. The effect of dietary PBB levels on weight gain was probably the result of reduced feed intake. This finding has been confirmed in chickens and Japanese quail as revealed in paired-feeding studies (4). The average biweekly weight gain response to dietary levels of PBB expressed as growth curves is shown in Figure 1 Week On Test diet grew significantly slower than the controls, and the difference increased as time progressed. Addition of 20 ppm of PBB to the diet did not significantly change the rate of growth until week 6 as compared to the controls. Three pigs, one from each treatment, are shown in Figure 2. As can be seen, there was no noticeable difference in their ap- pearance. The pig on the right side was fed the control diet, the one on the left side, the 20 ppm PBB diet and the pig in the center, the 200 ppm PBB diet. Likewise, pigs on either level of PBB did not have overt clinical signs of toxicity during the 16-week trial, with the exception of a dermatosis on the ventral surface of two of the pigs receiving 200 ppm of PBB.
Hematological data, RBC GSH concentrations and RBC GPx activities are summarized in Table 3. On the other hand, with addition of PBB to the diets the activity of RBC GPx tended to decrease. A possible explanation for these differences may be the refusal of food. Decreased RBC GSH concentrations and reduced RBC GPx activities have been observed in fasted animals (17,18). Total serum protein concentration and serum protein fractionation profies are presented in Table 4. There was no consistent effect of dietary PBB upon total serum protein concentration or electrophoretic profie.
The effect of dietary PBB levels on the activities of serum AP, GOT, LDH, and CPK are presented in Table 5. No significant influence of dietary PBB levels on these serum enzyme activities was observed, except that serum LDH activity was significantly higher in control pigs than in either PBB supplemented lots at week 16. Based on these enzyme assays, growing pigs fed either 20 or 200 ppm of PBB for 16 weeks showed no evidence of significant necrosis of liver, myocardium or skeletal muscle. It was confirmed later by gross examination at necropsy that no severe lesions due to PBB were present except that the glandular portion of the stomach appeared somewhat hyperplastic in pigs on 200 ppm of PBB.
A summary of the effects of dietary PBB levels upon selected organ weights is presented in Table 6. The PBB supplement at either level significantly increased the absolute weight of liver and kidneys.
The effect on heart was somewhat variable. When organ weights were expressed as a percentage of body weight, variations were observed. Liver, kidneys, and heart were a greater percentage of body weight in PBB-fed pigs. Adrenals tended to increase in weight either on 20 or 200 ppm levels. These alterations in organ or tissue size have also been observed in chickens, Japanese quail and rats (1,3,4,19). This is probably a normal detoxification response. It is similar to the known effects of a normal liver response to the presence of some known drugs (e.g., phenobarbitals) where the animal body attempts to detoxify potentially harmful chemicals. Based on these observations, it seems that the liver responds sensitively to PBB in the diet by increas- ing its functional mass. The effects of dietary treatment on PBB retention and excretion patterns at week 4 and at termination are combined in Table 7. Fecal excretion is a major excretory route for PBB. Dietary PBB levels had a significant effect on PBB retention in adipose tissue. These data suggest that the characteristics of PBB retention are similar to those of other halogenated organic compounds (e.g., PCB, DDT) in biological tissues (20).
It would appear that adipose tissue PBB levels are a sensitive reflection of the concentration of PBB in the diet. On the 20 ppm level, tissue PBB residue in muscle was lower than in liver, and the kidneys had the lowest concentration. On the 200 ppm level, the PBB residue was similar between muscle and liver and slightly lower in kidneys. The highest level of residue was found in adipose tissue, and it is apparent that the extent of accumulation of PBB in fat was directly proportional to the length of exposure. Since no PBB withdrawal study was conducted, a biological half-life of PBB in tissue could not be calculated. Table 8 provides information on one pig which was originally fed 200 ppm PBB and then was returned to a normal diet. The time on the normal diet was 102 days; the average daily gain was 0.70 kg.
Some organ weights of this pig expressed on both an absolute basis and as a percent of body weight are included in this table. Comparisons with the organ weights (as percent of body weight) obtained from pigs on the control diet and pigs studied by McMeekan (21) suggest that after PBB withdrawal, organ weights returned to a normal range. Figure 3 shows a gilt which was one of the pigs originally on 200 ppm of PBB. This gilt was returned to a normal diet for 14 weeks, grew at a normal rate, exhibited estrus, was bred and conceived. Four pigs were born at the end of the gestation period; three survived, grew normally, and showed no gross external abnormalities. They are shown in Figure 3 with their mother. Whether the one death at birth was related to PBB is not clear. The gross necropsy examination did not reveal changes in organ size or other tissue alterations. Hb and Hct values obtained from the sow and baby pigs were normal as compared to those reported by Miller et al. (22). Compared with the data of controls and others (Ku, unpublished data), total serum protein and the enzyme activities of serum AP, GOT, CPK, and LDH from the sow and baby pigs were also in the normal range, as were RBC GSH concentrations and RBC GPx activities (Parsons and Brady, unpublished data). | v3-fos |
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} | s2 | Effect of nitrogen source on nutritional management after small bowel resection in rats.
Nutritional effects of dietary nitrogen sources on rats subjected to 65% distal resection of the small bowel were compared at the 2.4% nitrogen level, i.e., by feeding the rats a diet which contains 2.4% nitrogen from various sources. The nitrogen sources used were casein (C), the casein-simulated amino acid mixture (F), the proposed amino acid mixture (T-2), which was devised in our laboratory, and the amino acid-casein mixture (R), in which 25% of the proposed amino acid mixture was replaced by isonitrogenous casein. All the experimental diets were made cellulose-free. Total weight gains of the resected groups during three weeks were less than those of the corresponding unresected groups because of the depressed gains in the first postoperative week. In the resected rats, as well as in the unresected rats, the maximum total weight gain was obtained with diet R and followed weight gains with diet C, diet F, and diet T-2. Fecal weight increased moderately in the resected rats but the degree of the increase was not influenced by the type of dietary nitrogen source. Dry weight of intestinal remnants in creased markedly within one week after resection. The extent of hyper plasia was not altered by the type of dietary nitrogen source. These results indicate that an amino acid mixture partially replaced with casein may be a useful dietary nitrogen source for short gut syndrome.
Summary
Nutritional effects of dietary nitrogen sources on rats subjected to 65% distal resection of the small bowel were compared at the 2.4% nitrogen level, i.e., by feeding the rats a diet which contains 2.4% nitrogen from various sources. The nitrogen sources used were casein (C), the casein-simulated amino acid mixture (F), the proposed amino acid mixture (T-2), which was devised in our laboratory, and the amino acid-casein mixture (R), in which 25% of the proposed amino acid mixture was replaced by isonitrogenous casein. All the experimental diets were made cellulose-free. Total weight gains of the resected groups during three weeks were less than those of the corresponding unresected groups because of the depressed gains in the first postoperative week. In the resected rats, as well as in the unresected rats, the maximum total weight gain was obtained with diet R and followed weight gains with diet C, diet F, and diet T-2. Fecal weight increased moderately in the resected rats but the degree of the increase was not influenced by the type of dietary nitrogen source. Dry weight of intestinal remnants in creased markedly within one week after resection. The extent of hyper plasia was not altered by the type of dietary nitrogen source. These results indicate that an amino acid mixture partially replaced with casein may be a useful dietary nitrogen source for short gut syndrome.
Synthetically formulated, chemically defined diets containing amino acid mixture as the sole source of nitrogen have opened new possibilities in the nutri tional management of intestinal disorders which are accompanied by impaired digestion and absorption (1)(2)(3)(4). However, the high osmolality of chemically defined diets results in a difficulty of successive intake in sufficient quantities (3,5 a The compositions of amino acid mixtures are shown in Table 2 . b Sucrose was added as required to complete the mixture to 100% . The compositions were the same as those used in the previous experiment (7).
RESULTS
Body weight change Body weight changes during the experimental period are shown in Table 3.
In the unresected rats, the body weight gain on the proposed amino acid diet (diet T-2) was slightly less than that on the casein-simulated amino acid diet (diet F), although the difference was not significant (p>0.05). When 25% of amino acids in the proposed amino acid diet was replaced by isonitrogenous casein, with unchanged amino acid pattern, the resultant weight gain of rats significantly (p<0.05) increased as compared with those of both the groups fed the diets F and T-2, and reached about the same level as in the casein diet group. Weight gains were directly related to food intakes. Food intake of the diet T-2 group was less than that of the diet F group, although the difference was not significant (p>0.05). On the other hand, food intake of the diet R group significantly (p<0.05) increased as compared with those of the diet F and T-2 groups and reached about the same level as in the diet C group.
In the resected groups, total weight gains were less than those of the corre E AFTER SMALL BOWEL RESECTION 171 2 Nitrogen sources of diets are described in Table 3 . hyperplasia occurred in proximal remnants after distal resection. The degree of hyperplasia was not influenced by the type of dietary nitrogen source.
DISCUSSION
A considerable amount of information has accumulated concerning the nutrition after small bowel resection. Postoperative weight gain of small bowel resected rats is influenced by the site and extent of intestine resected (11,12). Distal resection has greater effect on weight gain than proximal resection. The effect was more pronounced as the resected part was larger. However, the report of comparison of dietary nitrogen sources is very few. TOULOUKIAN et al. (13) compared the nutritional effect of a chemically defined diet containing an amino acid mixture as nitrogen source with that of a laboratory rat chow. Nutritional effect of an elemental diet containing casein hydrolysate supplemented with amino acids was also compared with that of a laboratory chow (14). The results ob tained from these rat experiments demonstrated that a chemically defined diet and an elemental diet could support nutrition in the adaptive phase of the short gut syndrome, but not so well as the laboratory chow. However, the diets com pared in these investigations were quite different in nitrogen levels and in the type of components other than nitrogen source. In the present study all the experi mental diets used were the same in the above dietary factors influencing on nutri tional comparison of them.
McCARTHY and KIM (15) found in the proximally resected rats that significant elevation in fecal excretion occurred in the second postoperative week, falling towards normal by the fourth week. NYGAARD (11) reported with a laboratory chow containing 23% of crude protein and 2.6% of fat that fecal nitrogen excretion did not change by 25% distal resection, but increased progressively as the extent of small bowel resected was larger. Similar effect of the small bowel resection was observed on fecal fat excretion. In our experiment, fecal weight in the first postoperative week could not be determined because of the occurrence of diarrhea in this period. Any fecal nitrogen and fat excreted was not also determined. No detailed information was, therefore, obtained on fecal excretion of ingested nitrogen and fat in the resected rats.
All the rats showed variable diarrhea for several days after distal resection; the diarrhea subsided within one week. Disappearance of diarrhea seemed to be in accord with development of hyperplasia in intestinal remnant. Regarding hyperplasia, it is known that the intestinal remnant after resection undergoes mucosal hyperplasia, particularly in the ileal remnant after proximal resection (12,15,16). Past experiments did not make it clear when adaptive hyperplasia occurred in the intestinal remnants since the measurements of intestinal remnants were made after a considerably longer time following resection. The present study revealed that a marked hyperplasia occurred in the proximal remnant within one week after distal resection (Fig. 1). Both the duration of diarrhea and the extent of intestinal hyperplasia were unchanged with the type of dietary nitrogen source; casein, amino acid mixture or a mixture of casein and amino acids (Fig. 1). After intestinal hyperplasia occurred, rats were found to tolerate the experimental diets well, and an optimal diet for normal rats was found to be suitable for the resected rats.
A chemically defined diet and an elemental diet help to overcome some of the limitations associated with the use of customary foodstuffs in various gastrointesti nal disorders. One of the interesting characteristics of the diets is that the diets are composed of simple or elemental form of nutrients which minimize the digestive process. The other is that the diets contain no indigestible bulk of fibrous materi als which leads to striking diminution in fecal excretion (15,17). All the diets used in the present study contained no cellulose component. In the preliminary experiment, these diets were found to reduce the fecal weight below one-tenth as compared with a commercial laboratory chow containing 6% of crude fiber. Therefore, even a diet which contains intact casein instead of amino acids as nitrogen source is a low residue diet, if indigestive and nonabsorptive components such as cellulose are not contained. A moderate elevation of fecal excretion in the resected rats, as shown in Table 4, indicates a reduction of the ability of digestion and absorption as compared with the unresected rats. The extent of fecal weight DIETARY NITROGEN SOURCE AFTER SMALL BOWEL RESECTION 175 both in the unresected groups and in the resected groups was unaltered with the type of dietary nitrogen source.
A use of amino acid mixtures requiring no digestion prior to absorption as the sole source of nitrogen seems to be advantageous only when digestive ability in intestinal remnant is evaluated. The classical view that dietary proteins were completely hydrolyzed to amino acids in the lumen prior to absorption had been believed. However, the present predominant view is that there appears to be at least two modes of absorption of protein digestion products: absorption of free amino acids and intestinal mucosal uptake of oligopeptides with intracellular hydrolysis (18,19). In the present experiment, an amino acid-casein mixture in which 25% of amino acids was replaced by isonitrogenous casein was tolerated by the 65% small bowel-resected rats as well as by normal rats.
From the results, a well-balanced amino acid-casein mixture is expected to be applied to dietary nitrogen source for various gastrointestinal disorders.
The authors are grateful to Mr. K. Tanida for his excellent technical assistance. | v3-fos |
2017-06-30T11:09:51.274Z | {
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} | s2 | A possible genetic interpretation of the colour variants in the fleece of the Gotland and Goth sheep
Summary The paper describes colours of 79 adults and 1 lambs in 6 flocks of the ancient Swedish short-tailed, horned Goth breed of sheep. Of the adults, 3were grey and 43 of a near-white colour, while 8 of the lambs were grey and 99 light-coloured. The light coloured lambs with limited amount of white markings showed two colour patterns, one a lightgrey with white belly, termed a grey mouflon », and the other a mixture of light steel grey and tan, termed a agouti ». White markings were common in all flocks, and extensive white markings interfered with the classification of the main colours in some of the flocks. On the basis of the observations, on the Goth sheep which contained some pedigree information, and on available observations from crosses of improved grey Gotland sheep and Shetland sheep in Scotland, together with a review of the literature, the suggestion is put forward that the grey colour in the ancient Goth sheep and in the improved Gotland sheep is caused by a new allele at the Agouti locus, A(Gotland grey), if not Ao (icelandic grey). The « grey mouflon » colour of the Goth sheep is caused by another, previously unknown allele, A i(lg for light grey) at the A gouti locus. Some white animals may be A or even carry the !4 ""’ gene for tan or white.
analysis of the extension locus (E) (I,AUV!RGN!, 1975 ). The studies support the conclusion of S!AR!! ( 19 68) that different mammals have homologous loci.
The colour of sheep, however, presents certain features that have not yet been elucidated, in particular the existence of several kinds of grey, the extent to which these are attributable to the Agouti locus not being known (ADALST!INSSON,ig 7 o).
Colour inheritance studies in the improved Gotland breed were first carried our by L6 FVENBER G and J OH A N SSO N ( 195 2), and later by S!L!RUD ( 1955 ), S K A R - MAN ( 19 6 1 , I g63a, 1963b), and Ess!ERST!N and S K A RMAN (Ig!6). All these studies showed a wide range of shades of grey colour, although selection against the lightest and darkest extremes have been carried out for several decades.
The predecessor of the improved grey Gotland breed is the old horned Goth breed, which was on the verge of extinction in the nineteen forties, but is now being preserved. The Goth sheep show a still wider range of grey shades than the improved Gotland breed.
The aim of the present study was to examine the type of grey in the improved Gotland breed and relate it to grey colour observed in other breeds, and at the same time to attempt a genetic interpretation of the wide range of grey shades in the Goth sheep. For this purpose the authors visited Gotland in June, I g 7 6 and made observations on the colour variation in the Goth sheep. At the same time visits were made to some breeders of the improved Gotland breed for visual inspection of the colour shades present there. These observations, together with observations on crosses between improved Gotland sheep and Shetland sheep in Scotlandform the basis for the conclusions of the present paper.
II. -Review of the grey coloration in domestic sheep, with reference to the Gotland breed and its various colours A. -The grey colour in breeds other than the Gotland If one regards grey as deriving from a mixture of black and white fibres, it is possible at the moment to distinguish three genetically controlled kinds of grey: lethal grey, progressive juvenile whitening, and grey as an Agouti factor. Lethal grey (in which the animals are grey at birth) was first studied in the Turcana breed of Romania by C ONS T AN T IN E SCU ( 192 6) then in the Karakul (G LEM -B oTSK y et al., I g 34 ). Among numerous works devoted to it are reviews by SERRA ( 194 8) and R A E ( 195 6).This is a mutation which when heterozygous is epistatic to other colour genes, producing grey through an admixture of white fibres, and when homozygous is lethal. In this, gastric troubles about the time of weaning are followed by death. On a black base this gene gives grey, while on a red base it gives a kind of lilac, both colours being exploited in Karakul furriery.
Studies on progressive whitening were summarised by SERRA ( I94 8) who listed several types under the names progressive whitening or silvering, which develop in breeds with dominant (Karakul) as well as recessive (black Wensleydale) pigment.
This whitening occurs regularly in red breeds (Solognote) and often in black breeds (Bizet and Karakul), but not in all animals. Z AHARIA ( 1939 ) for example showed that certain Turcana sheep became grey, while other remained black all their life. The way in which this difference is inherited has not been studied in detail.
The Agouti grey factor has been demonstrated by ADA!,sTW NssoN (ig6o and 1970 ) in the Icelandic breed. At birth, the grey animals are distinguished from all black ones by light marks around the muzzle and the eyes, as well as the presence of a white underwool on the scrotum, and in the inguinal region. The underwool develops with age to give a grey fleece at about four to five months. The expression of the gene A g is variable, and ADe!,sTW NssoN ( 1970 ) distinguishes seven degrees at birth. The homozygous A g A g lambs were found to be born light grey, while the heterozygous lambs usually were born dark grey.
Observations and test mating carried our by R YDER and al. ( 1974 ) with O y kney sheep (thought to have affinities with the Scandinavian type) accorded with the interpretation that the grey gene A 9, originally designated A 2 by A DA r,sTW Nsso N was also present in this breed.
In the review of Norwegian work by B ER G E ( 19 6 4 ) (summarised in English by R YDER and S'r!rxENSON, 19 68) the mixture of black and white fibres (grey) caused by this gene is termed &dquo; black skimlet & d q u o ; , and the mixture of brown and white fibres &dquo; brown skimlet &dquo;.
B. -History of the Gotland breed Until the nineteenth century there was in Sweden, as elswhere in Scandinavia, a population of primitive small Sheep, with coarse wool, and a short tail, which were often horned and showed a variety of colours. This type is named the northern short-tail group of Sheep. In Sweden this original population has been gradually replaced by meat breeds mainly from Britain, so that by the nineteen forties the ancient type was found mainly on the island of Gotland (E KMAN , 1 93 8, 19 64; HAI,LAND!R, 1976;RYDER, 1968, ig74).
Starting immediately after World War II the unimproved breed changed gradually into the improved Gotland which has the dual purposes of meat and furskin.
Fashion in furriery created a demand for grey skins from lambs four or five months old, and a system or performance testing and skin marketing has developed with the help of advisers from the Ministry of Agriculture. The methods used and the results obtained were described by S K A RMAN ( 19 6 1 , 19 6 3 a and b) and were summarised by G AH NE el al. (i975).
The number of Improved Gotland sheep has therefore increased from 170 ooo in 19 62 to 300 00 o today, and it now forms 90 p. 100 of the sheep population of Sweden (R YDER , 1970 ). In comparison with the original type, the improvement has involved a loss of horns, and the production of a fleece that has uniformity of structure and colour. Such success has been achieved in the replacement of the old breed with the new variety that the ancient type, horned and variegated was on the verge of extinction when several enthusiasts endeavoured to safeguard it in the nineteen sixties (EDB!RG, 1974).
In 1973 , according to the last author, the number of mature individuals of this relic breed was around 400 animals, of which two hundred are in the island of Gotland in which it originated. These sheep retain the horns and a great variety of colour shades. Except at Skansen Zoo and in the Jakobsson flock on I,illa Karlso small unimproved flocks often run alongside improved animals.
For the relic variety, E DBERG ( 1974 ) proposes the Swedish name Guteldr which in English would be called the Goth Sheep, instead of the previously used name beho y nade gotlkndska utegangsl d ren i.e. horned Gotland sheep kept outside all the time. The name given to the improved type is: Gotlands ulegangsfdr, i.e. outdoor Gotland Sheep, or more simply Gotlands fdr. In English, the improved variety is termed simply &dquo; Gotldnd Sheep &dquo; (Mason,ig6g). In French we propose Gotlandais for the improved variety, and Gothique for the relic type.
C. -The colour types of Gotland sheep as described by previous authors 1 . Descriptive F, KMAN ( 193 8, 19 6 4 ) was able to place the animals in three colour categories: black, grey and white. The grey sheep were moderately variable, and the author distinguished two types; he was not, however precise about the age at which the classification was made, nor the detail of the marks on the hairy parts of the body (the face and legs). L6F V E NB E R G and Joxarrssorr ( 195 2) recognised the three main classes of E KMAN and inclined towards a scale for grey by noting that only black and white colours were definitive at birth. Grey animals on the contrary were born black, or nearly black, and did not become grey until later. In seeking criteria for distinguishing grey animals from black animals at an early age, S!r.!xun ( 1955 ) investigated the tongue (which is black in black animals) and the underwool which is sometimes white at birth in grey animals. Later the fleece becomes lighter in the adult, becoming almost white in some individuals.
All the analysis of genetic segregation carried out by L6F V E NB E R G and JOHANS-SON , however, relied on classifications made at or before the age of sale (between two and five months) without taking account of the colour on the head and feet.
Following these authors, S K A RMAN recorded the colour shade on the scrotum and tongue, the presence of white underwool at birth and studied their correlation with different shades of grey at four to five months, but as with the previous authors his colour description at four to fives months referred to the colour on the woolly parts of the animals only. In the interval, however, the scale of grey had become extended; L6 FV E NB E R G and JoxArrssoN ( 195 2) recognised three grades : light grey, grey and dark grey; there are now five: white-grey, light-grey, grey, dark-grey and grey-black.
2 . Genetic interpretation L ÖFVENBERG and Joxarrssorr ( 1952 ) thought at first that the recessive black which they observed was due to a mutant (g) at the locus for wild coloration which they named G; and that the white was given by an allele (W) at another locus. They proposed two alternatives to explain grey: an allele or several alleles either at the G locus or the W, without being able to distinguish between the two. Later genetic interpretations by F,BB!RSTEN and S K A R MAN ( 197 6), can be summarised as follows: i) A quantitative inheritance was suggested for the shade of grey with a heritability of shade varying from 0 . 35 to 0 . 7 o depending on the method of estimation. ii) A marked effect was found of genes for white markings on fleece colour, increased white markings being in general accompanied by a lighter wool colour. ' . iii) The white colour, which is sometimes observed among the Gotland sheep is most likely due to the combined effects of genes for white markings and lack of darkening modifiers. iv) Black colour in the Gotland sheep may be either due to homozygosity for a gene which is recessive to the gene for grey, or it may be the result of a darkening modifier, which suppresses grey colour.
v) The grey colour of the Gotland breed was found to be recessive to the white colour of the Swedish Landrace sheep.
III. -Material and methods
Following the preceding analyses we approached the problem as follows : animals of the improved type were examined to see what colours or colour shades the improved stock had in common with the ancient stock and animals of the latter were examined to see which colour variants could be identified within this breed and which of the variants described by L6FVENBERG and J OHAN S S O N ( 195 2) were still in existence.
The flocks of Goth sheep available are generally small and dispersed, with a greater concentration in Gotland than on the Swedish mainland, those on the island of their origin were therefore naturally chosen for investigation; this also allowed improved flocks to be visited.
Flocks of the Goth breed surveyed are shown in Table i.
In the Lilla Karls6 flock all the lambs were described with respect to colour, but the colour of individual ewes in that flock was not recorded. In the Gullgarde flock the colour of both ewes and lambs was recorded, and pedigree records were available on all lambs in that flock. They were all sired by the same sire. In the remaining four flocks, ewes and lambs were claissified into the two groups grey and « light » only. Several of these classifications were made at a distance in the pasture.
A. -The observations i. The Gotland sheep In the Gahne flock, which according to Swedish experts is a good average example of present fur producers, the adults appear as follows: the head and legs are black, with lighter areas around the muzzle and the eyes (cf. fig. l a). White markings on the head are present in only few animals. A high proportion of the wool fibres is white, giving the body a near white appearance, but an admixture of black hairs is invariably found.
Lambs observed some time after birth had the same coloration on the feet and the head as their mothers. The coat was grey, the lightness depending on age, consisting basically of a black outer coat with a white underwool. The same descriptions apply equally well to the flock in Scotland. due to a coarser outercoat than is found in the improved type. The other type is light grey or near white. The distribution of the adult sheep by these colour classes is shown in Table i. See also pictures in fig. ia White markings were common in both types. The white markings were limited to head and feet only in most of the dark types, but covered larger areas in the lighter types. In some of the lightcoloured adults with the least developed white markings one could distinguish and indistinct type of a grey mouflon pattern (or grey reversed badger face). In light-coloured animals with extensive white markings this pattern was possibly present, but obscured. Some of the white sheep like those recorded by L6 FV E NB E R G and JoxeNSSOrr were perhaps of the lightgrey type with extensive white markings ( fig. I d).
b) It was possible to identify three colour classes in lambs which showed limited amount of white markings. The first of these was a dark grey type, similar to the improved type, but sometimes showing white kemp on the belly ( fig. Z b). A second type was a distinct type of a grey mouflon pattern with the following characteristics ( fig. If) : the upper part of the body was light grey or steel grey in colour, the head showed white colour inside the ear, above the eyebrow and under the chin, and the belly colour was white, all borderlines between grey and white were rather diffuse, which distinguishes this type of grey mouflon from the grey mouflon described in Iceland. The third type of lambs colour was termed « agouti » by the present observers ( fi g.tf). This type showed a steel grey colour on the shoulders, which sometimes extended downwards to the flanks.
The sides were tan-coloured at the wool tips, while the staple colour varied from steel grey to very light grey. The belly was white with no clearcut borderline between the sides and the belly.
When the lambs showed extensive white markings, it became very difficult to distinguish between the grey mouflon and the « agouti » patterns. They were then only termed « light ».
The distribution of the lambs in all the flocks visited on the two classes grey and « light » is shown in Table I , and Table 2 shows the distribution of the lambs at Lilla Karlsõ by sex and the three colour classes grey, grey mouflon and « agouti ». It is evident from Table i that the frequency of grey lambs is significantly higher in the Lifla Kar1s6 flock than in the other flocks (x , 21 = 12 . 0 8; P < o.ooi).
By assuming that the grey colour in the lambs is due to homozygosity for a recessive gene, the grey mouflon being the heterozygote and the « agouti » the other homozygote, the frequency of the gene for grey is found to be 0 . 77 in the Lilla Karls6 flock and 0 . 57 in the other flocks combined. For the Lilla Karlso flock the expected numbers of grey, grey mouflon and « agouti » lambs are 5 8. 1 , 34 . 7 and 5. 2 respectively, which is in good agreement with the observed numbers ( Z 2 , = 0 . 45 ; P > 0 . 05 ).
The grey mouflon and « agouti patterns were not observed among the improved Gotland sheep. No black animals were seen.
c) The white markings observed in darkgrey lambs and adults were usually a star or a blaze, and in several instances white socks on the feet and a white tail tip was also observed. Extensive piebaldness was rare among the dark greys. In the lighter coloured types the white markings were more extended. The head was sometimes almost completely white, except for some dark around the eyes, and white markings on feet and tail were common.
White markings on head (H), feet (S), tail (T) and body (P) are shown in Tables 2 and 3 for the I,illa Karlsõ flock, and inTables 4 and 5 for the Gullgarde flock. Only 2 lambs were found without white markings, both on Lilla Karls6. This gives the frequency of s, the recessive gene for white markings, of o. 99 at Lilla Karls6, and i.oo elsewhere within the Goth breed.
The main colour of the lambs on Lilla Karlsõ was found to affect the extent of the white markings significantly, the HST-combination (white head spot socks white tail tip) occuring among only 5 grey lambs out of 55 , compared to 12 HST lambs out of 41 with the colours grey mouflon and « agouti » ( X 2 1 = 6.56; P < 0 . 05 ).
The same was found to hold for the Gullgdrde flock, where 3 grey lambs out of 7 showed the HST combination compared to 2 6 HST lambs out of the remaining 30 ( X 2 i = 6.43; I' < 0.05). On the other hand the HST combination was significantly more frequent at GullgArde than at Lilla Karls6 within the same main colour. For the grey lambs the HST combination occured among 5 out of 5 5 at Lilla Karls6, but among 3 out of 7 grey at GullgArde ( X 2 1 = 6. 30 ; P < 0 . 05 ), and among the non-grey lambs, i2 out of 41 at Lilla Karls6 and 2 6 out of 30 at Gullgarde showed the HST combination ( X 2 1 = 22 . 94 ; P < o.ooi). The concurrence of piebaldness was also found to be significantly more frequent in the Gullgdrde flock ( Z 2 1 = 59!45! P < 0 . 001 ). This is consistent with a high frequency of modifiers which limit the extent of white markings in the Lilla Karls6 flock and a high frequency of modifiers which increase the extent of white markings in the Gullgarde flock. This is in agreement with the adopted selection practices in the two flocks, emphasis being placed on the dark types on Lilla Karls6 and on the piebald types in the Gullgdrde flock.
B. -The segregations The classification of the colour of the lambs at the GullgArde farm by colour classes of the dam is shown in Tables 4 and 5 . The ram used was most likely of the grey mouflon type, but it was dead by the time of the visit, and only a colour slide was available for inspection. The classification of the ewes was uncertain, as seen from the tables. The most clearcut category was the dark grey colour. The 12 lambs out of the darkgrey ewes showed only two colours, i.e. 6 were grey and 6 grey mouflon, which is a perfect fit under the assumption that the ram used was a grey /grey mouflon heterozygote. The other results in Tables 4 and 5 are in general agreement with this hypothesis, except for the lack of grey lambs in the lower parts of Tables 4 and 5.
Observations in the MacDonald flock in Scotland suggested that the relatively dark grey Gotland rams used for breeding there were homozygous grey, because no black lambs were born. A cross made between Gotland rams and 102 black or brown (moorit) Shetland ewes produced only grey lambs. The lambs were mostly dark, but some were light.
105 white Shetland ewes mated with a Gotland ram produced white and grey lambs roughly in the proportion I : I, indicating that a high proportion of the ewes were heterozygous white.
Discussion
It was shown by ADa!,sTW :·rsso:r (i 97 o) that nonwhite Icelandic sheep possessed either black or brown pigment (presumably eumelanin), the black pigment being produced by a dominant gene B and the brown pigment by the recessive allele, b. The recessive brown genotype bb, is found in the brown Icelandic sheep and in the moorit Shetland sheep (for the link between past and present gene symbols see A D A L STEINSSON, 1974).
Genes at the Agouti locus modify the pigment produced by the genes at the B-locus to a greater or less extent. The top dominant allele at the A-locus, Aw h , produces white or tan (red) colour, the intermediate alleles A b , Aw and A9 9 produce the patterns or colours badgerface, mouflon (reversed badgerface) and grey, and the bottom recessive allele, a, has no effect on black or brown pigment, producing selfcolour (black or brown) when homozygous.
It will be assumed that the above rules hold for the breeds under discussion in the present study except where otherwise stated. I . -The Gotland sheep The grey of the improved type has several of the characteristics of the grey at the Agouti locus found in Iceland, but differs from it some aspects.
The results from the cross made in Scotland between grey Gotland rams and black and brown Shetland ewes show that the grey gene is dominant to lack of grey in the black or brown Shetland ewes, that the rams used were most likely homozygous, BB, for the gene for black pigment, and that the relatively dark grey Gotland rams were most likely homozygous for the gene producing the Gotland grey colour. This last piece of evidence possibly indicates that the Gotland grey is not produced by A a (A 2 ) found in Iceland, because one the characteristic features of AD is the much lighter colour of the homozygotes than the heterozygotes (A D aI,s- TRINSSON, 1970). E BB E RS TE N and S K A RMAN ( 197 6) inferred that they had found rams which were heterozygous for the grey gene within the Gotland breed. These rams were themselves darker than other rams, and there grey progeny was also on the average darker than the progeny of other rams. This indicates that the effect of the grey gene within the improved Gotland breed is comparable to the A g -gene within the Icelandic breed, i.e. that neither gene is completely dominant to the recessive a, which in homozygous form gives black colour.
E BBERST E N and S K A RMAN further showed that the Gotland grey colour was recessive to the white colour of the short-tailed Swedish Landrace sheep. Their backcross of white (Sze!edish-Landrace x Gotland) ewes to grey Gotland rams produced only two colours, i.e. white (or tan piebald) and grey in the ratio I : I. Two independant pieces of evidence suggest that selection for darkening modifiers of grey has been carried out whithin the improved Gotland breed. F, BB E RS TI:, N and S K A RMAN (IC!!6) found that grey lambs from the cross white (Landrace x Gotland) ewes X Gotland rams were of an appreciably lighter shade than the average within the improved Gotland. In the MacDonald flock in Scotland it was also observed that the backcross white (Shetland X Gotland) X Gotland produced grey lambs which were mainly light grey. This, viewed in light of the selection against light types described by E BBE x-STE N and S K Å RMAN ( 197 6), together with the relatively high heritability they found for shade of grey, mentioned previously, alos points to selection for darkening modifiers. From the evidence presented here one feels that the following conclusions are permissible.
i) The Gotland grey is situated at the Agouti locus. ii) It may and often does produce a dark grey colour in the homozygote. iii) Selection for darkening modifiers has been carried out within the Gotland breed.
iv) The Gotland grey is produced by an allele which is very similar from A (A2) found in the Icelandic sheep and possibly also in Corsica (I,AUV!RGN! and A DAI , S T I ; INSSON I(!!6) but perhaps distinct. Provisionally we propose the name A gg (gg for (improved) Gotland grey) for this apparently new allele at the Agouti locus.
2 . -The Goth sheep The Goth sheep shows a considerably greater variation than the improved type, as mentioned earlier. The three phenotypes observed among the lambs at Lilla Karlsõ are consistent with a two-allele case, the dark grey lambs being homozygous AaeAae, the grey mouflon lambs being AoaA t a (lg for light grey) and the « agouti » lambs being homozygous A t aAia.
It is obvious from the studies on the improved Gotland sheep cited earlier that a more pronounced colour variation was encountered in the study of L 6FVEN -BERG and JO HAN SSO N ( 1952 ) than one finds today. The grey mouflon pattern found in the Goth sheep of today might for example have existed in the flock that L6FVENBERG and JoxArrsso N studied, but selection against unfavourable, highly beritable traits would soon have eradicated such a gene if it had occurred at a low frequency.
The segregation of the two alleles postulated in the Goth sheep at Gulgarde near Roma is not clearcut. One should bear in mind that white markings were found to be more pronounced in the Gulgirde flock than in the Lilla Karls6 flock, and this may have obscurred the picture.
The existence of a separate allele producing the light colour which the present authors have termed grey mouflon is not at all firmly established, but the type of grey mouflon pattern observed in the Lilla Karls6 lambs is consistent with the hypothesis that this light colour is produced by a single gene. It is also possible that the gene for tan or white A w h quite common in Iceland which probably was in the genetic formula of the white animals described by L 6 F V E NB E R G and J OHANSSON ( 1952 ) is still present in some flocks we have checked even if sometimes A w h it is mimicried by (A i eAle).
Sur la base d'observations sur le mouton Gothique avec quelques examens de croisés Gotlandais gris et Shetland faits en Écosse et d'une revue de la littérature on pense que la couleur grise de l'ancien mouton Gothique et du Gotlandais amélioré est due à un nouvel allèle en Agouti A fl9 (gg pour Gotland grey) à moins que ce ne soit le même allèle que A g (icelandic grey). La couleur mouflon gris du mouton Gothique serait due à un autre allèle encore non décrit A !e (lg pour Light grey) au locus Agouti. Quelques animaux blancs peuvent être A l a A w ou même porter le gène A w n pour le fauve ou le blanc. ' | v3-fos |
2017-07-29T04:25:16.245Z | {
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} | s2 | The economic values of various traits in pig breeding
. The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years. On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competitors and they are often at high risk of getting
in terms of goods rather than in money terms. Taking account of inflation the average cost of borrowing in real terms in the U.K. over the past 32 years ( 1945 -197 6) is estimated as 2 -3 p. 100 . This also provides the best available estimate for discounting future returns, and it brings the opportunity cost rate into line with the social time preference rate often proposed for public investments. , . The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years.
On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competitors and they are often at high risk of getting no returns. Implications to the form and amount of investment in animal improvement are discussed. Using examples from sheep populations, the relative importance of male, female and slaughter traits is discussed in relation to different ways of obtaining replacement animals. Three types of population are distinguished, namely nucleus populations with self-supply of breeding animals of both sexes, subnucleus populations with self-supply of females only, and commercial populations buying all breeding animals. Nucleus populations which supply various other populations with breeding animals are also considered.
SENSITIVITY
The examples given indicate that the appropriate breeding objectives need not be identical for both sexes, even if the animals selected are to be used in the same population. The way in which replacement is recruited influences the relative importance of the various traits to be considered within the same sex as well as the differences in appropriate breeding objectives for the two sexes. Dpt of Animal Breeding, Agricultural College, S. 750 0 7 Uppsala, Sweden An investigation concerning the economic influence on the production cost of various traits in pig breeding has been carried out. The terms " economic influence " or " economic value " of a trait in this work have been used to indicate the effect on the production cost per kg lean meat. To compute the production cost an equation was constructed, where the traits were included as variables. Using this equation the production cost per kg lean meat can be computed at different levels of the variables. To obtain the proper mathematical expression of the financial effect of changes in the variables, the partial derivative of the equation was computed for each of the traits referred to. These expressions directly show the financial value of each trait per unit of change at different levels of the variables. The stability of the economic weights has been investigated. The frequence and points of time of the monetary returns from breeding as well as the influence on the genetic gain of different or incorrect economic weights have been discussed. Hybridisation programmes are mostly realized by means of three levels of herds where breeding animals used in elite herds are deciding. In the consequence of the high reproduction rate in the pig it is possible to produce 700 -8 00 final hybrids per sow and year in elite herds. In Czechoslovak conditions 73 p. 100 of the total cost is expended on the own fattening, 21 p. 100 on sow breeding in commercial herds (i.e. 94 p. 100 on the third level of final hybrids production), 5 p. 100 on multiplier herds and i p. 100 only on elite herds. It is therefore possible to evaluate the effectiveness of the hybridisation programme on the basis of results achieved in the last production level.
ESTIMATION OF ECONOMIC VALUES OF PERFORMANCE CHARACTERS IN
In the sheep with much lower reproduction rate is the situation qualitatively different. In a specialized large scale unit for lamb production within the closed system of breeding about 20 p. 100 of the total cost is expended on the breeding of pure lines, 45 p. 100 on the breeding of F t females and 35 p. 100 on the own fattening. The calculation of a profit function is here therefore much more complicated. An example of the construction of a multifactorial function is presented. It includes four groups of basic production parameters and five categories of animals only compared with the practical situation where 22 categories are necessary.
THE USE OF PRODUCTION SYSTEMS ANALYSIS IN DEVELOPING SELECTION GOALS AND METHODS
J. W. WILTON Department of Animal and Poultvy Science, University of Guelph, Guelph, Ontario, Canada Production systems analysis is described, its present applications are discussed and its uses in determining selection goals and methods are discussed. The key features of systems analysis are the statement of a well-defined objective, the accurate representative of real-life production programs and the use of alternative procedures by decision makers. Measurements that have been suggested to describe selection goals are discussed and compared with the objectives specified in systems analysis. Selection procedures that have been used for these various measurements are also discussed and compared with methods that could be used in systems analysis. L'objectif d'une sélection est un ensemble complexe de nombreuses exigences exprimées sous forme soit de pondérations liant les caractères, soit de gains génétiques à réaliser pour certains caractères ou ensembles de caractères. Un index doit représenter le meilleur compromis possible | v3-fos |
2018-12-21T12:23:32.074Z | {
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} | s2 | How Burning and Other Methods of Removing Irrigated Crop Residues Affect Yields and Soils
This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1978 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.
Burning crop residues has long been discouraged for many reasons . It pollutes t he air, leaves the soil surface exposed to wind and water erosion and possibly volatilizes some nitrogen at the time of burning. Now, with crop residues proposed as a source of energy, we need to know how residue removal affects not only crop yields but also soil physical and chemica l characterist ics.
Several methods of managing residues have been studied f or ten years at the Garden City Experiment Station. The treatments included removing the residue: 1) by burning; and 2) by physi cally removing as much top growth as possible; and incorporating either normal or twice no rmal quantities of res idue. In addition, nitrogen was applied at 50 and 100 lb/A rates.
Yields did not differ significantly the first eight years of the experiment (Table 1}. However, in the past three years removal and burning t reatments have begun to produce lower yields than the other treatments. In 1979, yields from the two plots with residue removed were lower than from plots with residues incorporated. T hese results are consistent with other experiments which indicate that it may take as long as ten years to begin to observe yield reductions from burning.
The plots were sampled to a depth of 6-feet in the fall of 1979 to evaluate the soil chemical properties under these management practices. Chemical anal yses included pH, organic mater, P, K, Zn, Na, Mg, and Ca on t he surface 6 inches and residual N03and total Non the entire 6 foot profile. Analyses of the data showed no differences in P, Zn, Na, Mg, or Ca due to the residue management treatments. However, there were significant differences in pH, organic matter (O.M.) and potassium (K) (Tabl e 2). Continuously removing residues (physically or by burning) d ecreased soil O .M. as expected because resi· dues were not being returned to the soil. In addition, pH increased where the res idue was removed. Both the pH and O.M. are very important in managing soi ls. O rganic matter helps to maintain stability of soils by acting as a cementing agent for soil part icles so granular structure of the surface soil is maintained . Organic matter also supplies s.ome micronutrients needed for p lant growth.
Soil pH affects the availability of some nutrients; as pH increases, the availability of nutrients such as P, Fe and Zn decreases. In addition, pH and O.M. are critical in some herbic ide programs. The levels found after ten years' burning and removal (Table 2) are approaching these critical levels, so they may affect the rate herbic ides are degraded.
Potassium is also declining where residues have been removed (Table 2). This is expected since residues are high in K. However, the concentrations observed in this experiment are still in the very high soil test category and will no t limit c rop production.
Nit ra te-nitrogen (NO)-NJ analyses showed no statistical differences in the total quant ity of this nutrient accumu lated in the six-foot profile. However a higher percentage of NO)-N has been leached deeper in the physical removal and burning treatments, (See figure below). This may reduce N03availability to plants if it is leachedbelow the zone of greatest root activity. We attribute the greater leaching to incorporating reduced quantities of residues on t he two removal treatments. In these situations there is little Ue-up of N during residue decomposition and it remains susceptible to leaching as N03during high rainfall or irrigation. %of total N03 accumulated Although no immediate deleterio us effects on crop yields or soil properties were observed due to residue burning or removal, the continual long-term practice of these residue management treatments w ill have negative effects on soi l pH, O .M ., K and N03-N . These c hanges may eventually have negative effects on c rop yields. This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. | v3-fos |
2016-05-12T22:15:10.714Z | {
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} | s2 | Evaluation and performance tested beef bulls by blup
The Friesian breed makes a substantial contribution to beef production in Great Britain. The value of selection for beefing characteristics has been studied based on the discounted gene flow technique (M CLIN TOCK and CUNNINGHAM ). The technique has been adapted to consider the use of crossbred females as beef suckler cows. The selection for beef can rarely be justified at a level greater than I in ¢ the use of suckler cows making little difference. The parameter under selection and economic values have a large effect. As an initial step visual assessments of beef shape are being recorded and will be reported for progeny tested milk bulls.
The Friesian breed makes a substantial contribution to beef production in Great Britain. The value of selection for beefing characteristics has been studied based on the discounted gene flow technique (M C C LIN T OCK and C UNNINGHAM ). The technique has been adapted to consider the use of crossbred females as beef suckler cows. The selection for beef can rarely be justified at a level greater than I in & c e n t ; -the use of suckler cows making little difference. The parameter under selection and economic values have a large effect. As an initial step visual assessments of beef shape are being recorded and will be reported for progeny tested milk bulls. The method used in Sweden for performance testing of dual purpose bulls has, especially during recent years, shown some limitations due to many and small testing stations and an intense use of sires over a short time period. To overcome these problems, at least partly, and to get a more efficient estimate of the breeding value for growth rate an application of best linear unbiased prediction (BI,UP) in performance testing has been made.
In this procedure the daily gain of a bull on test is corrected for the effect of test station, year and season and the sire effect (the sire's predicted difference) and bull effect are estimated by BLUP. The bull's estimated breeding value is then calculated as the sum of these two effects and finally expressed as a relative breeding value.
The advantages of the BI,UP procedure in comparison with the officially used method can be summarized as follows, i. Bulls can be compared across test stations and across years and season, which means a more efficient utilization of data available.
2 . Information on half-sibs of the same sire is used to obtain estimated breeding values of bulls. 3 . Sire and station effects are independent of one another. The heritability of growth rate at station between 1 8 0 -3 6 5 days of age has been computed by maximum likelihood procedure for Cha y olais and Hereford. The estimates were 0 . 4 8 and 0 . 43 respectively. The variance components differed and the phenotypic standard deviations were 123 and 9 6 g per day for Charolais and Hereford respectively. BI,UP procedure for evaluation of breeding values of bulls was applied to the same data as was used for variance component estimation, namely 272 Cha y olais and 8 05 Herefo y d bulls by roo and 27 6 sires, respectively. The BLUP procedure was compared to two simplified proce-procedures was quite high.
However, because correlations were less than one and of anticipated increased genetic trends the model which shold be used for evaluation of performance tested bulls, should take into consideration the genetic trend in both sires and dams and should include information on half-sibs. The first phase of developing selection indexes for beef cattle is described. This phase involve the theoretical consideration of the factors that must be considered in development.
SCHÄTZUNG PHÄNOTYPISCHER PARAMETER
Six factors are discussed. First, the definition of the goal of selection is discussed. A monetary goal at the farm level appears suitable. Second, the goals must be expressed in such a way that selection indexes can be derived. The association between profit equations, models of aggregate genotype and selection indexes are described, including considerations of nonlinearity. Third, the association between selection goals and both commercial production programs and crossbreeding programs are discussed. Fourth, the sources of genetic parameters are briefly discussed. Fifth, the sources of economic parameters and their prediction for the future are briefly discussed. Sixth, the association between selection indexes and times of selection for both males and females are discussed. The total and relative number of discounted expressions for various traits in beef production differed between rotational and terminal crossing and between sexes within terminal crossing. Differences between relative number of expressions of traits were most evident within nucleus | v3-fos |
2018-12-17T20:43:17.784Z | {
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} | s2 | The Effects of Sevin (1-Naphthyl N-Methylcarbamate) on Morphology and Root Anatomy of Glycine max (L.) Merrill
Thi s study i nvest i gated the effects Sevi n (1-naphthyl N-methylcarbamate) had on the morphology .and root anatomy of soybeans (Glyc i ne m (L) Merr.). Morphological observations revealed that as the concentrati on of Sevin i ncreased, the cotyledons, hypocotyl and epicotyl decreased in s i ze. Absence of a pi gment (presumably anthocyanin) normally present i n the petioles and hypocotyl, was noticed at all applications of Sevin. Emergence of primary and trif oliate leaves was delayed as the concentration of Sevi n increased. Increased Sevi n concentrations produced thi n, white roots wi th numerous laterals. With i ncreased concentrat i ons, purple pigment appeared on both taproots and lateral roots. Growth rates of roots, when analyzed by Duncan's Modified Multiple Range Test, were not sign ificant between 2, 9 and 19 ppm of Sevi n, but were signifi cant at t he 1% level for 21, 25 and 55 ppm of Sevi n. Roots of treated soybeans exhibited development of lateral r oo t pr irnord i a close to the root apex . Thi s suggests t hat t he cells f ailed to e l ongate i n advance of differentiat i on. There appeared to be no change i n the quiescent center for all Sevi n concentrations. Noticeable vacuolizat i on of cort i cal, stelar , epidermal and root cap cells occurred at 21, 25 and 55 ppm of Sevi n. At i ncr ased Sevin concentrat i ons, t he root cap was usual ly sloughed off
AC KNOWLEDGE!Y£ENTS •••••••••••••••••••••••• ,. ••••••••••••••• i i i
W ithi n the last few decades, increasing de pendence upon the use of pest i c i des which are part i cularly hazardous to animal and human life, has led to laws that re gulate their production and distribution. Biologists are justly concerned with the e f fects of pestic i des on ani mals and the i r long term effects on future generations, but t h ere is less concern when an insectic i de i s a ppl i ed to a crop , unless i t i s hi ghly phy totoxic.
Research has been conducted on translocation, absorpti on, metabolism and retenti on of i nsectic i des i n plants, but few stud i es have addressed the problem of t he eff ects of insect i cides on the anatomy of t he plants. There i s no question that t he heavy use of i nsecticides has resulted in serious res i due problems i n many a gr i c ul tural areas, so i t is hi ghly des i rable to determine what levels of res i dues affect plant growth, and h ow t hi s e f fect i s expressed anatomically .
Sevi n i s t he most wi dely used carbamate i nsecti c i de i n the Uni ted States today (von Ruraker et al., 1974 ). I t i s a broad s pectrum i nsectic i de t hat controls many economically important insects, some of whi ch are the Painted Lady Butterfly larvae (Vanessa cardui), Green Cloverworm ( Plathypena scalira) and Mexican Bean Beetle (Epilachna var i vestis).
It is unique in having a low mammalian toxicity (560 mg/ kg in rats) as well as low persistanc·e in the envi ronment ( 8-135 days, dep ending on t he substrate) (Caro et al., 1974).
Soybeans are the most imp ortant cash crop i n t he United States and are an imp ortant food product for human and livestock consumpt ion (Howell, 1975). A review of the literature has revealed no s p ecifi c stud i es on the effects of Sev i n on soybeans (while applications of Sevin at 2 l b s/ acre of act i ve ingredient have been observed to be phytotoxic under field conditions). Therefore, thi s thes i s is concerned wi th the morpholo gi cal and anatom i cal effects of vari ous concentrations of Sevin on t h e soybean.
The pertinent l i terature on t he effects of i nsecti cides on p lant anatomy i s concerned wi t h the V isomer of hex achlorocyclohexane, comm only known by the trade name Li ndane.
Increased numbers of rnult i nucleate cells in the root a p ex were observed i n the roots of monocoty ledons, d i cotyledons and gymnosperms.
Lichtenste i n et al. (1962) rep orted that when corn and peas were sub j ected to 30 ppm Sev i n, t h e roots were shorter and t hick er t han the control roots, and f or most species, there was an overall reduct i on i n p l ant s i ze. Lateral roots formed wi t hi n 0.5 cm of t he root a pex a nd were shorter t han those i n the plants grown i n untreated sand. A dark purplish color appeared on t he lower p ort i ons of t h e roots.
The pr imary metaboli te of Sevi n, 1-naphthol, produced no effects on t he length of corn and pea roots, although t hey did exhibit t h e purp le color on i ts lower surfaces.
Li chtenste i n cate gor i zed t he anatomical changes as a 11 growth i nhi b i t i on" . The observat i on of d ifferent i ated P~otoxylem , metaxylem, protophloem and endoder.mis close to the root a pex, suggested t hat development of cel l s close to the root apex was 11 precoc i ous 11 • Sevi n induced growth i n-3. h ib it ion wi th not i ceable trac heary elements d ifferent i ated close t o i:he r oot ap ex i n a unique wedge-shap ed pa ttern opposite t h e proxylem poles.
Lichtenste i n also ob served proli fi c develo pm ent of lateral roots in both peas and corn treated wi t h Sevin .
Anatomi cal c hanges i n corn were d ifferent i at i on of tracheary elements close t o t he root a pex and s ignificant cell wall change i llustrated b y retention of saf r a n i n stai n in a peripheral band of cells i n t h e va scular c yl i nder t hat i n- The anatomy of soybeans has been stud i ed by ~Ji i k sche ( 1961), W eaver ( 1960), and Mitchell and Russell ( 1971).
It i s an upri ght plant, and depend i n g on the vari ety, has Notice lack of anthocyanin in hypocotyl and epicotyl. There is a de.lay of foliar expansion and prolif'eration of .lateral roots at 21 ppm. At 2 ppm Sevin, there appears to be little effect on the soybean plant growth.
Ef feet on Root
With increased concentrations of Sevin, both tap roots and lateral roots exhibited marked difference in size and number rather than difference in the day of emergence.
The roots were white and thin in comparison with the control roots (Figs. 2, 3). Many of the lateral roots emerged only a short distance from the root apex, and this distance decreased with increased concentrations of Sevin.
At lower concentrations of Sevin, the lateral roots were longer and more prolific than at higher concentrations.
After 10 days growth, the tap roots and lateral roots con-
RADIOASSAY OBSERVATIONS
The symplast consists of the interconnected living elements of plants (e.g., protoplasm) while the apoplast is the nonliving portion of plants (e.g., intercellular spaces, xylem vessels). "Specific Activity"(corrected counts per minute-:-weight of sample) and "Total Activity " (specific activity X sample weight) were computed and used to derive percent distribution values (individual plant part total activity.;.~ of total activity for the entire plant) of 1 4c-sevin applied to various plant organs (Fig, 6, since very little 1 4c-sevin was translocated to the meristematic areas of the soybean (i.e., shoot and root apices and pods) (arrows in Fig. 7). There appeared to be some 14 concentration of the C-Sevin in the symplast, but true symplastic movement is doubtful.
The primary leaf treatment of 1 .6 ~Ci 1 4c-Sevin per plant showed that 49% of the radioactivity remained on the treated leaf with the nex t higher percentage in the other leaves (31 %). The roots, stems and pods accounted for the remaining 20% of the 14c-sevin uptake (Figs. 6, 8, Table 4).
The faint outline of the stem (arrows) could possibly be due to aphids which had fed on the area of the leaf treated with 1 4c-sevin and transmitted this to other plant parts at subsequent feedings.
As with the primary leaf treatment, the other leaves received 13%, while the remaining 17% of the 1 4c-sevin might be attributed to insect transport (arrows). The evidence suggests that no symplastic movement had taken place since the meristematic areas (m) were deviod of labelled 14c-sevin.
ANATOMICAL ANALYSES
For purposes of discussion, the description of the tissue systems in the root tips of Glycine ~ are discussed as follows: 1) Quiescent center, 2) Root cap and epidermis, 3) Cortex and 4) Vascular cylinder.
The Effects of Sevin on the Quiescent Center
Examination of the quiescent center of the plants treated with 2, 9 and 19 ppm of Sevin revealed little or no appreciable differences from the control (Fig 10). The quiescent centers of plants treated with 21, 25 and 55 ppm did not appear appreciably different from the controls, although the hemispherical outline of the quiescent centers was somewhat less well defi ned than those of the controls (Figs. 11,13).
The Effects of Sevin ..9E. the Root Cap and Epidermis The root cap was not affected at 2, 9 and 19 ppm of Sevin. At 21 ppm, hypoplasia of the root cap and epidermal tissues, previously reported by Lichtenstein et al~ (1962), was observed (Fig . 11). In many instances, the root cap was partially sloughed off or w~s completely absent. The cells of the root cap appeared to be more compressed than those of the controls and were also rounded in appearance difference from the controls while at 21, 25 and 55 ppm, a noticeable difference in structure had occurred.
At 21 ppm, the cells of the cortex were apparently compressed. At 25 ppm the apical meristem was compressed and the cortex appeared to bulge out at approximately 300 ).ll11 from the quiescent center (Fig. 12). The cortical cells at 55 ppm were difficult to distinguish from the vascular cylinder (Fig. 13). The cells appeared shorter, more compressed and more vacuolate than in the controls.
Effects of Sevin .£!! the Vascular Cylinder
The vascular cylinder was affected primarily at higher dosages of Sevin. These caused an appearance of maturing tracheary elements and a development of lateral root primordia opposite the protoxylem poles close to the quiescent center. At 2, 9 and 19 ppm there were no readily observable differences between the treated roots and the controls.
Higher dosages of 21, 25 and 55 ppm of Sevin produced short, flat, maturing tracheary elements approxiamtely 300 µm from the quiescent center (Figs. 11,12). This suggests that the root apex did not advance before dif'ferentiation had taken place or dif'ferentiation occurs more rapidly than meristematic growth. Lichtenstein et al. (1962) labelled this process as a "growth inhibition " .
DISCUSSION
One of the most noticeable differences in the morphology of soyb eans fol lowing the application of Sevin was the absence of pigment (presumably anthocyanin) which is normally present in the stem and hypocotyl.
Hydroxylation is the primary means of oxidation of Sevin (Kuhr, 1970). The hydrolysis of Sevin to its aromatic r ing components, 1-naphthol, or to its N-methyl carbamate Auxin (Indol Acetic Acid) is produced in the apical meristem of the shoot and is transported toward the root apex (Galston and :Davies, 1970 Sevin is applied to the foliage of soybeans at concentrations ranging from 1 .O to 2.5 lbs Active Ingredient/ acre or 1.0 to 1 .5 Actual Ingredient/ 100 gallons of water (Thompson, 1967). These concentrations range from 1 to 9 ppm in the field environment. Actual grower applications 2) In all treated plants a pigment, presumably anthocyanin, that is normally present in untreated plants was absent from the hypocotyl and epicotyl.
3) Emergence of primary leaves was delayed with increasing concentrations of Sevin. At higher concentrations the trifoliate leaves never fully expanded even though primordia were present.
4) The r oots were abnormally whi te and thin. As Sevi n concentration increased, numerous lateral roots app eared close to ,the root apex. A purple color on the ro ots was noticed at 21, 25 and 55 ppm of Sevin.
58.
RAD I OASSAY OBSERVATIO NS 1) When 1 4c-sevin was applied to the root 64% of the radioactivity was translocated into the primary and trifoliate leaves. The autoradiographs suggest that transport of 1 4c-sevin is primarily apoplastic and possibly concentration of 1 4c-sevin into the symplast occurs.
2) With foliar application to the primary and trifoliate leaves, 49 and 70% respectively, of the 1 4c-sevin remained at the application site. As a result of infestations with aphids, some 1 4c-sevin may have been translocated to other plant parts.
ANATOMICAL ANALYSES
1) The quiescent center for all concentrations of Sevin showed little or no difference from the controls, but at doses of Sevin of 21 ppm and above, the hemispherical outline of the quiescent center appeared less well defined than those at 2, 9 and 19 ppm of Sevin.
2) At increased concentrations, noticeable hypoplasia of the root cap and epidermal tissues occurred. At 21, 25 and 55 ppm of Sevin, the epidermis was a flattened, roughened area against the cortex.
3) At 21 ppm of Sevin, the cortex was a shortened and flattened into compressed files of cells, and at 2~ ppm, the cortex appeared to bulge at approximately JOO )J!ll from the quiescent center. At 55 ppm, the cells of the cortex were difficult to distinguish from the vascular cylinder, and appeared more compressed and vacuolate than the controls. 4) Higher concentrations of Sevin affected the vascular cylinder by possibly inhibiting the rate of elongation, therefore, causing the appearance of maturing tracheary elements close to the quiescent center.
Lateral root primordia were similarly affected at higher concentrations of Sevin.
5)
The phloem, endodermis and parenchyma of the vascular cylinder showed no change in appearance in any of the treated plants. 60. | v3-fos |
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} | s2 | SAP frequency in Saskatchewan Charolais cattle
A survey among 350 breeders of Charolais in Saskatchewan, Canada, was undertaken in the fall of 1977 . Useable replies were received from 124 breeders on 320 8 cows producing 3 zi8 calves. Among these there were 19 cases of hereditary SAP (Syndrome of Arthrogryposis and Palatoschisis) in neonates for a frequency of 0.59 p. 100 . Following adjustments for percentage of Charolais breeding in the dams it was apparent from the complete data that the frequency of defectives calves was not significantly different from that observed in the pure breedinFrance. It is concluded that the frequency and penetrance of the SAP gene among Chavolais in Canada
Summary
A survey among 350 breeders of Charolais in Saskatchewan, Canada, was undertaken in the fall of 1977 . Useable replies were received from 124 breeders on 3 20 8 cows producing 3 zi8 calves. Among these there were 19 cases of hereditary SAP (Syndrome of Arthrogryposis and Palatoschisis) in neonates for a frequency of 0 . 59 p. 100 . Following adjustments for percentage of Charolais breeding in the dams it was apparent from the complete data that the frequency of defectives calves was not significantly different from that observed in the pure breedinFrance. It is concluded that the frequency and penetrance of the SAP gene among Chavolais in Canada are the same as in France : q = 0 . 20 , w = 0 . 12 (mid sex) and that the latter is not influenced by the percentage of Chavollais blood (which is contrary to former belief).
I. -Introduction
Studies of SAP (Syndrome of Arthrogryposis and Palatoschisis) in Charolais cattle have been mainly conducted in France and Canada and have been reviewed by IrAUV!RGN! and FAUCON (1976). This syndrome has been demonstrated to be conditionned by a single autosomal recessive gene with a penetrance apparently varying with the percentage (i) A Franco-Canadian project supported by FIAERBC (International Charolais Association, Nevers, France) and the CIES of the French Minisbry of Foreign Affairs, for travelling and lodging.
of Charolais blood : quite low in the pure breed in France but approaching unity in the early generations of repeated back crossing with Charolais in Canada.
In France the data were obtained by methodical survey (Ir!xoRT et al., 1977 ) whereas in Canada initial studies were conducted with data obtained only from affected herds voluntarily submitted by their owners (Saskatchewan work of N AWRO T, 1973 ) or from breeding studies in an experimental herd of known carriers (work in Alberta by BERG and GOO N E W A R DENE, 1974).
In order to make comparisons with French data and to establish a base line for future monitoring comparisons of genetic load, it seemed necessary that a survey be conducted among Charolais breeders in Canada.
The present Saskatchewan survey was designed as a pilot study for that purpose.
II. -Materials and methods
A survey of calving results in ig!7 was conducted by mail among 350 randomly chosen members of the Canadian Charolais Association resident in Saskatchewan.
Survey kits were distributed in September 1977 and the data forms recovered by the agricultural advisers in 42 of the districts of the province.
Information for all mating's involving some Charolais in both parents was requested as follows: -breed composition, age and relationship of sires and dams, -sex, breeding system (natural service or AI) birth condition and nature of defects (if any) of their calves.
The breeders were also asked to indicate whether they had completed the forms from recorded data or from memory.
Each participant in the survey was provided with complete detailed instructions and data sheets. Pictorial and verbal descriptions of conditions associated with SAP were furnished from which the breeders could indicate the exact nature of the defects.
The survey was anonymous that is, no information was requested as to the identity of breeders or animals.
III. -Results
Returns were received from 15 6 breeders. Of these 99 provided complete information, 25 were incomplete for minor items such as percentage Cha y olais of dams, breeding system (AI or natural), ages and distribution of defects other than SAP, twenty five were sufficiently incomplete to be unuseable and seven respondents had abandoned the cattle business because of continuing depressed prices.
A summary of the information received in completed forms is presented in table 1 .
Nine of the ig reported SAP calves were described as having their hind legs stretched towards the rear when in a cumbent position with the fetlock joints bent sharply forward. These were described as poorly muscled particularly in the shoulders, hips and thighs. Three of these also had their front legs bent and twisted inward at the knee and fetlock joints so that the soles of the feet were directed toward the head and one of these had a cleft palate.
Ten of the 19 reported cases of SAP displayed defective development of the front legs but not of the hind legs. In 7 of these, both front legs were bent and twisted inward at the knee and fetlock joints so the soles of the feet were directed toward the head. In three of them, only one front leg displayed the malformation and one of these had a cleft palate.
The 15 abnormalities not classified as SAP (on completed forms only) were of various types reported in many breeds with variable genetic components such as blindness, hip dislocation, polydactyly and swollen head (hydrocephalus?).
In table 2 are shown the frequencies of SAP according to the percentage of Charolais blood in the dams ( 311 of the sires were purebred Charolais and 9 were i5 /i6 Charolais).
Only forty five percent of the breeders ( 15 6 out of 350 ) replied at least partially. This may be due to a lack of time to complete the detailed form in large herds with many calves. As one can see (table i) the average herd size was 52 cows for the returns classified as incomplete compared with 10 cows in herds for which the returns were classified as complete. This alone would probably not introduce a bias to the data but some other factors may explain non compliance such as having too many abnormals or none at all, lack of interest, peer influence... It is impossible to determine the effect of such factors in our case but a future survey must take account of these possibilities and their circumvention.
It is worth noting that 94 p. 100 of the respondants utilized written records of herd and animal performance (table i). Although 6 p. 100 indicated the use of memory in completing the data form, these few averaged only 10 Cha y olais matings none of which resulted in an SAP defective. It is also possible that records were available but had been thoroughly committed to memory. It is evident from herd ratios of sires used relative to number of Charolais calvings that the fewer such calvings the proportionately greater was the use of AI sires. Although an estimate of AI useage could not be derived from the incomplete data for the larger herds, i 3 p. ioo of Charolais calvings in the complete data file were reported as AI.
The secondary sex ratio (at birth) in this sample of ig2i calvings for which data were complete was 52 aa for 4 8 Y? which is exactly the same as the sex ratio observed in French Cha y olais, L!xoxT et al. ( 1977 ).
Twinning frequency varies greatly among families within breeds but the 9 sets within this sample of 1921 calvings (. 0047 ) is in close agreement with estimates for other breeds of I set per 200 births.
The respondents furnishing complete data indicated in total the occurence of 25 defects among igzi neonates for a frequency of . 0130 or z 3 in I ooo births. Among these were 10 identifiable cases of SAP giving a frequency of . oo52 or 5 defective arthrogrypotic calves among i ooo births. The leg and muscular defects reported were very similar to descriptions by L AUVER G N E and BLIN ( 19 6 7 ), GR!!!,Ey et al. ( 19 68), I, W ror,n et al. (ig6g) but the frequency of cleft palate among our cases ( 2 : 19 ) is rather low being only 11 p. 100 compared with 70 p. 100 in no SAP cases reported by L AUV E R G. N E (ig 75 ). This may be due to the fact that the survey was conducted after calving and many breeders had not checked the mouth for palate development before disposing of the calf.
Although there was reported in French data a significant sex effect (I,!!'ORT et al., 1977 ) on the incidence of SAP there is no such indication in our data but the sample is too small to verify such a discrepancy. The simplest hypothesis with which to test our data is whether the frequency of the gene in the French cattle imported into Canada are the same as in French Charolais cattle studied by I,!xoxT et al. ( 1977 ) and also whether the penetrance is the same for each percentage of Charolais blood in recurrent back cross generations as in purebred French Charolais.
Let r be the percentage of Charolais blood in the females, q the frequency of the gene for SAP and w the mid sex penetrance of the abnormality among homozygotes.
The probability of observing one SAP calf in a cross between a Charolais bull and a female with per cent of Charolais blood is then rq 2 w. Since rq 2 w is small the frequency of SAP in n calvings from such crosses follows a Poisson distribution with parameter nrq 2 w.
The expected values for the Poisson variables with q = . 2 o and w = . z 2 from L EFORT et al. 's paper ( 1977 ) among the various matings are given in table 3 with the corresponding observed values for the complete data.
One can see that there is no significant discrepancy between observed and expected values in complete data. There is perhaps a difficulty due to the abnormally high number of SAP in 3 / 4 category among incomplete data if one assumes that the distribution of dams is the same as in complete data. But, in any cases, the penetrance among the calves born from dams with 50 p. ioo of Charolais blood or less cannot be very high. In the complete data we have one SAP among 5 r 9 a births, this observation is the value of a Poisson variable of zo.o 5 w and the values of w equal to or greater than . 5 are rejected by statistical tests, the value of . 12 giving, on the contrary, a vey good agreement with the observed value. This result does not fit with the conclusions of N AWROT ( 1973 ) and of BERG and G OON E WARDENE ( 1974 ) of a penetrance increasing to unity as the percentage of Charolais blood was decreasing to 75 p. ioo in the calf (dams being half Cha y olais).
The explanation of this discrepancy could possibly be connected to the following : 1 0 The inclusion of sampling bias, especially in the data analysed by N AWROT ( 1973 ). As seen consistantly in human genetical data the probability of sibship with abnormalities to be reported when more than one sib is affected is much higher than when only one sib is affected (see L I ( 19 6 1 ) on probability of ascertainment). N AWROT ' S cases were mainly multiple and voluntarily submitted by breeders. Morever, some of the parents of SAP calves among this cases may have been homozygous normal overlap for the offending gene.
2 0 The results obtained in the experimental herd at Kinsella (Alberta) reported by BERG and G OON E WARDENE ( 1974 ) could be based on the effects of selecting breeding stock proven to be carriers thereby indirectly selecting for greater penetrance (reduction of suppressor modifiers).
V. -Conclusion According to our findings, the frequency and penetrance of the SAP gene in Canadian Charolais is becoming very similar to that of France especially as the gradingup carries the population toward an increasing proportion of purebred animals (see in table I the proportions based on percentage Charolais).
We have shown in a recent paper (I,auv!RGNE and H OW E LL , 1977 ) that this situation precludes eradication of the gene at least in the Charolais population in France where there are relatively few progeny per bull. The Saskatchewan situation is even less amenable to such a process especially for the bulls used in natural service (less than 30 calves per bull per year, table i). And, even if all the AI bulls were proven non carriers, reduction in frequency of the gene would be very slow and eradication impossible since AI represents only 13 percent of all services in this sample (table i).
Application of expensive ared time-consuming test mating procedures to reduce the frequency of the offending gene is contra-indicated in Canada as in France, see L EFORT and LAUVERGNE ( 1974 ) for population genetic discussion. Some practical conclusions in the more general field of monitoring defects in cattle by a field survey may be extracted from this study.
-It is necessary to modify the survey data form in order to accomodate larger herds by requesting summarized rather than detailed data.
-It is necessary to code the form for confidentiality but to retain a method of access to the respondent for clarification.
-Personal contact rather than a mail-in survey would be desirable to avoid the introduction of biases resulting from incomplete information and nonrespondents.
-Better results would be obtained if breeders were briefed before the calving period from which information is to be gathered.
-A method of assuring the ability of breeders to accurately identify defects is necessary.
-The participation of existing networks of district agricultural advisors is invaluable for this type of survey.
-Based on this pilot study and the foregoing comments an improved model can be structured for application to various of the breeds of the principal classes of domestic livestock for defect monitoring.
Aknowledgments
The valuable cooperation of the Agricultural Representatives of the Saskatchewan Department of Agricultuve in conducting the survey is greatly appreciated.
We would like to thank also Dick KurTxEr., student assistant, Department of Animal and Poultry Science, University of Saskatchewan, for his technical assistance in designing the survey and assembling the data.
Professor G. L!roxT, Institut Natioiaal A gronomique Pavis-Grignon, France, has given valuable council in the mathematical treatment of the data. | v3-fos |
2022-02-24T16:04:36.072Z | {
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} | 0 | [] | 1979-10-01T00:00:00.000Z | 247071825 | {
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} | s2 | Estimation ofsoil moisture at deeper depth from surface layer data
I n s;tl£ measurements of soil moisture at deeper layers is very difficult e~p~ci:t llr in heavy soils. Regression equations have, . therefore, been developed to ,estimate J!1 the c1~a r s:~son 5011mOlstu~e vafl~.t l ~ns atdeeper layersfrom fluctuationof 5011moisture JO the surface layer. [ stl.mateO values Me compared with observed ones. The estimates are seen to be reasonably accurate.
Introduction
A knowledge of the patte rn of soil mo isture distr ibut ion and its accumulation at various depths in different ty pes of ra infall year s is vita l for designing effective agronomic practices. The assessment of th e march of the productive soil moi sture by ph ysical measure ment , s pee i all~in .the deeper layers is very difficult WIthout d isturbing sod and plan t roots. M oreover,. taking samp les from deep er layers are also labourious and : time con surnmg. A lar ge nu mber of samples per unit a rea arc requ ired te be takcn for obtaining a represe ntative va lue of soil mo isture. On e of the ways to avoid th ese difficult ies is to formu late some meth od of estimation of soil mo isture at the deepe r layers from para meters which can be measured easily.
Stud ies o n some of the characteristics of soi l moisture varia tion s in the sur face layer and th e movemen t of moi stu re through the soil have been made by R arndas and Mallik (1942), Ramdas (1951), Baier a nd Rob ert so n (1966) and Ba ier ( 1969). Biswas ( 1978) stu died the flyctuall on s of soil moisture at the different layers In relation to ra infall for a few stations.
Atte mpts have been made in this study to esti: mate soil mois ture at deeper dept hs from soil moistu re at or ncar the surface layer by fittin g suitable regressio n equat ions using soil moi sture data record ed at various stations of different climati c and soil zones.
Data
Three stations, Delhi, Surat and Dharw ar, where reliable soil moist ure data were nvaiiable for a long period , were taken. for the study. Ob servation s were recorded for tn igh tly for th e four depths, 15 cm 30 Col , 45 cm and 60 ern , At Dharwar ob servati ons were avai lable on bare soil only and fo r one addit ion al dept h 7.5 em, At Surat ob servation s were taken on bare soi l a nd soil covered with two varieties of cotton crop, the varieties being 'S uyog' a nd '2087' henceforth referred 10 as cotton VI and cotton V2 respective ly. At Delhi observations were taken on so il covered with two varieties of wheat, namely, 'N P 4' and 'NP 7 10' henceforth referred to as wheat VI a nd wheat V2 respectively . The o bservatio ns over bare so il were round the year. The ob servation s in crop were avai lab le at Delhi taken during November to April of next year and Su rat from th e end of Sep tember to March. The soil moisture values for a particular fortni ght for all the yea rs for which data were available were averaged o ut. This was done mai nly to get rid of small ob servat ional err o rs and also to avoid situatio ns where ob servation s were takenjust after the occurrence of heavy rainfall since under such condition the soi l moisture profi le will be con siderably d isturbed.
I\'1ethod
. Whe n plotted as a graph th e soil m oistu re values sho wed the followin g features ; (i) The variation of so il moistu re at a particular depth was more or less linear with the soil moisture near th e surface layer (Figs . I to 6). (ii) The variation of soil moistu re with depth was curvilinear, but approached linearity with decrease in so il moi sture near the surface layer (Fig. 7) to qu ite low values.
Incorpo rating these two features l :L~eq uation suggested to estirmt e soil m oisture at deeper depth fro m that at or Dear t he surface layer was of tho for m, where S is thc soil moisture at depth d and Su th e soil moi sture at or near the surface layer whose depth is do; A, B a nd S, arc constants in the equation. So, d and do being kno wn S was estimated from th em. | v3-fos |
2019-04-25T13:10:18.365Z | {
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} | 0 | [] | 1979-01-01T00:00:00.000Z | 130717985 | {
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} | s2 | Yield and quality of six summer annual forages
In 1977, all summer annual forages studied produced excellent yields. Based on leafiness and regrowth ability, sudangrasses and pearl millet appeared to be best for early vegetative and boot cutting management. The sorghum-sudan hybrids had suitable yields and quality at all harvest stages. The hybrid forage sorghum appeared best suited for soft-dough-stage harvest although yields of pearl millet and sorghum-Sudan hybrids were also excellent.
Summary
In 1977, all summer annual forages studied produced excellent yields. Based on leafiness and regrowth ability, sudangrasses and pearl millet appeared to be best for early vegetative and boot cutting management. The sorghumsudan hybrids had suitable yields and quality at all harvest stages. The hybrid forage sorghum appeared best suited for soft-dough-stage harvest although yields of pearl millet and sorghum-Sudan hybrids were also excellent.
Many summer annual crops can provide excellent forage during the hot, dry summers in Kansas when other pasture grasses have declined in production and quality. Summer annuals, including sudangrasses, hybrid sudangrasses, sorghum-sudangrass hybrids, sorgos, hybrid forage sorghums, and pearl millets, may be used for pasture, hay, silage, and greenchopping. Differences in their anatomy and growth characteristics reward producers who carefully select the proper crop to match their livestock needs.
Materials and Methods
The hybrid forage sorghum was planted in 30-inch rows; all others, in 6-inch rows. Plots were 5 x 20 feet for the narrow spacing and 10 x 20 feet for the wide spacing. The center 3 feet or 2 rows were harvested for yield, leaving a 6-inch stubble. Harvests were by stage of growth, not calendar date. At Hutchinson, forages were cut 3 times at the early vegetative stage, 2 times at the boot stage, and 1 time at the dough stage. One additional early vegetative cutting was obtained at Manhattan. Samples were taken from the flail-chopped material for dry matter and quality analyses.
As shown in Tables 12.1 and 12.2, mean forage yields were similar at Hutchinson and Manhattan for the early vegetative stage, greater at Hutchinson for the boot stage, and greater at Manhattan for the soft-dough stage. The forages sometimes responded differently at the two locations. The most difference was noted for Millex at the soft dough stage; it yielded much better at Manhattan. Cuttings were at different calendar dates, and rainfall patterns differed between locations, but such differences are expected and would be expected in other years.
Crude protein content and in vitro digestible dry matter (IVDDM) declined with advancing maturity. Crude protein was always lower at Hutchinson, particularly at the soft dough stage, probably partly because of near-record August rainfall, unusually high yields, and moderate nitrogen fertilization.
Piper sudangrass and Trudan hybrid sudangrass performed best for early vegetative and boot harvests. The FS 25A hybrid forage sorghum, as expected, performed poorly under early vegetative management, and its yield was quite low at the boot stage at Manhattan. At Hutchinson, it yielded well despite being cut only once, while the others were cut twice. Yields of the two sorghum-sudan hybrids and pearl millet varied most at the various stages and locations. Additional years of data are needed to better estimate the forages' true yielding abilities.
Summer annual forages vary in growth characteristics. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | 0 | [] | 1979-02-01T00:00:00.000Z | 12433245 | {
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} | s2 | Effects of nutritional factors on metabolism of dietary cadmium at levels similar to those of man.
Several nutrients are known to affect cadmium toxicity, but little is known about the effect of dietary nutrient levels on absorption and tissue retention of cadmium at low dietary levels, similar to those of man. Feeding gradedlevels of zinc in a casein-gelatin diet to young Japanese quail with 109Cd (as the chloride) and 0.062 ppm added cadmium decreased the cadmium concentrations in the proventriculus-ventriculus, duodenum, jejunum-ileum, and the liver, but not in the kidney. Zinc also affected some zinc, iron, manganese, and copper tissue levels. Different tissue concentration patterns of cadmium and essential minerals were obtained with two purified control diets, one based on casein-gelatin and the other on soy isolate as the principal protein sources. The data show that relatively small dietary changes can markedly affect tissue levels of cadmium and that a low intake of zinc may increase the risk to dietary cadmium exposure. The complexity of the nutrient interrelationships and their effects on cadmium require further study to define mechanisms, which may be similar to those produced by low cadmium intakes in man.
Introduction
Many investigators have shown that the nutrient composition of the diet can markedly influence the severity of toxic manifestations that occur after a few days or weeks of feeding high levels of cadmium. Single deficiencies of zinc, copper, iron, and calcium, and combined deficiencies of calcium, zinc, and protein markedly exacerbated the toxic effects of cadmium. Supplements of zinc, copper, iron (primarily the divalent form as ferrous sulfate), ascorbic acid, and L-cysteine were shown to be protective. These nutrient-cadmium interactions have been reviewed (1)(2)(3)(4).
The practical problems of cadmium toxicity in man are not due to high levels of cadmium intake, but rather to very long-term cadmium accretion in the kidney until levels that can cause kidney damage occur (5). Experimental data that are most pertinent to man include those from studies on the effects of dietary nutrient levels on absorption and long-term retention of very low levels of dietary cadmium, similar to the intakes of man (6). Functional and morphological changes in the kidney under these conditions need to be investigated. The tissue retention of single oral doses of radioactive cadmium with low total cadmium intakes has been elevated by deficiencies of iron (7,8), calcium (9), and protein (10). Hamilton and Smith (11) produced a change in the distribution of a tracer dose of "5MCd between the liver and kidneys by feeding a low calcium diet; however, calcium level had no effect on the total amount of ll5mCd in the two organs.
Jacobs et al. (12) found that simultaneously doubling dietary levels of zinc, copper, and manganese in a soy isolate diet caused a marked decrease in the retention of "09Cd (fed as the chloride to Japanese quail between 7 and 14 days of age) in the liver, kidneys, and jejunum-ileum. Cadmium in the duodenum was not affected. The total dietary levels of cadmium, 0.020, 0.082, 0.145, 0.270, 0.520, and 1.020 ppm, bracketed dietary concentrations equivalent to the intake of man (approximately 0.08-0.10 ppm), assuming the absence of moisture and fiber for similarity to the type of diet fed in the bird experiment. With both the basal and supplemented diets, cadmium accumulated in the duodenum, liver, and kidney in a linear log-dose, log-response relationship. Within each of the two dietary matrices, the percentage retention of the "09Cd dose in each tissue was the same at each cadmium dose level. The same supplement also had a beneficial effect on the long-term whole body retention curve in accelerating the loss of "15mCd (total 1 ppm cadmium) that had been fed in the diet to Japanese quail between 7 and 14 days of age (13). Zinc was shown to be the primary component of the three-element supplement responsible for lower tissue cadmium concentrations following the 7-day feeding regime (14). Copper and manganese had small effects, which included increases in some tissue concentrations of cadmium. Jacobs et al. (15) showed that in birds fed a soy diet, concentrations of a dietary tracer of "09Cd (total 0.145 ppm cadmium) in the jejunum-ileum, liver, and kidneys declined as dietary zinc was increased from 15 to 30 and 60 ppm in the diet; 120 ppm zinc had no further effect.
The capacity of the small intestine of young Japanese quail to accumulate cadmium results in significant concentrations of cadmium in both the duodenum and the jejunum-ileum of birds fed basal diets. The cadmium level is sufficiently high to permit assay by flame atomic absorption spectrophotometry. The high correlation between decreases in jejunal-ileal concentrations of cadmium and decreases in liver and kidney cadmium led Fox et al. (16) to use the concentration of cadmium in the jejunum-ileum as an index of cadmium bioavailability from human foods. With the soy isolate diet, cadmium (as the chloride) fed to give total dietary concentrations of 0.082, 0.145, and 0.270 ppm cadmium resulted in linear relationships between the logs of dietary cadmium concentrations and logs of duodenal and jejunal-ileal cadmium concentrations. The concentrations of cadmium in the two small intestinal segments were significantly lower with the casein-gelatin diet than with the soy isolate diet. The relative bioavailability value (as compared with cadmium as the chloride) for oysters was similar with the two diets, 38 + 18% and 48 + 13% with the soy and casein-gelatin diets, respectively. Earlier studies by Fox et al. (17) had shown marked differences in cadmium toxicity and tissue mineral levels when soy isolate, casein-gelatin, or dried egg white was the dietary protein source. From these varied data, it was concluded that investigations of single nutrient variables and of foods (plant and animal) intrinsically labeled with "09Cd during growth are needed to identify dietary components (singly and together) that can affect accumulation of cadmium in the liver and kidneys and to establish the relationships between cadmium concentrations in the jejunum-ileum and those in the liver and kidneys.
Casein and gelatin typically contain lower levels of contaminant minerals and other complicating factors, such as phytate, and thus contribute to a better dietary matrix for studying mineral interactions than does soy isolate. Variations in mineral levels between batches are usually less for casein and gelatin than for soy isolate. The purpose of the present study was to determine the effect of variable dietary zinc concentrations in a casein-gelatin diet upon tissue cadmium levels derived from low dietary cadmium intake and to compare soy isolate with casein-gelatin as protein sources when the zinc concentrations were at the required level for each diet.
Experimental Procedures
Day-old Japanese quail (Coturnix coturnix japonica) of both sexes from our stock colony were housed in heated, continuously lighted, suspended stainless steel cages. Precautions to avoid environmental and dietary contamination with trace elements were observed. The birds were housed one group per cage except for birds 7-14 days of age in experiment 1, when they were housed individually to permit measurement of food intake. Diet and deionized drinking water were available at all times. The birds were wing-banded at 7 days of age and were weighed at weekly intervals. In experiment 1, groups of 10 birds each were fed with either a soy isolate or a casein-gelatin diet (Tables 1 and 2). In Na2SIO3, Alfa Inorganics, Beverly, Mass. See Table 2 for amounts of cadmium, zinc, iron, manganese, copper, and magnesium. bEstimated from analysis of other lots of soy isolate. c Amounts of components other than purified protein and chemical source of individual elements. Due to the higher dietary levels of manganese and copper in the soy diet, the small amounts of these elements in other components were not included in the dietary calculations. d It was not possible to assay the calcium salts. experiment 2, 40 day-old birds were fed the caseingelatin diet (Tables 1 and 2) for 1 week and 20 birds were fed the soy diet. On day 7, the birds were redistributed by body weight into 4 groups of 10 birds each; 3 groups were fed the casein-gelatin diet and one group was fed the soy diet. Excess birds at the weight extremes were eliminated so that the mean body weights + SE were 20.0 + 0.47 g and 20.3 + 0.30 g for the casein-gelatin and soy isolate diets, respectively. The birds continued to receive the same diets except that the zinc concentrations in the casein-gelatin diets were adjusted to totals of 12 and 60 ppm for 2 of the groups.
Accelerator-produced carrier-free '09Cd (as the chloride) in 0. IN HCl (New England Nuclear Corp., Boston, Mass.) was premixed with glucose, freeze-dried, finely ground in a mortar, and mixed with each diet to provide 100 gCi '09Cd/kg diet. The labeled diet was fed to the birds from 7 to 14 days of age. Total food intake of each bird during the second week was measured in experiment 1.
The birds were decapitated on day 14 without a prior fast. The liver was removed, washed in 0.75% NaCl solution, and blotted with tissue. In experiment 1, the intestinal tract was closed with a hemostat placed immediately distal to the ventriculus to prevent contamination of tissues with '09Cd in the digesta, and the entire intestinal tract was removed. The duodenum was defined as the loop encircling the pancreas and the jejunumileum was the next section of small intestine terminating at the cecal juncture. The sections of small intestine were placed on pieces of rigid plastic, opened longitudinally, washed thoroughly with a stream of 0.75% NaCl solution to remove all intestinal contents, and blotted with tissue. The kidneys were blotted in situ to remove any blood. They were removed with bent stainless steel microspatulas with sharpened edges. All tissues were immediately placed in preweighed vials with tightly fitting caps. In experiment 2, tissues were removed identically except that the proventriculus and ventriculus were also excised, opened, and washed with saline to remove all contents.
The tissue weights were obtained and the whole tissues were solubilized in 5 ml concentrated nitric acid (redistilled, G. F. Smith, Columbus, Ohio) and diluted with deionized water to a final volume of 10 ml. Except for some residual fat, each tissue was completely solubilized. Radioactivity of the tissues and 0.5 g samples (in triplicate) of diets similarly solubilized were measured in a Nal (Tl) crystal scintillation detector (Model 5285, Packard Instruments, Des Plaines, Ill.). An integral window was employed to maximize efficiency and the tissues were counted to an error of less than 2%. All samples had identical geometry.
After measurement of "09Cd in experiment 2, the solubilized tissues were transferred to large test tubes; 20 ml of a 5:1 mixture (volume:volume) of nitric and perchloric (70%, double distilled, G. F. Smith) acids were added. The tissues were wetdigested and diluted to volume. To control viscosity and minimize phosphate interference, the final solution contained 10% glycerol and 0.7% perchloric acid by volume (21). Half-gram samples of diet and 0.1 g samples of mineral premixes (in quintuplicate) were similarly digested. Zinc, iron, manganese, and copper were determined by atomic absorption spectrophotometry (Model 503, Perkin-Elmer Corp., Norwalk, Conn.). Cadmium in the cadmium premix and magnesium in the diets and magnesium premix were similarly determined. By these techniques, analytical values for the same elements in Bovine Liver, Standard Reference Material 1577 (National Bureau of Standards, Washington, DC) fell within the certified ranges.
Triplicate 3 g samples of casein, gelatin, and soy isolate were wet digested with 20 ml nitric and perchloric acids (5:1, volume:volume) and 1 ml concentrated sulfuric acid (G. F. Smith) and were assayed for cadmium by differential pulse anodic stripping voltammetry (22). The cadmium concentration of all remaining dietary components minus protein, calcium phosphate, and calcium carbonate was similarly determined. Formation of precipitates with the calcium salts prevented their analysis for cadmium. The cadmium concentrations were 37.3 ng/g soy isolate and 3 ng/g gelatin. The cadmium in casein and other dietary components (exclusive of protein and calcium salts) was below the detection limits (<3 ng/g).
The tissue cadmium concentrations derived from the diet fed between 7 and 14 days of age were calculated from the specific activity of the added cadmium, 62 ,ug/kg. We have repeatedly observed linear log-dose, log-tissue concentrations and identical tissue retentions of cadmium within this dietary range of cadmium fed in one of several dietary matrices (12, 16, unpublished data). The use of added cadmium provided a constant base level so that comparisons could be made between the two types of diets. Statistically significant differences between group means were based on use of Student's t test (23a). Evaluation of response magnitude in relation to dietary zinc level was based on correlation coefficients (23b).
Results
Type of dietary protein did not affect body weight or cadmium intake (i.e., food intake) of the birds (Table 3). Birds fed the soy diet retained in the four selected tissues almost half of the cadmium consumed between 7 and 14 days of age, whereas birds fed the casein-gelatin diet retained only about onesixth. The amounts in the small intestine were by far the largest and accounted for most of this difference (Table 4). Birds fed the soy diet had greater amounts of cadmium in the liver.
There were no effects of diet on tissue weights, except that the jejunum-ileum was significantly larger in birds fed the soy diet (Table 4). Similar results were obtained in experiment 2 ( Table 5). The level of dietary zinc in the casein-gelatin diet affected neither body weight nor tissue weights.
The mineral concentrations in five tissues from birds in experiment 2 are presented in Table 6. Birds fed 20 ppm zinc were the normal requirement controls for the casein-gelatin diet. There were some differences in absolute concentrations of cadmium in some tissues between experiments 1 and 2. The relative effects of diet on cadmium in the intestinal tract segments were the same; however, the values for the liver in experiment 2 were not different from those in experiment 1. With respect to other elements, the diet did not cause marked differences except that birds fed the soy diet had markedly lower amounts of iron in the duodenum and liver, and higher amounts of manganese and copper in all tissues.
With increasing levels of zinc in the casein-gelatin diet, there were increased concentrations of zinc in all tissues ( Table 6). As dietary zinc increased, there was a general decrease in cadmium concentrations for all tissues except the kidneys. These relationships are all supported by statistically significant correlation coefficients ( Table 7). The linear correlations between tissue zinc concentration and tissue cadmium concentration were statistically significant only between the duodenum and jejunum-ileum. The highest correlation coefficient for tissue zinc versus tissue cadmium was obtained for the jejunum-ileum and the range in cadmium concentrations was the highest for any tissue. Since the changes in liver cadmium represented the most important effect in this study, the correlation coefficient for jejunum-ileum versus liver cadmium was calculated. It was statistically significant (p < 0.05).
The concentrations of iron in the two small intes-tinal segments were generally inversely related to dietary zinc concentration ( c Not significant. with respect to the level of specific nuti feasibility of adding conventional hur future experiments. Cadmium was adn feeding at levels comparable to those man (6). Essential nutrients were pres required by the quail insofar as they Excesses of essential nutrients were av as possible and no substances were; diet that were not required.
Requirements for the quail have beei by feeding the same graded dietary lev eral for either 2 or 4 weeks. It was four that the minimal level required after o' less than that required during the first growth data in Table 5 illustrate for zin ing the dietary zinc from the require( ppm by 40%, to 12 ppm, growth and gr ment during the second week were no the minimal adequate level of zinc fo week. It is likely, therefore, that as growth rate slowed during the secon levels of other nutrients were present requirements. Changes in nutrient requ human beings have been established i age and growth rates (24); however, f for experimental animals have been lii set of requirements for the growth peri practice of determining animal requ stepwise doubling the nutrient concent diet and forcing maximal weight gain m resulted in establishing requirements t] atively higher than the requirements: These traditional types of control diet, have probably influenced experiment indicate lesser problems with cadmium n in birds fed the be true for human populations, whose nutrient ina takes may not exceed or even meet requirements r p (4 The tissue concentrations of cadmium at 14 days of age result from a spectrum of metabolic prorients and the cesses that probably proceeded at different rates nan foods in during the final 7-day period. Most of our data inninistered by clude changes that seem to be due to nutritional in the diet of effects upon intestinal absorption of cadmium. With sent at levels higher cadmium levels, feeding cadmium for 48 hr are known. immediately prior to killing at 14 days of age reoided insofar sulted in a high cadmium uptake by the duodenum added to the and marked decreases in duodenal iron concentrations (26), and morphological abnormalities of the n determined villi (27). It is thought, therefore, that changes at the ,els of a min-intestinal level occurred early in the week of these nd, however, experiments. Since 109Cd was fed for the last 7 days ne week was of this experiment, the distribution of 109Cd among week, as the the various organs, particularly that in the liver and ic. By lower-kidneys, is the sum of cadmium consumed at differd level of 20 ent times and of metabolic and transport processes ross develop-that have not been defined with respect to time.
irmal. This is r the second Effects of Dietary Zinc on the normal Tissue Cadmium Levels Id week, the in excess of It is significant that supplemental zinc protects iirements for against dietary cadmium over a range of intakes in relation to from less than 0.1 ppm in this experiment to 80 ppm requirements in the first study by Supplee (28). High levels of mited to one cadmium interfere with zinc absorption and proiod (25). The duce signs of deficiency; however, the levels of irements by cadmium of importance to man are far below the tration in the levels that interfere with zinc as an essential nuiay also have trient. More subtle interactions at binding sites inhat were relvolved in transport and residence in tissues are unset for man. doubtedly involved in the latter case. sfor animals The best dose-response, to zinc was found in the al results to jejunal-ileal section of the small intestine. Dethan would creases of cadmium in this section have been cor-Environmental Health Perspectives related with decreases in the liver and/or kidney (12,15). The greater difference between cadmium concentrations in the jejunal-ileal section from birds fed 12 versus 20 ppm zinc, as compared with 20 versus 60 ppm zinc, may be indicative of a more significant effect of zinc at deficient than at excess levels. This places emphasis on correcting zinc deficiency in human beings exposed to typical background levels of cadmium rather than administering zinc in excess of requirement, where safety of the zinc supplements is not well defined. In other persons exposed to high levels of cadmium, either industrially or via environmental contamination, supplements of zinc in excess of requirement should be useful. A controlled study of zinc supplements in such a population is needed. As zinc concentrations in the diet increased, not only did zinc concentrations in the tissues increase, but the concentrations of iron, manganese, and copper decreased in one or more tissues. Single supplements of manganese and copper had some small effects in increasing tissue cadmium concentrations from low dietary intakes (14). It is not known what effects deficiencies of these two elements might have upon uptake under these conditions. Flanagan et al. (8) showed that low iron status, as indicated by low serum ferritin values, was associated with high absorption of 25 gg cadmium with ll5mCd as the chloride consumed in a single meal by human volunteers (8). Flanagan et al. (8) also showed that in mice a tracer of '09Cd as the chloride given with 1.12 ppm cadmium in the drinking water was bound primarily to larger proteins in the liver and kidneys rather than to metallothionein. With a high intake of cadmium the reverse distribution has been found. Additional studies are needed on the absorption and metabolism of low levels of cadmium, and the manner in which cadmium is influenced by other essential elements, both individually and interacting with each other. From a practical viewpoint, differentiation is needed between the effects of mineral status (deficient and excess) and the mineral levels consumed directly with cadmium.
Effects of Diet Type on Tissue Cadmium Levels
The lower levels of cadmium in the duodenal and the jejunal-ileal sections of birds fed the control casein-gelatin diet (20 ppm zinc) were not associated with similarly lower levels of cadmium in the liver as compared with birds fed the soy diet. Although increasing the zinc content of each diet decreased cadmium in both the jejunum-ileum and the liver, the use of cadmium content of the jejunum-ileum as an index to bioassay cadmium in natural foods now appears equivocal because of the differences between the two types of control diets. It is possible, but unproven, that given dietary supplements would influence cadmium uptake by the jejunum-ileum and liver similarly with each diet. This was true for jejunal-ileal cadmium uptake from oysters (16). The correlation in this study of jejunal-ileal cadmium concentrations with liver concentrations supports the validity of the approach. The range in liver values with the graded zinc levels was relatively small (Table 6), much smaller than would be expected in a bioassay with cadmium fed at three levels (12); therefore, correlations of cadmium in the two tissues in a bioassay range should be good.
The differences in jejunal-ileal weight and the concentrations of essential minerals in intestinal tissue all may have affected tissue cadmium levels apart from characteristics of the dietary proteins. Table 2 shows that much higher proportions of dietary iron, manganese, and copper were supplied by the soy protein than by the casein-gelatin. Although total dietary levels of each element except copper were established at levels to meet requirements with no excess, the relative decreases in requirement during the second week may have been different for each diet. Phytate in soy may affect cadmium directly or indirectly by its effect on zinc. Some more minor components of the soy isolate may also be involved.
The complexity of the apparent interrelations among the essential elements themselves, and particularly in relation to cadmium, makes one cautious in drawing conclusions regarding the best dietary mineral levels to minimize adverse effects of cadmium. The relative hazard of cadmium in an individual food must still be evaluated on the basis of total cadmium content until data are obtained on the bioavailability of cadmium in human foods. | v3-fos |
2019-03-22T16:13:25.998Z | {
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} | s2 | Effects of ambient temperatures on clinical and immune responses of pigs infected with transmissible gastro-enteritis virus
Two- to three-months-old pigs infected with transmissible gastroenteritis (TGE) virus showed no clinical response when housed at 30°C, but comparable infected pigs exposed to temperature changes from 30°C to 4°C following infection showed typical signs of TGE. Development of TGE-specific immune responses, as measured by blastogenic response of tissue lymphocytes, occurred at 3 days post-inoculation (DPI) in pigs held at 30°C, but not until 7 DPI in infected pigs held under the adverse conditions. Immunosuppression with corticosteroids resulted in a fall in circulatory T cells, lowering of non-specific blastogenic response of circulatory lymphocytes, and clinical signs of disease when immunosuppressed pigs were infected with TGE virus and held at 30°C. It is suggested that clinical responses to TGE virus infection may be affected by the influence of ambient temperatures on the immune responses of pigs.
INTRODUCTION
Transmissible gastroenteritis (TGE) of pigs is a highly contagious, enteric, viral disease, affecting pigs of all ages but causing severe death losses only in piglets less than 2 weeks old. The disease, characterized by severe diarrhoea and vomition in piglets, is caused by a coronavirus (Tajima, 1970).
One of the interesting epizootiological features of TGE is its seasonal appearance. It is recognized that the majority of herd outbreaks of TGE occur during the colder months of the year (Ferris, 1973). In a previous report (Shimizu et al., 1978), the effects of ambient temperatures on infection of TGE in feeder pigs 2 to 3 months old were examined. It was shown that a high ambient temperature resulted in increased resistance of pigs to clinical disease, whilst a low ambient temperature, and temperature changes, caused a dramatic enhancement in clinical sequelae. These results might explain in part the seasonal occurrence of TGE outbreaks. However, the mechanism of adverse effects of low temperature and temperature changes in induction of clinical disease in feeder pigs remains obscure.
The purpose of the present paper is to investigate the mechanism by which adverse ambient temperature enhances the severity of clinical disease following infection with TGE virus.
MATERIALS AND METHODS
Viruses. The virulent Shizuoka strain (Sasahara et al., 1958) of TGE virus was used for oral inoculation of pigs. The strain had been passaged 17 times in piglets and stored at -80°C in the form of a 10% suspension of infected small intestine.
The TO strain of an attenuated TGE virus was used for production of antigen to be used for lymphocyte stimulation in vitro. The strain had been serially passaged in pig kidney cell cultures (Harada et al., 1969), and was used at passage 168 in the present studies.
Vesicular stomatitis virus (New Jersey type) was used as challenge virus in an interferon (IF) assay.
Experimental pigs. A total of 28 Yorkshire pigs 2 to 3 months old, serologically negative for TGE antibody, were obtained from a farm where no outbreaks of TGE had been reported.
Experimental design. Two experiments were carried out. In the first experiment, effects of the ambient temperatures on induction of immune responses and on production of IF in the intestinal tract in pigs inoculated orally with TGE virus were investigated. Twenty-two pigs were kept at the high temperature (30 + 2°C) for 4 days and then divided into two groups. Eleven pigs (Group 1) were transferred to the low temperature (4 ± I°C) room. Nine of them were infected orally with 104 50% pig infectious doses (IDs0) of virulent TGE virus and kept at 4°C. The remaining two pigs kept at 4°C were not infected and served as controls. Eleven pigs (Group 2) were maintained at the high temperature throughout the experiment. Nine of these pigs were exposed orally to 104 IDs0 of virulent TGE virus on the fourth day of the experiment, and the remaining two pigs served as uninfected controls.
Three pigs inoculated with virus in each group were sacrificed at 3, 5 and 7 days post-inoculation (DPI), respectively. All the control pigs were killed on the seventh day of the experiment. Lymphocytes were collected from Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood of these pigs, and tested for proliferative reactivity to TGE viral antigen in vitro Shimizu, 1979a, 1979b).
IF activity of the intestinal contents and washings, obtained from all the pigs, was also examined.
In the second experiment, the influence of an immunosuppressive drug on the protective effect induced in pigs by high ambient temperatures was investigated. Six pigs were kept at the high temperature. Three of them were given dexamethasone (DM), a synthetic corticosteroid, subcutaneously in doses of 5 mg/kg daily for 5 days, except on the third day when this dose of the drug was administered intravenously. On the fifth day of the experiment, all pigs were exposed orally to 104 IDso of virulent TGE virus, and maintained continuously at 30°C. The concentration of circulatory T lymphocytes and non-specific blastogenesis of peripheral lymphocytes to phytohaemagglutinin-P (PHA) were determined on the first and the fifth day of the experiment.
Preparation of lymphocyte suspensions. Lymphocyte suspensions to be used in a lymphocyte proliferative assay were prepared by methods described previously Shimizu, 1979a, 1979b).
TGE viral antigen. TGE viral antigen used for lymphocyte stimulation in vitro
was prepared from pig kidney cell cultures infected with the attenuated TO strain of TGE virus Shimizu, 1979a, 1979b).
Nonspecific mitogen. PHA was purchased from the Difco Laboratories, Inc.
Lymphocyte proliferative assay. Lymphocyte proliferative reactivity to TGE viral antigen and PHA was determined by measuring the amount of [3H]thymidine incorporated into nucleoprotein of cells Shimizu, 1979a, 1979b). The results were expressed as a stimulation index, which was defined as the mean count per minute of cultures incubated with mitogen, divided by the mean count per minute of cultures incubated with control antigen.
Detection of T lymphocytes. The number of T lymphocytes was determined by rosette-formation of cells with sheep erythrocytes. The details of the rosette-formation test have been described elsewhere (Shimizu et al., 1976).
Preparation of intestinal samples for IF and its assay. The small intestine, 1 m in length, was removed from all pigs in the first experiment and washed with 300 ml of phospate buffered saline solution (PBSS). The intestinal contents were mixed with the corresponding intestinal washings and centrifuged at 2000 X g for 30 min. Resulting supernatants were heat-inactivated at 56°C for 30 min. Zinc acetate was added to the samples at a concentration of 0.02 M. Precipitates formed were collected by centrifugation at 2000 X g for 1 h and resuspended in 0.2 N solution of hydrochloric acid to 1/30 of the original volume. After dialysis against several changes of a large volume of PBSS, the solutions were tested for IF activity. Tubes containing pig testicle cell cultures were treated overnight with 0.5 ml of serial dilutions of the intestinal samples, using Eagle's minimal essential medium supplemented with 10% bovine serum. After overnight incubation at 37°C, the samples were removed, and the cultures were challenged with 100, 50% tissue culture infectious doses of vesicular stomatitis virus. The cultures were incubated at 37°C for 40 h. The samples which inhibited development of cytopathic effect in the assay were regarded as positive samples for IF.
Effects of ambient temperatures on early immune responses in pigs inoculated with TGE virus.
Results of the first experiment are shown in Table I. All pigs inoculated with TGE virus immediately after transference to the low temperature room showed profuse diarrhoea and vomition after an incubation period of 2 days. On the other hand, pigs infected and held at 30°C showed no clinical signs of the disease.
Development of immune responses, as measured by blastogenic responses of lymphocytes from Peyer's patches and mesenteric lymph nodes, was first noted at 3 DPI in pigs held at 30°C, whilst infected pigs held at 4°C showed no comparable response until 7 DPI (convalescent stage).
In both groups of uninfected control pigs no blastogenic responses of lymphocytes were noted when these animals were killed on the seventh day of the experiment.
Effects of an immunosuppressive drug on infection with TGE virus.
In comparison to untreated pigs (Table II), T lymphocytes of the peripheral blood of pigs treated with DM and held at 30°C were significantly reduced in number and reactivity to PHA by the fifth day of treatment.
Infection with TGE at this stage of apparent immunosuppression was associated with profuse diarrhoea which continued for 3--4 days after incubation periods of 1--2 days. Control pigs, not treated with DM, infected at the same time, and held at the same temperature (30°C), showed no clinical evidence of TGE infection, their faeces remaining normal throughout the experiment.
DISCUSSION
There are many reports dealing with effects of ambient temperatures on the pathogenesis of various virus diseases (Boring et al., 1956;Marshal, 1959;Carmichael et al., 1969;Lycke et al., 1971;Bell and Moore, 1974;Kiorpes and Yuill, 1975;Bell et ah, 1977). Lower temperatures usually intensified the severity of the particular disease, whereas higher temperatures often resulted in an increased resistance to the clinical syndrome. In a previous report (Shimizu et al., 1978), it was shown that feeder pigs 2 to 3 months old, kept at a high temperature, endured infection with TGE virus without showing clinical signs, and that both temperature change, and low temperature, exacerbated the infection in pigs. The change from the high to low temperature was considered to be an important factor in the induction of TGE in feeder pigs. At present, however, the mechanism by which the adverse temperature enhances the severity of various virus diseases is not fully understood. One possible explanation for the mechanism might be that an elevated body temperature, induced by the high ambient temperature, enhances the resistance of animals to the infection. The elevated body temperature may directly inhibit intracellular replication of virus, or may induce abortive infection. In rabies virus infection of mice (Bell and Moore, 1974) and canine herpesvirus infection of puppies (Carmichael et al., 1969), resistance of animals to infection was associated with an elevated body temperature. It remains obscure, however, whether resistance induced by elevated body temperatures results from direct effects on viruses, or from indirect effects through protective responses of the host.
According to Furuuchi et al. (1975), the optimum temperature for growth of virulent TGE virus ranges from 37°C to 40°C. Bustad and Book (1975) have shown that the body temperature of pigs weighing 30--65 kg, and kept at 4.4°C, was about 39°C and was about 40°C in pigs held at 32.2°C. It seems unlikely, therefore, that pigs kept at 30°C showed subclinical disease as a result of direct effects of the elevated body temperature on TGE virus replication in vivo.
Based on observations that mice inoculated with Coxsackie B1 virus, and kept at 25°C, developed IF with reduced virus levels in their livers, compared with the absence of IF and high levels of virus when kept at 4°C, Ruiz-Gomez and Sosa-Martinez (1965) suggested that ambient temperatures play an important role in virus infections by the regulation of IF production. In the present studies, however, IF was not detected in the intestinal contents and washings obtained from pigs with and without clinical signs of TGE, suggesting that the effects of ambient temperatures may not operate through regulation of IF production in this situation.
Results from the present studies suggest that the nonspecific cell-mediated immune response of pigs may be reduced at lower temperatures. The delay in initiation of the immune response in infected pigs held at 4°C, compared with that in comparable pigs held at 30°C, was in the order of 4 days. It will be noted that clinical disease in the pigs kept at 4°C resulted after an incubation period of 1--2 days. If suppression of cell-mediated responses is the cause of clinical disease in pigs held at 4°C, it would imply that such cell-mediated responses in pigs with subclinical disease held at 30°C, occur within 48 h. From the experiment with an immunosuppressive drug, there would seem no doubt that immunosuppression was associated with clinical disease even at 30°C, and the tentative conclusion is drawn that low ambient temperatures may interfere with the early initiation of local cell-mediated responses in the TGE model. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Analysis of the genetic variation for adaptation to a short thermal stress on young « Bos taurus » cattle
In the present work, an attempt was made to determine the adaptation to heat of some cattle breeds, especially French ones. A total of 5 8 2 young male and female cattle, about 14 months old and coming from 41 elementary genetic combinations analyzed in 5 different non bioclimatological experiments, were subjected to a heat stress for 8 hours during which the room temperature was increased from 1 8° to 3 8 °C. The usual reaction were observed i.e. increase in the rectal temperature during the stress (+ 0 , 54 °C), increase in the respiratory rate (X 3), increase in the sweating rate, heart rate and skin temperature. The statistical analyses were made at three stages of stress (beginning, middle, end). In all cases, a very strong environmental effect was noticed, ie effect of the year and of experimental errors in the management of the theoretical schedule involving temperature and relative hygro-metry. The data adjusted for environmental effects rather clearly show the genetic variability. However, a more accurate analysis shows that it is mainly proceeded from a between group variability when defining the groups according to « animal husbandry » parameters (dairy purpose , beef purpose, and local breed group). As a matter of fact, in most cases, there was no significant variability between the reactions of the genotypes belonging to the same group, whereas this variability existed between the groups..The satisfactory performance of the local breed group clearly appeared in comparison with the group including the improved dairy or beef breeds exhibiting only minor differences in the parameters analyzed. In the discussion, emphasis in laid ov the possible influence of thermogenesis on the results. Although our findings could have been afrected by disturbing events such as emotional stress, they agree rather well with data obtained is practice on the behaviour of the local French breeds in some hot countries.
Introduction
Animal breeding improvement of milk and meat productions in tropical countries gives rise to problems depending on the practical conditions encountered in these countries (P AYN E, 1970 ;Me DO W E L L, I9!2; HAI,IPR! and BIBA, Ig73; Me Dow!LL, I g 7 6). They are in connection with the direct effects of the climate, especially the temperature and its indirect effects : low average level and large variations in the quantity and quality of feed, parasitic attacks (F RIS C H ,197 6).
The peculiarity of these conditions is so strong that the breeding methods used often take advantage of crossbreeding between Bos taurus and Zebu cattle, for milk production as well as meat production. Many experimental results show that in the present tropical conditions, there is an optimal proportion of « Bos taurus » genes (cc productive » genes) between 5 o and 70 p. 100 , the remaining proportion corresponding to « resistance » genes necessary for expressing productive genes (AMBLE and J AIN , 19 65; CIrNDI!x, 1970 ;P AYN E, 1970 ). These results are mostly derived from comparisons between a local zebu breed, a European breed and some corresponding crosses. However, it is not often possible to obtain a direct comparison between the European breeds, pure or crossbred, concerning their production abilities in tropical conditions and their components.
Most of the results of the comparison between lines or breeds of « Bos taurus » cattle were obtained in British breeds but the technical references on typically French breeds are scarce, although some of them are used in hot climates : the Ta y entaise breed in North Africa, the Norman breed in South America and the Charolais and Limousin breeds all over the world. Thus, between 1971 and1975 , an experiment was carried out in order to compare the resistance to a short thermal stress of various animals from the running experiments managed by the Animal Breeding Department of the Institute.
Unfortunately, no Zebu « control », was introduced into the test nor were tested the same genetic combinations outside, in real hot conditions. However the range of the studied animals was very large, all the types of common « Bos Taurus » breed being represented.
The purpose of this paper is to give the final result and conclusions, after a rather long list of preliminary surveys written by scientists or students (H A -L I PR A, 1971, 1973GUII,LARD, 1971;CORBEL, 1972;VIZINE, 1973;SINGH, 1976;COL L EA U , 1977).
Material and methods
A. -The hot room The room used is closed and measures 4 . 7 x 4 . 2 X 2 . 2 m i.e. 43 m 3 . Heating is performed by three electric radiators with a total capacity of 6 00 o W.
Four animals may be recorded at the same time in the room. They are separated by bails and are not allowed to lie on the concrete floor. They have not access to feed and water during the thermal stress.
A direct radiative heat load is brought to the animals since they are fairly close to the radiators. Because of geometrical considerations, this radiative load is expected to differ slightly from bail to bail. However we never observed any significant influence of the site ( I to 4 ) inside the hot room, on the studied variates, as reported subsequently.
The thermal programme applied during the test is transmitted to the radiators by means of the following method. It is drawn as a diagram on a paper disk, turning at a constant speed. The inner part of the diagram has been covered previously with graphit using a soft pencil. A conducting stylet is kept close against the disk and connected with a metallic serpentine pipe located in the room. The moves of the stylet are controlled by the dilatation of a gas inside the serpentine pipe. The dilatation is proportional to temperature. When the temperature of the room is higher than the programmed one, the stylet gets off the graphit area and the current in the radiators is switched off. The actual temperature in the room is recorded continuously on a second paper disk.
Relative humidity is continuously registred by means of a hair hygrograph.
A device for regulating moisture, working according to the same principle and connected with a fan was used. A mobile button allows the releasing of the fan, extracting air when relative humidity is higher than a previously chosen threshold.
The ordinary ventilation in the hot room was static and obtained by means of a trap in the wall, opposite the door.
The thermal programme used is shown in figure i. The animals are introduced into the hot room at 8. 3 o a.m. at room temperature ( 1 8°C). The trial was finished at 1 6. 30 p.m. when the programmed temperature was 3 8 °C. The increase is temperature is sharp till i2.oo a.m. Then a plateau was maintained at 34 °C for 2 hrs and at 3 8 °C for 3 hrs. This programme was intended to be applied under high humidity ( 70 -8 0 p. 100 ) in order to enhance the thermal load.
Measurements -Rectal temperature was recorded by a medical thermometer introduced into the rectum of the animal at constant deepness ( 13 cm), during 1 .5 mn. The rectal temperature during normal conditions, when the animals were tied in the stall, was taken in the morning, 24 hours before the stress.
-Respiratory rate was evaluated visually from the number of beats during 30 seconds. This measurement was generally made two times consecutively.
-Heart rate was derived from the tracing of an electrocardiograph with 4 electrods, within 10 seconds. During the last two years, it was derived from the number of heart beats in the coccygean artery, during I minute.
-« Sweating rate o was evaluated from a small previously shaved area located on the hips, according to the method of Srxr,!GEx and BEAN ( 1971 ) : three pellets of dried chromatographic paper, previously moistered with a 20 p. 100 cobalt chloride solution were put on this area. For each pellet, the time to change thoroughly into pink was measured and then averaged over the three measurements. The more the animal sweated, the faster the paper turned from blue to pink.
-Skin temperature was evaluated by a thermocouple positioned in the area of the last ribs, on the side turned towards the middle of the room.
B. -Variates
The experiment lasted five years ( 1971 -1975 ) The animals originated from the fattening cattle herd in La Minière near the Research Centre of Jouy-en-Josas. As hot room was constructed in the cattle shed, almost no transport of animals was required. Every young cattle fattened in this shed whatever the sex or the genotype entered the hot room at least once.
The period of entering the hot room ranged from February to June, each year.
The animals actually come from five experiments : -an experiment comparing three French meat breeds (Charolais, Limousin, Maine-Anjou) composed of purebreds and crossbreds, with a Hereford control sample; -an experiment comparing local breeds (Aubrac, Gasconne) known as hardy breeds, purebred and crossbred with meat breeds such as Charolais and Blond d'Aquitaine, -an experiment comparing a specialized dairy breed (Holstein), a dualpurpose breed (Norman), a specialized meat breed (Charolais) and several crosses between these breeds; -an experiment comparing the effect of sex an type of sire (normal, double muscled) on Charolais X French Friesian crossbreds; -a specific heat resistance experiment devoted to comparing three local French breeds (Sale y s, Abondance and Tarentaise) and a French dairy breed (Montbéliarde), all these breeds coming from mountainous regions in France.
D. -Statistical methods All analyses were made according to linear models involving factors like genotype X sex combination, year and covariates.
The effect of years was introduced because of the changing of operators. The combinaison genotype X sex was introduced as a factor instead of two factors, because of the few genotypes in the females.
A possible « seasonal » effect was never tested, and that is perhaps a shortcoming, because of the already complex statistical model and mostly because of the problem of summing up efficiently the thermic « history » of each animal before the test, from the observations of temperature and hygrometry in the cow shed.
On the other hand, genotypes are in fact primary groups (small groupings) according to the following rules : -no distinction between the reciprocal crossbreedings, -backcrosses are considered as purebreds.
The interactions between year and genotype were never tested because of the high number of empty cells. Nor was tested the « experimental » factor because of the nearly total confusion between « experiment and « genotype ». Despite the fact that each experiment had its own system of rearing before fattening, it appeared that the long stay in I,a Miniere (approximately 5 months) before the test at the age of 14 months was large enough to cancel any expected experimental effect.
Within these defined linear models, the least square solutions, analysis of variance and residual correlations were obtained. Owing to a special computer programme, additional hypotheses were tested in reference to large secondary groups of genotype-sex combination (differences between and within groups).
The group means were then calculated from the estimates of each member weighted according to the inverse of their sampling variance. That expression is only one approximation since the best estimation of the group means should be obtained by generalized least squares procedure (S!nR!,!, 1971 ), involving not only the sampling variance of the individual estimates of the tl ' genotype of the i th group but also their sampling covariance. The calculations were simplified because the off diagonals term of the variance-covariance matrix between the genotype effects
Results
Since the measurements collected were not always the same, the results will be shown in two parts : the last three years (complete data) and the whole five year period for the variates collected all over these years (incomplete data).
The genetic types and the number of animals involved are reported in table 2 .
1 . -Analysis of the complete data a) Overall range of the climatic room measurements Table 3 shows the averages and standard errors for five variates related to the animals and for two variates related to the hot room, at three phases : beginning, middle and end of stress. Rectal temperature rose steadily, by 0 . 54 °C during the whole stress. At beginning of the stress, rectal temperature was already considerably higher than in the barn, 24 hours before : -!-0 . 3 8 °C.
The rise in respiratory rate was drastic, i.e. nearly three times higher than normal. The rise in heart rate was low : from 88 to 90 pulses per min. The rise in the sweating rate was shown by a distinct drop ( 5 o p. 100 ) of the turning time of the pellets. The rise in skin temperature was marked (+ 2 °C).
The high final temperature of the room ( 39 °C) and the regular fall in relative humidity ( 70 p. 100 at the end of the stress) can be noticed. Temperature and hygrometry curves obtained on different days are far from overlapping each others : the standard errors for temperature and hygrometry at a fixed hour are large, for example 0 . 7 °C for temperature at the end of the stress.
b) I'ariation factors at a given time
The results of the analysis of variance are given in table 4 and the estimates for the end of the stress only are shown in table 5 .
Generally there exists significant differences between c· genotype X sex » combinations. Further comments will be made about this factor.
It can be noticed that the « year » effect has always a large influence, probably due to the changes of operators from year to year.
Rectal temperature (R.T.) and respiratory rate (R.R.) at a given hour strongly depended on the actual temperatures and hygrometries. This is even more marked as the end of the stress is approaching. At that time, if the hot room is i °C warmer than planned, the rectal temperature is enhanced by o.2 °C and the respiratory rate by 13 beats. Thus a higher hygrometry at constant temperature enhances the stress effect as expected. As far as R.T. is concerned, as increase of 8 p. 100 in relative humidity at a given temperature is equivalent to a i °C increase in room temperature. R.R. seems less sensitive to hygrometry, since an increase of 13 p. 100 in relative hygrometry has the same effect as a increase of 1°C.
Skin temperature (S.T.) closely depends on external temperature : the final regression coefficient is even higher than calculated for rectal temperature. This is in agreement with what can be observed during the overall stress. Skin temperature increases more than rectal temperature ( 2 °C relatively to 0 .5 °C). At a given time, sweating rate (S.R.) seems to depend on R.H. rather than on temperature.
Heart rate in practically independent on fluctuations when applying the programme and this is in agreement with the overall evolution of H.R. during the stress. c) Effect of the « genotype-sex » factor It is a combined factor that has no specific meaning. In fact, it groups 1 6 classes of males and one of females (CH X FF).
-Comparison between males and females. Application of contrast analysis shows that females CH X FF are more resistant than males CH X FF to heat stress. Their R.T., R.R., S.R. and H.R. are lower.
-Overall comparison between males. the differences observed are particulary large and significant for heart rate and sweating rate. The differences are also significant for rectal temperature. The results are however fluctuating for respiratory rate and skin temperature.
These males can be divided into four groups according to animal husbandry criteria : -The « beef » group : Cha y olais, Limousin, Maine-Anjou, Hereford, Cha y olais X Limousin and reciprocals, Charolais X Maine-Anjou and reciprocals, Limousin X Maine-Anjou and reciprocals, Hereford X Limousin, He y eford X Maine-Anjou. In these conditions, the between-male variation observed was also divided into between and within-group variation. The results of table 4 show that the variability observed at the end of the stress is located primarily at the group level, the within group variability being non significant. S.R. is an exception since the contrary is observed : non significant between-group variation and significant within-group variations. The details on the individual estimates for each genotype are shown in table 5 . Figure 2 to 6 allow to locate the between-group differences. When comparing the « dairy » and « beef » group, it appears that the differences are small (except for H.R. and S.T.) but all in the same direction. Beef animals seem to show a less favourable heat balance, although appealing to thermolytic mechanisms (stronger respiration and sweating). The heart rates are definitely different : all over the test, the number of pulses per minute was approximately 8 points higher for beef animals. At the beginning of the stress, a much lower skin temperature was observed in the « dairy » animals. The dairy X beef animals, when compared to the former groups are generally located at an intermediate level.
The local group exhibited the best heat resistance measured by R.T. However, as regards the other variates, it does not differ very much from the « dairy » group except for sweating rate.
d) Repeatabilities
This kind of measurement was not made during the first four years. They were carried out in 1975 in animals of experiment 5 , ( 3 8 animals recorded two times at i month interval). But that year, incomplete data for H.R. and S.T. were obtained. So the repeatability was not calculated for these criteria. Table 6 shows the within breed, between-months, within-year repeatability values, before and after correcting for temperature and relative hygrometry in the room. This correction does not seem to affect very much the repeatability values. They are fairly good and range between 0 . 35 and o.66 according to time and trait. At the end of the stress, the R.R. seems to be the most repeatable ( 0 .66), followed by the sweating rate ( 0 -5 3 ) and the rectal temperature ( 0 . 37 ). But it must be kept in mind that all these values are derived from means of elementary measurements. The repeatability r for one measurement can be extracted from the repeatability Rn for a mean of n measurements : According to this, r is almost constant for RR and S.R. whereas it decreases for R.T. as stress rises (from 0 . 41 to 0.13).
e) Correlations between variates obtained in the hot room
Within genotypes, the variates R.T., R.R., H.R., S.T. are positively correlated ( 0 . 2 -0 . 4 ) especially the first two ones (table 7 ). It can be therefore assumed that the unfitness to stress is described by an overall increase of these variates. The sweating rate seems to be a specific variate independent of the others. Considering that it is somewhat subjective, it might be associated with a bad repeatability. However, the results of table 8 show that the S.R. is as repeatable on the same day as R.T. or R.R. This lack of correlation with the other variates describing the heat stress is surprizing since the impact of thermolysis by evaporation is recognized to be very important at high environmental temperatures. The between genotype analysis does not differ very much from the forementionned results. Once more, S.R. does not seem to be seriously bound to any other variate. The positive correlations between all these other variates are found again, at a somewhat higher level than for the within-genotypic analyses (r = 0 . 4 -o.8).
f) Correlations between the final rectal temperature and other variates not measured in the hot room In order to understand better the origin of the variations between and within genotypes, some parameters were investigated : live weight before test, some body measurements before slaughter, feed intake before test, consumption drop after test and carcass composition. The general means for these variates are shown in table 9 as well as the weighted group effects (their sum is not necessarily zero), the values of the F tests for differences between and within group.
As expected, at the moment of the test when the animals were approximately 14 months old, the males of the « beef » group were the heaviest followed by the crossbred &dquo; Beef X Dairy » males, the « Dairy » males, the « local » males and finally the « Beef x Dairy » females. The males of the « Beef » group were the widest but not the tallest. The differences in body conformation were obvious from these figures. Compacity was maximum for « Beef » and « Beef X Dairy » anirnals and minimum for «Dairy » and ((Local)) animals. The body measurements were collected just before slaughter, they were somewhat biased by the differences in age at slaughter, which were significant between groups because of the differences in fattening procedures. However the maximum difference ranged around o. 5 months.
Measurements of the feed intake show that despite their heavier weight, the cc Beef » or « Beef X Dairy » males ate less than the other males, the lowest consumption being of course found in the females. As the animals generally reduced their consumption after the test, this drop was calculated and estimated as approximately 12 p. 100 of the initial consumption, when measures were made three days after the test. The differences between groups in this respect seemed to be somewhat large but they were not significant. The figures concerning carcass composition were classical, except perhaps for the « Local » males, which seemed to be intermediate between the « Beef » and the « Dairy » group. Before going further, it must be stressed that the so-called « groups » are obviously not homogenous for these « zootechnical » variates, contrary to the « hot room » variates, despite the fact that the groups were precisely based on animal husbandry considerations. This is the reason why we did not try to correlate the differences between groups for resistance to heat to any other betweengroup differences but rather to calculate the correlation directly at the genotype level. The results of the corresponding calculations are shown in table io, together with the calculations at the within-genotype level. The highest correlation coefficients between genotypes and the final rectal temperature were obtained with weight and body composition criteria. From a statistical point of view, the less resistant the genotypes are to heat, the bigger, the compacter, the leaner they are. The correlations with compacity may be evaluated from the correlation between final rectal temperature and height at withers at a given weight (— 0 . 50 ) or between rectal-temperature and width at schoulders on the same basis ( 0 . 71 ). The positive statistical association between rectal temperature and weight or compacity is found again at the within genotypic level, though considerally lower. From an energetical point of view, the concept of metabolic weight is more pertinent than that of weight. However, that transformation is not expected to have affected significantly the result, since within the range of the observed weights, the metabolic weight is a practically linear function of the actual weight. As far as body composition is concerned, the between and within genotypic results do not agree, as no relationship between rectal temperature and body composition was found within genotype. Good agreement at the two levels was obtained for the association between rectal temperature and consumption : it seems to be clear that the more the animals eat, the less resistant they are to heat. The association is moderate (r = 0 . 30 ), but it is to be mentioned that the within genotype variation coefficient for the before test consumption is important ( 12 p. 100 ).
. -Analysis of the incomplete data (five years period)
The only variates regularly measured during these years were rectal temperature and respiratory rate. Table 2 shows that working over a period of five years instead of three introduces supplementary groups (males « beef X local », females « beef », females cc local X cc beef ») and supplementary genotypes in the groups already present such as Blonde d'Aquitaine for the group cc male beef » or such as Charolais X Norman crosses for the group « male beef X dairy ».
The results of the analysis of variance are shown in table II . Although the data were not adjusted for variations in external temperature and relative hygrometry, the significance results of the previously analyzed factors do not vary very much. Besides it has to be noticed that there was never any significant influence of the site ( I to 4 ) inside the hot room.
Analysis of the incidence of genotype-sex factor shows again that there are significant differences between males and females, between males from different genotypes and between females from different genotypes. Between groups of males (cc dairy », « beef X dairy », « beef X local », «local&dquo;) differences are mostly significant. Within the group of males, differences are significant for rectal temperature. This results differs from that obtained during the last three years, but is has to be recalled that the data are not corrected for temperature and hygrometry variations on the hot room. Between groups of females, differences are significantly for rectal temperature. The differences within group for the females have a fluctuating significance level. In figure 7 , comparison between groups of males for rectal temperature shows that the groups « dairy », « beef », « dairy X beef &dquo; differ little. The « local » and « beef X local » groups differ clearly with a lower rectal temperature. Figure 8 confirms that the differences between (( dairy », « beef », « beef X dairy » groups for respiratory rate are not very large.
As to the females, the between-group differences are very large for rectal temperatures. A very high difference can be noticed between the « beef X dairy » and « beef » females while that is not true for the corresponding males. This proceeds probably from the fact that 56 p. I oo of these « beef » females have at least 5 0 p. I oo Blond d'Aquitaine blood, since the results for pure Blond d'Aquitaine animals are better. The « local » females have a lower rectal temperature than that of the « beef » females, what agrees with the results obtained on males.
Discussion
A. -Inter!yetation of the variates In order to evaluate the possible correlation between measurements taken in a hot room and the same measurements in current hot conditions, it would be useful to gather some information about the physiological mechanisms involved in the overall control of the variates as well as about their variability.
It is well known that at high ambient temperatures, the processes of heat loss by conduction and convection are somewhat reduced, because of a decrease in the gradient skin to air temperature when the air temperature increases (K IBLER and B R O DY , 195 2; Me LEAN, I(!63;M ON TE I T H , 1973 ;BI;R BIGZ1;R, 1978). As a consequence, only a small direct effect of variates related to conduction and convection processes can be expected in relation to the thermal equilibrium measured by rectal temperature. In our opinion, these direct effects can safely be measured from the variability occurring within genotypes as they can be confounded with other effects reasoning on a between-genotypic level. Conduction processes as predicted by the percentage of highly insulating tissues (fat) in the carcass are obviously unrelated to rectal temperature (table 10 ) but one estimator of the subcutaneous fat would have been by for more pertinent (B IAN C A , 19 65). The convection processes may be accelerated by a high surface ratio, estimated for weight t I ! ! height at withers ...... j ! . f --i example by the height at withers ratio if it is assumed that surfaces primarily weight t depend on the size of skeleton at least at a within-breed level. Table 10 shows an estimation of -0 . 13 for the correlation coefficient between this ratio and final rectal temperature. Thus, our results seem to confirm a small influence of conduction and convection processes on rectal temperature.
Contrary to sensible heat losses, latent heat losses are expected to increase at high environmental temperatures, especially by sweating and panting (KiBrrEx and B R O DY , rg52; B IAN C A , 19 65). Present results show that these phenomena considerably increase from the beginning to the end of the stress and that, at a given hour of the stress, variations of hygrometries influenced the R.T. by affecting the S.R., a high hygrometry being associated with a high S.R. These overall results are in accordance with those found in the literature (S CHRODE and al., 19 6 0 ; Kisz,!x 1964; BIANCA, 19 65). However, the wide variability of the S.R., within breed and within hour, is never related to the variability in rectal temperature.
This lack of relation cannot be attributed to the lack of accuracy in S.R. measurements as they are highly repeatable from one hour to another. This apparently surprising result may most likely be related to the high hygrometries in the hot room which in addition to high external temperatures involved a high water vapour pressure of 35 mm Hg approximately. In this respect, results of Me LEaN (rg6 3 ) clearly show that an increase of the water vapour pressure (from 8 mm Hg to 35 mm Hg) induces a significant drop (from 150 to 6 0 gm /m 2 /h) in the sweating rate of calves. This physical limitation to evaporation may have adjusted between animal variability of true evaporation.
The heat production processes have also to be considered. They might be at the origin of the clearly positive within genotype correlation on the one hand between final rectal temperature and weight, and between final rectal temperature and feed consumption before test on the other. A high weight should theoretically be associated with a high rectal temperature since the surface /weight ratio and the thermolysis processes relatively to thermogenesis tend to decrease (Bzarrca, ig68). The fact that this relation is found to be positive ( 0 .6 3 ) within and between genotypes demonstrates the incidence of thermogenesis on variability of the results of heat balance. As expected, even more obvious is the influence of the level of feed consumption, demonstrated by the statistical relationship between feed consumption and weight i.e. the level of maintenance needs for energy, and by the direct effect of the amount of feed consumed, through the processes of digestion and absorption of nutrients (I.N.R.A., 197 8). All these figures show the incidence of thermogenesis on the individual variability of heat balance but they are probably underestimated as thermogenesis also depends on other factors than weight and feed consumption. It has been shown that heat production during a short heat stress may be considerably increased by the stress of adaptation (KI B LE R and B R O D Y, 1952; BIANCA, 19 65; Mc LEAN, 19 63), contrary to long term heat stress where heat production slows down (B IAN C A , 19 65; BOND and Mc DOWELL, 1972;SINGH and NEWTON, 197 8) mostly through feed intake decrease (B IANCA , zg65; A LLEN and Dorr!GaN, 1974 ). This phenomenon might have affected our mean results since at the beginning of the stress, the rise in R.T. is nearly as high as during the whole stress. Another indication of that is that for every group of animals (except for the « beef » group) the H.R. decreases during the first part of the experiment resulting in a calming effect, before increasing because of the real heat stress. The share of thermogenesis in stress effects in the individual variability is difficult to establish, but it can be assumed that it is somewhat large as experimental conditions are disturbing enough for aspects other that heat: transfer from the shed to the hot room, lack of feed and water and tiredness due to the standing position. This could explain the better correlation between final R.T. and initial H.R. ( 0 . 30 ) rather than final (o.18), the initial H.R. being probably influenced by the stress.
B. -The between breed differences
The most striking result of our studies is that between genotypes the withingroup differences (dairy, beef or local breeds) are generally non significant (except for S.R.) whereas the between-group differences are significant.
As emphasized for the thermogenesis, such a result has first to be examined in the light of the o animal husbandry differences like weight and feed consumption.
In a first approach, the differences in rectal temperature between the three main groups may be explained by a corresponding difference in weight, the ranking being the same. But this is probably not so easy since the position of the dairy group with respect to R.T. is clearly half way between the beef group and the local group, while it is closer to the local group with respect to weight and feed intake.
It is difficult to speculate on this. Two hypotheses, not exclusive, can be forward.
First, it has been established that in normal heat conditions and at the same weight, the heat production of the dairy breeds is higher than that of beef breeds (V ER -nzoR!r&dquo; Ig76; CO LL UA U , Ig 7 8; LN.R.A.,197 8). Secondly, the beef group could have been more stressed in addition of the heat stress as it is clear that their R.T.
differs from that of the other two groups from the beginning of the experiment. In this experiment, the local breeds are characterized by a low weight and a high S.R. This second factor had probably a minor incidence on the heat balance as shown by the previous figures. Thus, a first approximation suggested that the good resistance of the French local breeds to heat as indicated by a low final R.T. is related to their low weight that reduced the thermogenesis. Contrary to the dairy breeds in the North, it may be objected that these local breeds originate from the central and southern parts of the country, where climate is somewhat hotter in summer, and this might have led to some natural selection. In our opinion, the effect of these hot climates of Southern France must be not be exagerated and it must be recalled that the local breeds are mainly located in hilly and mountainous areas: altitude may cancel the effect of latitude.
C. -Possibility of extrapolation to normal hot conditions . First of all, the present results cannot be extrapolated to any type of cattle production. When we are speaking of dairy breeds, it is for systems (fattening of males, rearing of females) where the animals are not expressing milk production, because at the onset of milk secretion, production of heat is highly increased (LN.R.A.,197 8) and therefore the body core temperature (W OLFF and M ONTY , 1974 ;M ONTY and GARBARENO, 1978).
The intensity and shortness of our test are characteristics that must be stressed upon. It is most likely that factors such as emotive stress have affected some animals, especially those from the beef group. The mechanisms usually developed for long-term adaptation such as the control of feed intake could not be evaluated at all. Some information can be drawn from the decrease in feed intake observed after the test: it is rather confusing that the group resisting the best to stress (the local group) showed the largest decrease, in absolute and relative values, as if it was beginning to adapt its feed consumption to hot conditions. However, this observation could not be proved statistically.
The between-group differences are large enough to be maintained in normal hot conditions, especially the good position of the local group. The real ranking of the beef group relative to the dairy group is somewhat more questionable as shown in our previous discussions.
A.s already mentioned, no significant within group differences between breeds could be found showing that there is no effects of heterosis either, according to the rather large fraction of crossbred animals studied. Such an observation can probably be made in actual hot conditions. If not, it may be due to differences in S.R. for the groups are not homogenous in this respect and these differences did not have any efficient impact in our experimental conditions.
Conclusion
It appears from the current analyse that in our experimental conditions some French local breeds may be resistant to heat, that is to say maintain their core temperature at a lower level. This supports frequent observations made in that field. Improved breeds for either milk or beef production seem to have difficulties in keeping constant their mid body temperature, what is usually considered as a bad adaptation to heat. Another very important result of the present study is that within each large group (improved for dairy or beef and local) neither between breed variability nor heterotic effects seem to occur, at least within our system of testing and the statistical efficiency of our data analysis. Though the test might be largely improved by using for example a pre-experimental in the non heated room, long enough to prevent any emotional stress, the general pattern of our results, between group speaking, could well be observed in practice because in our conditions the heat balance variability was primarily related to the variability in heat production. That factor was really demonstrated to be predominant for explaining some differences in heat tolerance in actual conditions, for example between Bos Taurus and Zebus (J OHNS T ON and al., i 95 6; F RI SC H and VE R C O E, 1977 ). However, our test is probably too inaccurate to discriminate possible heat resistant genetic types from other contemporary types from the same group.
Unfortunately French local breeds are generally small and less productive than the commonly used European cattle breeds and their poor production ability must be considered together with their ability to resist to heat. Furthermore, the capacities of an animal to maintain low core temperature under hot conditions are usually considered as criteria of heat tolerance but they are not necessarily criteria of economic profitability under hot conditions. Thus, the local breeds have to demonstrate their overall economic superiority, which may be the case at least for some peculiar cases, as shown for example by C ASU and al. ( 1975 ).
These authors reported that the crossbreds between Charolais and Sardinian breed (a local Italian breed) could be superior to pure Charolais. In very hot and dry conditions, it is likely that the European local breeds should have to be compared not with the improved European breeds, but with some special genetic combination, « Bos taurus x Zebu for example. If they do not lead to better results, it is not even sure that they would be valuable in these genetic combinations, as FxiscH ( 1977 ) showed that the ranking of European breeds in hot conditions might vary according that they are pure or crossed with Zebu cattle. | v3-fos |
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} | s2 | Effect of low protein diets on free amino acids in plasma of young men: effect of wheat gluten diet.
Studies were made on alterations in plasma amino acids in young men fed a diet containing graded levels of wheat gluten. After one week on a standard diet containing 200 mgN/kg of mixed protein (animal protein content 45%), 38 young men were given a wheat gluten diet containing 170, 100, 60, 30, 15 or zero mgN/kg for 2 weeks. Blood samples measuring plasma free amino acids were taken before breakfast at the end of the periods on a standard diet and an experimental diet. In subjects on diets containing 170 to 30 mgN/kg the plasma concentrations of threonine, valine, methionine, leucine, tyrosine, phenylalanine, serine, histidine and arginine fell significantly with decrease in protein intake, but the concentration of alanine increased significantly. On the other hand, in subjects on diets containing 15 or zero mgN/kg, the plasma concentrations of the essential amino acids did not decrease, but increased to slightly more than in subjects on a diet containing 30 mgN/kg, and the alanine and glycine concentrations increased steadily. Values for plasma lysine varied from 146 +/- 22 to 194 +/- 31 mumoles/liter with gluten intakes of 170 to zero mgN/kg, but were comparable with that of 186 +/- 33 mumules/liter in subjects on a standard diet, showing that the plasma lysine concentration did not clearly reflect the dietary concentration of lysine in young men on a wheat gluten diet.
Summary
Studies were made on alterations in plasma amino acids in young men fed a diet containing graded levels of wheat gluten. After one week on a standard diet containing 200mgN/kg of mixed protein (animal protein content 45%), 38 young men were given a wheat gluten diet containing 170, 100, 60, 30, 15 or zero mgN/kg for 2 weeks. Blood samples measuring plasma free amino acids were taken before breakfast at the end of the periods on a standard diet and an experimental diet. In subjects on diets containing 170 to 30mgN/kg the plasma concentrations of threonine, valine, methionine, leucine, tyrosine, phenylalanine, serine, histidine and arginine fell significantly with decrease in protein intake, but When a diet deficient in an essential amino acid is given to animals, the concentration of the missing amino acid in plasma decreased (1)(2)(3)(4). JACOBS and CRANDALL (5) reported that administration of wheat gluten to growing rats caused an appreciable decrease in the free lysine concentration in the plasma and inhibited growth. GRAHAM et al. (6) showed that plasma lysine in children is greatly influenced by the dietary lysine level. As we reported previously (7), when young men were given a low egg or rice protein diet for three weeks, their plasma concentrations of lysine and other essential amino acids (EAAs) decreased significantly. Unpublished data from our laboratory, however, showed that the concentration of free lysine in the plasma and tissues did not decrease in adult rats fed diets containing different levels of wheat gluten for four weeks. Furthermore, in starving chicks (8,9) and sheep (10) and protein-deprived men (11) the plasma lysine concentration remains normal or increases. Moreover, EDWARDS et al. (12) showed that plasma free lysine was not affected significantly by supplying a wheat gluten diet to human adults. These conflicting results suggest that there is a difference in the responses of the plasma lysine concentration in growing and adult animals and humans to a wheat gluten diet.
The primary object in this study was to gain more detailed information on changes in the plasma amino acid concentrations, particularly lysine, in young men on diets containing various levels of wheat gluten. To clarify the results obtained with wheat gluten, we also examined plasma aminograms of subjects fed diets with adequate protein and a protein-free diet.
EXPERIMENTAL
The subjects were 38 healthy male university students 19 to 24 years of age. They were given a standard diet containing 200mgN/kg of mixed protein (animal protein content 45%) for one week and then diets containing graded levels of wheat gluten or a protein-free diet for 2 weeks. The level of wheat gluten in the diet was 170mgN/kg for 4 subjects, 100mgN/kg for 3, 60mgN/kg for 3,30mgN/kg for 10 and 15mgN/kg for 9 subjects. Nine subjects were given a protein-free diet. Throughout the experiment the energy intakes of the subjects were maintained at 47 to 50kcal/kg, which is sufficient to meet , energy requirements. The energy intakes of the subjects on a protein-free diet were excessively maintained at about 54kcal/kg to obtain the least level of endogenous N excretion, though the excess energy supply has little influence on N output of the subjects fed a protein-free diet (13). The compositions of one wheat gluten diet and the protein-free diet are shown in Table 1.
Plasma amino acid analysis was carried out at the end of the standard diet and at the end of the experimental period. Blood samples were withdrawn from the antecubital vein before breakfast. The procedures and methods for amino acid analysis were essentially as reported previously (7).
RESULTS
Body weight and nitrogen balance Results of body weight and nitrogen balance of the subjects fed graded levels of wheat gluten are shown in Table 2. Throughout the experiment the energy intakes of the subjects were maintained at about 54kcal/kg for the protein-free group and at 47 to 50kcal/kg for the other groups, which is sufficient to meet energy requirements. However, their body weights tended to decrease in the experimental periods the amounts being -0.89, -0.28, -0.75, -0.21, -0.60 and 0.97kg/2 weeks at N-intakes of 170, 100, 60, 30, 15 and zero mgN/kg, respectively. In addition, their nitrogen balances were negative irrespective of the dietary N levels, even in the 170mgN/kg group. Plasma amino acid pattern in subjects on a protein free diet The plasma amino acid pattern of subjects on a protein-free diet is shown in Table 4, with that for subjects on a standard diet for comparison. Characteristic changes appeared within one week and these alterations were then maintained. Findings were as follows: branched-chain amino acids, especially valine, decreased by about 25%. Alanine and glycine increased markedly by 73% and 37%, respectively, by the end of the 2-week experimental period. Lysine was maintained at nearly the standard level. As a result, after two weeks the value for total EAA had decreased by about 18% and that for total NEAA had increased by about 39%, resulting in a decrease of the E/N ratio from 0.98 in the control period to 0.58 in the period on a protein-free diet.
Effect of wheat gluten diet on the plasma amino acid pattern As described previously (7), the N balance was negative, even when the protein intake from wheat gluten was about 1.0g/kg. The dietary lysine intakes were calculated to be 1.6, 3.3, 6.5, 9.8 and 18.5mg/kg/day at N-intakes of 15, 30, 60, 100 and 170mg/kg, respectively. Regression analysis showed that the lysine require ment for zero maintenance was 24.3mg/kg, although from results with an amino acid mixture the safety intake was calculated as 11.4mg/kg by ROSE et al. Table 3, the plasma lysine concentration in subjects on a gluten diet were less than those in subjects on a standard diet. The concentration at N intakes of 100,60 and 15mgN/kg did not decrease significantly, the mean values remaining at about 90% of the standard value. There was no significant correlation between lysine intake and the plasma lysine concentration, as seen in Table 5. These findings suggest that in human adults receiving a wheat gluten diet, lysine deficiency is not reflected in a decrease in the lysine concentration of the plasma.
Apart from lysine, individual EAA tended to decrease with increase in protein deficiency and the decrease in valine was greatest, as observed previously. Total EAA also decreased significantly with decrease in protein intake, being about 30% lower than the standard value at N-intakes of 30 and 15mg/kg. Among the NEAAs, alanine increased most (about 50%) at N-intakes of less than 30mg/kg, although this change was less with higher N-intakes. The changes in serine, glycine and other NEAA were not marked. Thus, a significant increase in total NEAA was observed only in subjects on diets with very low gluten contents. The mean E/N ratio ranged from 0.74 to 0.55 in the experimental period and the values were significantly lower than in the standard period. The relationships between the plasma levels of free amino acids and the dietary N intakes for diets of 30 to 170mgN/kg were tested statistically (Table 5). Most EAAs except lysine changed significantly in parallel with the N intake, but a significant inverse correlation was observed for alanine. A highly significant correlation was also found for serine, although it was not observed with 200mgN/kg (standard diet). There was no significant correlation between the E/N ratio and gluten level, although the total EAA changed significantly in parallel with the N intake.
The following points are also of interest: The lowest values for total EAA and total NEAA were obtained with 30mgN/kg, and the values increased progressively with a further decrease in protein to 15 and zero mgN/kg, as seen in Fig. 2. The values for EAA and NEAR of the protein-free group differed significantly from those for the group on 30mgN/kg, but not from those for the group on 15mgN/kg, due to the high variations of threonine, valine, lysine, serine, glycine and alanine, in the latter group, which made its amino acid pattern similar to that of the protein-free group.
DISCUSSION
The standard aminogram for Japanese adults obtained in this study is comparable with those of WELLER et al. (11), ADIBI (15) and others (16)(17)(18) and with those for Japanese adults by OKUDA et al. (19), despite differences in the diets of the subjects examined. The A/E ratio calculated from this standard aminogram is different from that of egg proteins and from the provisional amino acid scoring pattern proposed by FAO/WHO 1973, which was estimated from human amino acid requirements (20) ( Table 6). We showed previously that in young men on a low egg or rice-protein diet, essential amino acids in the plasma tended to decrease, while some nonessential amino acids increased (7). Similar results were also found in subjects receiving low levels of wheat gluten. fi ndings are consistent with the report of YOUNG et al. (21) that the concentration of plasma lysine, unlike those of valine and other EAAs, was not correlated with a decrease in lysine intake below 7mg/kg. We also found that a protein-free diet had very little effect on the plasma concentration of lysine, as reported by others (9,11), and observed in young men on a diet containing a lysine-free amino acid mixture (21). As is well known, when growing animals were given a gluten diet, their plasma lysine concentration decreases to a very low level and increases again when lysine is added to the diet (5,22). MCLAVGHLAN and ILLMAN (23) reported that the plasma lysine level correlated with the dietary lysine level in estimating the amino acid requirements of growing rats. This indicates that the plasma-dietary lysine relationship observed in growing animals must be basically different from that observed in adults. FISHER et al. (16) recently found that the lysine requirement for adults was considerably lower than that reported in earlier studies. Moreover, the lysine requirement for maintenance is considerably lower than that for growth (24). In general, the plasma concentration of lysine was affected less than those of other EAA by protein deficiency. This may be explained by the following considerations. l) A normal or rather increased concentration of plasma lysine was demonstrated in starving chick (8,9) and sheep (10) and protein-deprived men (11), in which tissue breakdown was increased. In subjects on gluten diets, tissue proteins may be catabolized to compensate for the poor amino acid composition of the gluten, as indicated by BANKS et al. (22). Since, of the EAAs in tissue proteins, lysine is present in the highest concentration (25,26), breakdown of tissue proteins should resultt in a higher level of lysine than of other EAAs in the free amino acid pool. 2) Free lysine is metabolized in the tissues more slowly than other EAAs (27), and YAMASHITA and ASHIDA (28) found that in lysine deficiency, its breakdown decreased to half the control rate.
When protein intake is restricted to less than 30mgN/kg, plasma EAAs tend to increase gradually with increase in severity of protein depletion and total NEAAs increase further, as shown in Fig. 2. Changes in plasma free amino acids differed above and below 30mgN/kg of gluten intake. These findings for plasma free amino acids are consistent with our previous findings in nitrogen balance studies (29) that the slopes of regression equations between nitrogen balance and nitrogen intake become steeper below 30mgN/kg gluten intake. On the basis of data on plasma amino acids and results of nitrogen balance studies, we concluded that a protein intake of 30mgN/kg was of biological significance. Thus, the relationship between the increased concentration of plasma free amino acids and the efficiency of protein utilization at intakes of less than 30mgN/kg was studied further. The present study provides further evidence for the nutritional significance of plasma free amino acids in nitrogen balance studies reported previously (29,30). | v3-fos |
2018-12-07T03:20:13.624Z | {
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} | s2 | Using Foliar and Planting-time Insecticides to Control Chinch Bugs in Grain Sorghum
Gerald Wilde and Terry Mlze Chinch bug infestations have been more widespread and intense in eastern Kansas in recent yea rs than since the outbreaks of the 1930's and 50's. Field insecticide tests in I 977 indicated that correctly applying foliar insecticides significantly reduces chinch bug numbers, and greenhouse tests suggested that some granular treatments at planting time will control chinch bugs on seedling sorghum, so tests were continued in 1978. In the first test, planted June 5, a granular application at planting time was evaluated 8 and 21 days later (Table 1). The most effective treatment on day 8 (June 13) was an in-furrow treatment of carbofu ran. No band treatments gave good control. At 21 days (June 26) carbofuran in-furrow or a band gave some control, but left some chinch bugs surviving on all plants. In th'!l second test, planted June 29, planting-time applications were evaluated 14 days later (Table 2). Infurrow treatments of carbofuran and bands of or phorate significantly reduced chinch bug numbers. A 1.2-inch rain 1 I days after planting probably accounted for the effectiveness of the band treatments in the second test. 1. Mention of a compound and results do not constitute a recommendation by the Kansas Agricultural Experiment Station or the Kansas Cooperative Extension Service.
MARCH 1979
Using Foliar and Planting-t ime Insecticide s to Control Chinch Bugs in Grain Sorghum 1
Gerald Wilde and Terry Mlze
Chinch bug infestations have been more widespread and intense in eastern Kansas in recent yea rs than since the outbreaks of the 1930's and 50's. Field insecticide tests in I 977 indicated that correctly applying foliar insecticides significantly reduces chinch bug numbers, and greenhouse tests suggested that some granular treatments at planting time will control chinch bugs on seedling sorghum, so tests were continued in 1978.
In the first test, planted June 5, a granular application at pla nting time was evaluated 8 and 21 days later ( Table 1). The most effective treatment on day 8 (June 13) was an in-furrow treatment of ca rbofu ran. No band treatments gave good control. At 21 days (June 26) carbofuran in-furrow or a band gave some control, but left some chinch bugs surviving on all plants.
In th'!l second test, planted June 29, planting-time applications were evaluated 14 days later (Table 2). Infurrow treatments of carbofuran and bands of or phorate significantly reduced chinch bug numbers. A 1.2-inch rain 1 I d ays after planting probably accounted for the effectiveness of the band treatments in the second test.
1. Mention of a compound and results do not constitute a recommendati on by the Kansas Agricultural Experiment Station or the Kansas Cooperative Extension Service. Of the granular tests, the carbofuran in-furrow treatment is registered for use on sorghum for ch in'ch bug control. Both carbofuran and phorate band treatments are registe red for use on so rghum for greenbug contro l, but not fo r chinch bugs. Disu lfoton, reg iste re d as a band treatment for g reenbug control, does not affect chinch bugs. Phorate should not be applie d in-furrow a t pl anting time because of its phytotoxicity .
AGRICULTU RAL EXPERIMEN T STATION
Foliar a pplications of carbaryl, ca rbofuran, and e ndrin were tested on seedling sorghums, though endrin is not registered for sorghum crops. The other two are registe red for chinch bug control. We included it as a chlorinated hydrocarbon to which control with newer inse cti· cides can be compared. The fo liar sprays we re applied with a one-nozzle hand sprayer del ivering 16 gal. of water per acre. Results of two tests at Abilene (Table 3} and one test at Manhattan (Ta ble 4) indicate th at all those insecticides significantly reduced ch inch bug num- bers. ' But their residual poison does not extend more than 3 days, so chinch bugs migrating into a sorghum field could make repeated applications necessary.
The :most important point in controlling chinch bugs with foliar sprays is to use drop nozzles or other methods to direct sprays to the base of plants where bugs congregate and to use as much water as possible, preferably at least 16 to 20 gallons per acre.
Most Important In applying carbofuran at planting time is application In the seed furrow, which is more effective than the b and treatments because it does not require a rain for treatment to control ch inch bugs. | v3-fos |
2019-03-22T16:06:25.792Z | {
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} | s2 | Cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses
Cross-protection studies between the feline infectious peritonitis (FIP) and the porcine transmissible gastroenteritis (TGE) viruses were conducted in cats, pigs and pregnant gilts. Cats vaccinated with TGE virus developed neutralizing antibodies against TGE virus and low titer antibody against FIP virus detected by an indirect fluorescent antibody technique but were not protected against a virulent FIP virus challenge. Baby pigs and pregnant gilts vaccinated with FIP virus did not develop detectable antibodies to TGE virus. Nevertheless, it appeared that vaccination of swine with FIP virus conferred some immunity against TGE virus infection. Seventeen-day-old pigs vaccinated with two doses of FIP virus had a 67% survival rate following a virulent TGE virus challenge, and 75% of the 3-day-old pigs suckling either FIP or TGE-virus-vaccinated gilts survived virulent TGE virus infection in contrast to 0% survival of baby pigs suckling unvaccinated gilts.
INTRODUCTION
The porcine transmissible gastroenteritis (TGE) virus is serologically related to the canine corona virus (Binn et al., 1974), feline infectious peritonitis (FIP) virus (Osterhaus et al., 1977;Reynolds et al., 1977;Witte et al., 1977;Pedersen et al., 1978), and human corona virus 229E (Pedersen et al., 1978). Of these viruses, the antigenic relationship between TGE and FIP viruses is especially strong (Pedersen et al., 1978). Because of this strong antigenic relationship, it is logical to assume that infection with one virus might confer protection against infection with the other virus. Preliminary studies suggest that TGE virus will not protect cats against FIP virus infection (Witte et al., 1977), but no information is available on the protective effect of FIP virus vaccination on baby pigs and pregnant gilts infected with virulent TGE virus.
The purpose of this report is to present information on the cross-protective effect of TGE virus vaccination on FIP infection of cats and FIP virus vaccination on TGE infection of swine.
Viruses
The virulent Miller No. 3 strain of TGE virus (2.5 X lo6 plaque forming units (pfu)/ml) and an attenuated small plaque variant TGE virus (2 X lo6 pfu/ml) were used (Woods, 1978). The virulent UCD-1 strain of FIP virus was prepared as a homogenized suspension containing the equivalent of 2 g of liver per ml of suspension from kittens infected experimentally (Pedersen, 1976a). Bioassays have not been developed that permit an actual determination of the FIP virus content.
Experimental animals and vaccination
Two cats were given three doses of virulent TGE virus intraperitoneally (IP) according to the protocol given in Table I, and two cats were held as uninoculated controls. A total of 27 newborn pigs, less than 6 h old, were tested (Table II). Of those, three were given orally (O) 2 × 106 pfu of attenuated TGE virus. 15 were given 0.15 g-equivalents of FIP virus IP and 0.15 g-equivalents intranasally (IN); nine of those 15 pigs were given a secJnd FIP dose 7 days later by the same protocol. The other nine pigs were held as uninoculated controls.
Two gilts were given 2 g-equivalents of FIP virus 8 and 2 weeks before parturition; one half of the dose was given O/IN and one half was injected into the mammary glands at four sites (Table III). Two gilts were given 2 X lo6 pfu of attenuated TGE virus by the same vaccination protocol. Two gilts were held as uninoculated controls.
Challenge
Pigs were 0 challenged with about 2000 pig infectious doses of virulent TGE virus. Cats were challenged IP with 0.5 g-equivalents of FIP virus (Pedersen, 1976a).
Antibody titer
Blood sera from cats, pigs and gilts were tested for their TGE antibody content by a plaque reduction technique (Woods, 1978). The FIP antibody content was determined by an indirect fluorescent antibody technique (IFA) (Pedersen, 1976b).
Cats VFI and VD5, vaccinated with virulent TGE virus, developed virus neutralizing (VN) antibodies
(1:67-l: 309) against TGE on day 45, after three injections of vaccine (Table I). These cats also developed FIP reacting antibodies (1: 2-l: 8) before exposure to the FIP virus. Following inoculation with FIP virus, both TGE-virus-vaccinated and unvaccinated cats developed effusive FIP and were killed 18 days post-challenge.
Pigs vaccinated with attenuated TGE virus developed VN antibodies against TGE, but unvaccinated pigs and pigs infected with FIP virus did not demonstrate TGE antibodies (Table II). The survival rate of pigs vaccinated with a single dose of FIP or TGE virus was 33 and OS, respectively; that of pigs vaccinated with two doses of FIP virus was 67% and that of unvaccinated controls was 22%.
No clinical signs were observed in pregnant gilts vaccinated with either FIP of TGE virus. Two gilts vaccinated with attenuated TGE virus had VN antibodies against TGE virus, but the two gilts vaccinated with FIP virus and the two controls gilts did not have detectable VN antibodies against TGE virus (Table III). The survival rate of baby pigs suckling FIPvaccinated gilts was 75%; that of pigs suckling TGE-vaccinated gilts was 71% and that of pigs suckling unvaccinated control gilts was 0%. A mild diarrhea was observed in about 60% of the protected pigs, but vomiting and dehydration were not observed. FIP and TGE-virus-vaccinated gilts did not develop clinical signs of TGE after their suckling pigs were 0 challenged. However, one of the unvaccinated control gilts did develop typical signs of TGE following infection of her suckling offspring.
DISCUSSION
The serological relatedness of these two coronaviruses has been previously shown, but this is the first reported attempt to use these viruses as naturally occurring vaccine strains. Cats vaccinated with TGE virus developed high titers of VN antibody against TGE virus. TGE virus antibody cross-reacting with FIP virus was detectable at low titer only after three injections of TGE virus. The pronounced differences in the titers and speed of appearance of TGE VN antibody and FIP-virus-reacting antibody indicates that these two antibody activities differ from each other. TGE antibody that reacts with FIP virus in the IFA assay is probably not virus neutralizing, as cat sera with high IFA titers to FIP virus do not neutralize TGE virus (Osterhaus et al., 1977).
The fact that TGE-vaccinated cats developed antibodies that cross-reacted with FIP virus indicates that at least one antigen is shared by these two viruses. Not only did TGE-virus-vaccinated cats develop low levels of FIP virus reacting antibody, but they also underwent an anamnestic response when challenged with FIP virus. Cats vaccinated with TGE virus had high levels of FIP virus reacting antibodies 6 and 9 days post-challenge with FIP virus, while the unvaccinated control cats did not develop detectable levels of FIP antibodies until 12 days post-challenge.
Attempts to elicit active immunity in 6-h-old pigs with FIP or TGE virus produced inconclusive results. Protection, as measured by mortality, is difficult to evaluate in pigs over 14 days of age because of the rapid onset of resistance to TGE after this time (Moon et al., 1975). In this study, a single dose of FIP or TGE virus did not protect the pigs; whereas, two doses of FIP virus appeared to provide some protection against virulent TGE virus challenge. Because of the small number of pigs used in this study, these results need to be confirmed.
The survival rate of pigs suckling either FIP or TGE-vaccinated gilts was about 70%. Although this is lower than the survival rate of baby pigs born to sows that had undergone a natural infection, it is still high enough to merit further investigation. These results suggest that the FIP virus may confer some degree of colostral immunity against TGE virus infection of suckling pigs. It is hoped that these findings can be confirmed in larger numbers of pigs, and that methods can be developed that would allow quantitation of the FIP virus so more meaningful comparisons can be made.
The phenomena described in this report appears similar to that reported in swine with the Pestiviruses (bovine viral diarrhea (BVD) and hog cholera (HC)). The BVD--HC virus complex appears to be related to the strain of BVD immunizing virus and the Ames challenge strain of HC virus (Snowdon and French, 1968;Stewart et al., 1971). In unpublished studies using agar gel diffusion, at least one antigen shared by TGE and FIP was soluble. However, because this is a preliminary report and only one strain of FIP virus was used, much additional information needs to be generated to clarify the TGE--FIP complex. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Carcinogenicity of betel quid ingredients: feeding mice with aqueous extract and the polyphenol fraction of betel nut.
Male mice of inbred strains Swiss and C17 were fed daily 5 times a week by intragastric tube 0.1 ml of betel-nut aqueous extract, betel-leaf aqueous extract and the polyphenol fraction of betel nut. Male mice of corresponding strains fed 0.1 ml of distilled water served as controls. Treated and control mice were kept under observation and killed when moribund. Betel-nut aqueous extract induced tumours of the gastrointestinal tract in 58% Swiss mice and 25% C17 mice. The polyphenol fraction by the same route induced tumours at other sites in 17% of the mice. Betel-leaf aqueous extract failed to induce any tumour in the treated mice, which supports an earlier report of the lack of any carcinogenic principle in betel leaf, an essential constituent of betel quid. Results are discussed in relation to the relevant literature. ImagesFig. 1Fig. 2Fig. 3Fig. 4
IN INDIA and the Far East the habit of betel chewing is a major factor in the cause of oral cancer. Many attempts have been made to develop a theory of the origin of betel-chewer's cancer, based on the chemical constituents of the chew.
The chew or betel quid consists primarily of a few pieces of areca nut wrapped in the leaf of the betel vine, together with some lime. According to Tenneckoon & Bartlett (I 969), lime might have an irritant action, but was used in such small quantities that dilution by saliva rendered it innocuous. In some localities certain other ingredients such as catechu and tobacco may be added, but they are not essential constituents of betel quid. In any case, similar pathological changes have been found in the absence of these ingredients (Pindborg et al., 1968). Our preliminary studies on the aqueous extract of betel nut and its polyphenol fraction have shown that both produce a high percentage of fibrosarcomas at the site of injection in Swiss mice. Betel-leaf aqueous extract by s.c. injection, however, failed to produce any tumours (Ranadive et al., 1976;Ranadive & Gotboskar 1.978). To simulate human conditions more closely we have tested betel-leaf aqueous extract, betel-nut aqueous extract and polyphenol extract of betel nut by gavage feeding.
MATERIALS AND METHODS
Male mice of two inbred strains, Swiss and C17, were fed by intragastric tube 0-1 ml of aqueous extracts of betel nut and leaf, and also 0-1 ml of the polyphenol extract of betel nut, daily 5 times a week. Feeding -,A,as started at the age of 8-10 weeks and continued throuo,hout the life-span of the treated animals. The following experimental groups, were maintained. Betel-nut aqueous extract and the polyplienol extract (Shivapurkar et al., 1978) Betel-nut aqueous extract Of the 21 Swiss mice in this group, 7 developed liver tumours (33%), out of which 5 were hepatocellular carcinomas ( Fig. 1) and 2 haemangiomas. Five other mice developed tumotirs at other sites, 2 being lung adenocarcinomas, I a squamouscell carcinoma and I an adenocarcinoma of the stomach (Fig. 2), and I leukaemia.
Of the 30 C, 1 7 mice fed betel-nut aqueous extract, 3 developed squamouscell carcinoma of the fore-stomach (Figs. 3 and 4) and 2 adenocarcinomas of the glandular stomach. In addition I developed lung adenocarcinoma and 2 leukaemia.
repeatedly with 100ml aliquots of distilled -xA,ater on an automatic shaker. The combined extract was lyophilized and the dry residue -%A,as dissolved in 10 ml distilled -vN-ater. For quantitation of the extract, arecoline content ,%A,,as measured by the method described by Sharp (1931) and polyphenol content by the method described by SAan & Hills (1959).
0-1 ml of the aqueous extract was found to contain 1-5 mg of arecoline and 1-9 mg of polyphenol (measured as tannic acid). The polyphenol fraction was prepared by vigorously shaking 100 g of betel-nut powderwith 150 ml of ethyl acetate (containing 8 ml of ethanol/100 ml of ethyl acetate) for 4 h with an automatic shaker. The extraction was repeated several times and the combined extracts A%,ere treated NN-ith 0-IN HCI to reinove anv alkaloid impurities. The purified fraction N%,as lyophilized and the dry residue dissolved in 10 ml of distilled water. The purity of this preparation was checked by silica-gel thin-layer chromatography NNrith arecoline as the reference substance. I't was then diluted 10 times, for treatment. The amount of total polyphenols, measured as tannic acid, was 1-9 mg in 0-1 ml of diluted extract.
Pre,parations of betel-leaf extract.-100 g of bete] leaves were ground with 150 ml distilled AN-ater in a grinder and kept at 4'C for 24 h.
Polyphenolfraction
Of 18 Swiss male mice fed the polyphenol fraction, 2 developed tumours of the salivary gland and I haemangioma of the liver.
DISCUSSION
The present studies attempt to simulate the situation in humans, in which the oral and oesophageal squamous epithelium is in contact with betel nut before it reaches the glandular stomach. Rodent gastric mucosa is pre 'sumed to be the counterpart of the human oesophagus, in which large numbers of tumours are reported in betel-nut chewers (Jussawala & Deshpande, 1971).
The above data have shown that betelnut aqueous extract (BN) induced a sub-stantial number of tumours of visceral organs such as liver, lung and GI tract in treated mice. However, treated mice of the C17 strain failed to develop any liver tumours, whereas 33% of betel-nutextract-treated Swiss mice developed liver tumours. This may be because the liver tissue of Swiss mice is more susceptible to even weak carcinogenic activity than that of C 1 7 mice. We have reported a significant number of liver tumours in Swiss mice treated with relatively weak carcinogens, such as thioacetamide (Date et al., 1976). It is possible that C17 mice lack the necessary enzymes for activation of the carcinogens, or for the formation of proximal carcinogens from the betel-nut aqueous extract.
The tumorigenic effect of betel-nut extract injected s.e. in Swiss mice has already been reported from this laboratory (Randive et al., 1976). By contrast the feeding of aqueous betel-leaf extract was not able to induce any tumours in the present experiments. These observations support those of an earlier report from this group, in which it waa shown that betelleaf extract injected s.e. in Swiss mice failed to induce any tumours (Randive & Gothoskar, 1978). Further studies on these extracts have shown that betel-leaf extract even exerts a protective effect in Swiss mice when injected simultaneously with betel-nut extract (unpublished data). It is also interesting to note that feeding of betel-nut extract produced a significant number of tumours of the gastrointestinal could not detect any phenol degradation products in the urine of rats fed sericea grape tannins, and they tentatively concluded that there was little if any absorption of the anthocyanidin polymer per se or its degradation products from the intestinal tract. Tumours observed in the betel-nut-fed Swiss and C17 mice could be attributed to some constituents in betel nut other than tannins, e.g. alkaloids.
Alkaloids from different plants which are consumed either as food or folk medicine by the natives of various regions in the world are reported to be carcinogenic. Indepth studies on the alkaloids in betel nut (viz. arecoline) are under way using the oral route, and will be reported later. | v3-fos |
2020-06-04T09:08:57.345Z | {
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} | s2 | A PRELIMINARY STUDY OF VEGETATION ZONES AND WINTER BIRD DISTRIBUTION IN SASKATCHEWAN*
The distribution of winter birds varies considerably across the 11 degrees (and 1220 km) of latitude that encompass present day Saskat¬ chewan. This latitudinal spread in¬ cludes vegetation zones that range from short-grass prairie through deciduous and coniferous forests, to the sub-arctic transition. Compara¬ tive information on bird composition among these vegetation zones in all seasons is sparse. The best informa¬ tion for the early winter period is pro¬ vided by 946 Christmas Bird Counts conducted from 1940 to 1976.
The distribution of winter birds varies considerably across the 11 degrees (and 1220 km) of latitude that encompass present day Saskat¬ chewan. This latitudinal spread in¬ cludes vegetation zones that range from short-grass prairie through deciduous and coniferous forests, to the sub-arctic transition. Compara¬ tive information on bird composition among these vegetation zones in all seasons is sparse. The best informa¬ tion for the early winter period is pro¬ vided by 946 Christmas Bird Counts conducted from 1940 to 1976.
In an earlier analysis of Christmas counts, bird distribution maps based on geographic units (degree blocks) suggested that the frequency of occurrence of most species was markedly influenced by vegetation zones.5 6 In this paper we use these counts to assess apparent pre¬ ferences for specific vegetation zones exhibited by the more regular species, and characterize the species composition of each zone.
Methods
We assigned each count locality to one of the vegetation zones recog-* Part II of a 35 year review of Christmas Bird Counts in Saskatchewan. Part I appeared in the December 1977 Blue Jay 35: 224-239. nized in the Atlas of Saskatchewai (see Figure 1 ).7 In addition to these si: vegetation zones, we have includec the Cypress Hills region as a seventf zone since it has several unique physiographic and vegetationa1 features. Only one count (Fort Walsh has included extensive mixed wooc forest typical of the upper elevation.' of the hills. However, counts a Eastend, Maple Creek, Piapot anc Skull Creek are likely under the in fluence of the hills because they in elude streams and their associatec riparian habitats that originate in th< Cypress Hills; hence, these localities have been included in the Cypres! Hills region. We then calculated the frequency of occurrence, during count period for each species within each zom (i.e., the percent of total count! recording the species). Forty-six com mon species are shown in Figure 2 Given this information, it was possible to obtain an index of species rich ness within each zone (Table 1).
The index used is the number o species recorded on more than 25°/1 of the counts. We felt that, with the modest degree of coverage on mos counts, all of these species likeb would be recorded on at least half o the counts had the count circles beer« covered by larger numbers o observers (20+) as in the larger cities, )rt Walsh count, located in the West c ock of the Cypress Hills, has been ?! e only locality other than Saskatoon r id Regina where more than 30 \ )ecies have been recorded in a i ngle day. Twenty-one of the 46 ; )ecies represented in Figure 2 were I corded at least as frequently in the rl /press Hills as in any other vegeta-)n zone.
Somewhat lower species richness as exhibited by the aspen parkland id the mixed wood zones, each with ) index of 17 species. There are pmerous examples of small parties these areas recording relatively >od counts. In 1952, four people in o parties observed 25 species on •unt day and three other species during count period in the Nipawin-Fishing Lakes area on the aspen parkland/mixed wood boundary. In 1954, five observers recorded 17 species on count day and 13 addi¬ tional species during count period in the Nipawin-White Gull Creek area. And between 1951 and 1976, two people frequently recorded 20 to 25 species during count period at Somme, a farming community on the southern edge of the mixed wood forest.
Species richness appeared to be lowest in the sub-arctic forest tundra transition, the northern coniferous forest and in the prairie zones. Although the number of counts in the first two of these zones has been small (5 and 4, respectively), the small number of species observed is in general agreement with the depau¬ perate winter bird life reported for these areas by others.2 4 cember, 1979. 37(4) Ten of the 34 species in Table 1 were seen on more than 25% of the counts in only one vegetation zone. Five of these 10 restricted-distri¬ bution species were in the Cypress Hills (Tree Sparrow, Northern Shrike, Rough-legged Hawk, Prairie Falcon, American Robin), four were in the northern coniferous forest (Spruce Grouse, Pileated Woodpecker, Boreal Chickadee, Hoary Redpoll) and one was in the mixed wood forest (White¬ breasted Nuthatch). Only two species, Black-capped Chickadee and Common Redpoll, were included in the index for each of the vegetation zones. The Black-billed Magpie and House Sparrow were indexed in six of the seven zones (not in sub-arctic), and the Hairy Woodpecker was seen in all seven zones but was seen on more than 25% of the counts in only five zones.
Discussion
Within the province, the frequency of occurrence of species on Christ¬ mas counts differs among vegetation zones. This is probably at least in part a function of the habitat diversity present in each zone. The number of; habitats in an area depends upon both the natural environment and the nature and extent of man-inducedl environmental changes. As long as human activities do not destroy the last examples of any natural habitat, areas modified by human activities would provide a much greater variety: of habitat types in which an artificially high degree of species diversity would exist.
In recent years the greatest number of species has been consistently recorded on Christmas Bird Counts in the two largest cities, Regina (located in the mid-grass prairie) and Saskatoon (located on the fringe of the aspen parkland). In the 10-year period, 1967 to 1976, Regina aver¬ aged 39 species and Saskatoon aver¬ aged 34 species during count period. Saskatoon, with a river, and Regina, with a man-made lake, both provided open water during the winter. Open water was a factor explaining the local diversity of birds in these areas. This was especially evident at Regina where a maximum of 19 water-asso¬ ciated species and an average of 12 such species were recorded; at Saskatoon a maximum of 8 and an average of 4 species were recorded.
When water-associated species are excluded from each year's total, Saskatoon averaged 30 species and Regina averaged about 27 species during count period. The total at Saskatoon is similar to some of the : higher counts taken by smaller num¬ bers of people in typical areas of the parkland, mixed wood or Cypress ; j Hills vegetation zones. The unex¬ pectedly high total for Regina is per¬ haps attributable to the large number of observers, compared to other localities in the same vegetation zone, jjj and the relatively great diversity of T; micro-habitats afforded by an i "island" of woodland on the otherf wise largely treeless plain.
Ii
On a broader scale, one could suggest several obvious reasons for he large number of species recorded on individual counts and the higher species index found in the Cypress Hills, aspen parkland and mixed wood zones. The Cypress Hills and aspen parkland naturally encompass habitats ranging from grassland to orests. The mixed wood component of the Cypress Hills forest provides habitats for additional species not regularly found in aspen parkland. The area is a meeting place for species which have both boreal affiniies (e.g., White-winged Crossbill, Goshawk, Northern Three-toed '|Woodpecker) and montane affinities e.g., Townsend's Solitaire, Red ^Crossbill, Gray-crowned Rosy Finch) B s well as being a prime wintering area for the more typical prairie species. Most of the boreal and mon¬ tane species are still infrequently recorded (and hence absent from Table 1); however, as more counts in the Cypress Hills include the exten¬ sive mixed wood habitats of the Centre and East Blocks, these species will no doubt become in¬ creasingly regular.
The natural diversity of the Cypress Hills and aspen parkland have been further modified by agriculture and other cultural influences. Most counts in the mixed wood zone have been taken along its southern boundary where agricultural practices have resulted in clearing of a part of the forest cover, thus providing openings for species that formerly may have had to travel further south to find suit¬ able winter range (e.g., Snow Bunt¬ ing). There, as in other more souther¬ ly vegetation zones, the presence of farms and towns, with their associ¬ ated food supplies, have provided yet another micro-habitat which wintering birds have learned to exploit.
Climatic patterns within Saskat¬ chewan probably exert both direct and indirect effects on the number of species that winter in various parts of the province. Climate is an important determinant of the vegetation zones which, in turn, influence species rich¬ ness. Temperature also affects the distribution of many species through effects on food supplies and energy requirements. With a mean January temperature varying about 22°C. (40°F.) latitudinally across the province,7 temperature may deter¬ mine the limits at which a given species is able to balance its energy requirements with the available food resources, taking into account the north-to-south variation in daylight available to diurnal species.
The small number of species , December, 1979. 37 (4) observed on the prairies is probably the result of the combined effects of low vegetation diversity and ex¬ posure. Studies have shown that, even for hardy species like the Snowy Owl and Starling, wind chill signifi¬ cantly increases the energy require¬ ments of the bird.13 The wintering bird species of the primeval prairies undoubtedly includ¬ ed fewer species than are present today, since introduced speciesprimarily the House Sprarrow, Gray Partridge and Rock Dove-are now among the most conspicuous winter birds. There is no evidence that any naturally-occurring species have been seriously displaced by intro¬ duced species. The growing number of species now occurring locally in southern Saskatchewan (particularly in towns and cities) attest to the new "richness" of these areas.
Summary
The number of species recorded on more than 25% of counts in each of the seven vegetation zones (i.e., "species richness") was highest in the Cypress Hills, aspen parkland and mixed wood zones, where a high degree of natural and man-induced habitat diversity now exists. Species richness was lower in the northern coniferous forest, the mid-grass prairie, and the short-grass/mixed prairie. Within the mid-grass prairie and aspen parkland vegetation zones, counts in cities invariably recorded the largest number of species; contri¬ buting factors included the avail¬ ability of open water, a great variety of man-induced habitats (including concentrated natural and artificial food and shelter) and usually more observers. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Bioavailability of Cd to Food crops in relation to heavy metal content of sludge-amended soil.
Results of greenhouse and laboratory experiments on factors influencing uptake and accumulation of Cd by economic crops are summarized.Tolerance to Cd is highly crop-specific. For example, 21 different economic crops were grown in pots filled with a calcareous soil treated with increasing amounts of Cd. Yields versus Cd addition rate relations showed yield reductions to occur with Cd sensitive plants (spinach, soybean, curlycress, and lettuce) at addition rates varying from 5 to 15 mug Cd/g soil, whereas tolerant crops (tomato, squash, cabbage, and rice) did not suffer a yield reduction when treated at rates less than 150 mug Cd/g soil. Nutrient solution experiments likewise revealed marked differences in growth of crops. Corn, turnip, beets, bean, and tomato plants grown in solution cultures containing 0.1 mug Cd/ml accumulated different amounts of Cd in leaf tissue depending upon crop species; leaf Cd concentrations ranged from a low of 9 mug Cd/g leaf for beans to 200 mug Cd/g leaf for beets. Large differences also occur with regard to distribution of Cd within the plant. Fruit and seed tissue contain less Cd than leaves. Experiments comparing the toxicity of Cd to Cu, Ni, and Zn in an acid soil +/- lime showed Cd to be the most phytotoxic. While interactive effects occur with regard to metal uptake and accumulation by plants, Cd uptake is essentially dependent upon the Cd concentration of the soil. Studies of chemical speciation of Cd in relation to Cd availability indicate that the free Cd(2+) concentration correlates better with Cd uptake than Cd total of the soil solution.
Introduction
In the early 1970's, our laboratory group initiated a research program for identifying and evaluating various soil and plant factors having an influence on uptake and accumulation of Cd by economic crops. Experiments were designed to illustrate the importance of factors such as Cd concentration in soil, soil pH, other metals (Cu, Ni, and Zn), oxidative state of soil, and plant species. The principal results of such experiments serve as the basis for this review paper.
Substrate Concentration Relationship
A series of nutrient solution culture experiments were carried out with the objective of establishing Cd uptake and accumulation characteristics for a number of economic crops in relation to substrate * Department of Soil and Environmental Sciences, University of California, Riverside, California 92521.
February 1979
Cd concentrations ranging from 0.10 to 10.0 ,ug Cd/ml of nutrient solution. These experiments were also designed to indicate the minimum Cd concentration of the solution culture associated with a yield depression and visual symptoms of Cd injury. The results of the above experiments clearly showed that plants differed greatly with respect to being able to grow in the presence of Cd (Cd tolerance) and Cd accumulation characteristics. Table I contains a list of crop species tested under known levels of Cd in nutrient solution cultures, a ranking according to Cd tolerance as indicated by the nutrient culture level associated with a 25% reduction in growth [compared to yield of plants in the -Cd solutions (controls)], 'and corresponding leaf Cd values of plants showing a 25% yield reduction. Visual leaf injury symptoms consisted of chlorosis, similar to that due to Fe deficiency, and in some instances, leaf burn.
The influence of crop species is strongly reflected in response of test plants to Cd present in nutrient solutions. The lowest Cd concentration utilized as a treatment was excessive for three of the nine crop . The influence of substrate Cd concentration on growth characteristics of economic crop species was also carried out by growing a wide variety of food crops to maturity in pots containing soil treated with low to phytotoxic amounts of Cd. In these experiments, the procedure entailed amending 7 kg lots of a soil with a municipal sewage sludge (10 g sludge/kg soil) pretreated with different amounts of CdSO,. This procedure produced Cd addition rates of 0.0, 5.0, 10.0, 20.0, 40.0, 80.0, 160, 320, and 640 ,ug Cd/g soil. A calcareous soil (pH 7.5) was used for this series of experiments. Various crop species (14 in all) were grown to maturity, at which time yield weights were obtained and samples of leaf and edible tissue (fruit, seed, lettuce head, etc.) were collected for chemical analysis. Results of this series of experiments provide information pertaining to Cd tolerance as well as Cd uptake and accumulation characteristics; the principal results of the study are summarized in Table 2. The addition of 10 Ag Cd/g soil curtailed yields 25% of sensitive crops such as spinach, soybean, and curlycress. Crops such as tomato, zucchini squash, cabbage, and swiss chard were able to grow normally at Cd addition rates up to 100 ,ug Cd/g. Rice under flood management showed no decrease in yield at any of the Cd treatment rates (-640 Ag Cd/g). There were also large differences in the Cd content of leaf and edible tis- sue associated with the plant species under test. The Cd content of edible tissue is of particular concern from a public health consideration because of the toxic nature of Cd to animals. Part of the experiments, testing tolerance of crops to Cd, included treatments with low additions of Cd to soil, simulating levels of Cd that conceivably could exist in agricultural soils treated with large amounts of sewage sludge. Table 3 contains Cd concentrations in the edible portion (part harvested and consumed as food) of a number of important field and vegetable crops grown on a calcareous soil pretreated with sewage sludge enriched with CdSO, sufficient to Environmental Health Perspectives produce Cd concentrations of 0.1 and 5.0 ,ug Cd/g soil. In general, seed and fruit contain the lowest Cd concentrations and leafy tissue from plants such as chard, lettuce, curlycress and spinach contain the highest Cd concentrations. Leaf Cd values for the latter plants grown in soil treated at the 5 ,ug Cd/g rate vary from 21 to 91 ug Cd/g leaf tissue. Seed, however, contains much lower Cd concentrations.
As examples, plants under the 5 ,g Cd/g soil treatment contained the following Cd levels in seed: rice, 0.2; bean, 0.4; sweet corn, 0.5; wheat, 2.8; and soybean, 6.8 ,g Cd/g tissue. Detailed information on Cd content of food crops as influenced by soil Cd concentrations is given in publications by Bingham et al. (2,3).
Relative Toxicity of Cd to Food Crops
Toxicity of Cd was compared to that of Cu, Ni, and Zn using two widely different soils and indicator plant species. Our procedure entailed amending an acid soil (pH 5.2) and a calcareous soil (pH 7.5) with a municipal sewage sludge (10 g sludge/kg soil) which had been pretreated with different amounts of CdSO4, CUSO4, NiSO4, or ZnSO4. The metals were not combined; four sets of treated soil were prepared each for the acid and calcareous soils. Approximately 7 kg of treated soil was placed in pots and cropped with romaine lettuce and wheat. The plants were permitted to grow to maturity, at which time they were harvested for measurement of yields (lettuce head and wheat grain) and samples of lettuce and grain collected for analysis of heavy metals. By plotting yield as a function of metal addition rate, we were able to determine the addition rate (threshold rate) causing a 25% depression in yield for each metal-soil-plant combination. These threshold rates which were expressed relative to that for Zn are summarized in Table 4. In all combinations, Cd was more toxic than any of the other metals, ranging from 2 to 20 times more toxic than Zn. The publication by Mitchell, Bingham, and Page (4) gives a detailed discussion of the above relative toxicities of heavy metals. A multiple metal system was subsequently investigated to evaluate interactive effects of heavy metals. This experiment involved treating an acid soil (pH 5.2) with municipal sewage sludge (10 g sludge/kg soil) enriched with different amounts of CdSO4, CUSO4, NiSO4, and ZnSO4 such that each metal was present at a low, intermediate, and high concentration in complete factorial combination with the other metals. A total of 81 treatments were prepared (34 = 81 metal combinations). A duplicate set of treatments were applied to the acid soil limed to pH 6.7 making a total of 162 treatments. Wheat was grown to maturity, harvested, and weighed. Grain production per plant was related to metal addition rates through multiple regression analysis. Table 5 contains stepwise regression equations for the above yield-metal relationships. The best-fit equation for grain yield of wheat cultivated on an acid soil may be written as follows: y = 3.71 -0.0036(1.00 Zn + 1.44 Cu + 2.06 Ni + 4.03 Cd) where y is yield in g grain/plant and the metals are expressed as ug/g soil. If we consider yield reduction equated with toxicity, Cd is 4.03-fold more toxic than Zn, 2.8-fold more toxic than Cu, and 1.96-fold more toxic than Ni. Upon liming, Ni and Zn were not toxic at any levels tested (-80 Ag Ni and -200 ,g Zn); thus the best-fit multiple regres- gives an expression of Cd toxicity relative to Cu, i.e., Cd is 4.61 times more toxic than Cu. As mentioned earlier, the Ni and Zn addition rates did not extend into a toxic range for limed soil. Results of a current experiment with this limed soil indicate toxicity (wheat) at treatment rates of 200 ,ug Ni/g and 600 ug Zn/g. Accordingly, toxicities for heavy metals relative to Cd for wheat are as shown in Table 6. Liming reduces the toxicity of Cu, Ni, and Zn expressed relative to that for Cd. As example, Cd was 4.0 times more toxic than Zn in the unlimed soil and after liming, Cd becomes 7.1 times more toxic than Zn.
Although information pertaining to soil Cd-yield relationships is of interest, the likelihood of Cd attaining a phytotoxic concentration in agricultural soils is low. However, pollution of soils by Cd to levels permitting uptake and accumulation of Cd in food crops to concentrations rendering the food crop unsafe for consumption is a real possibility. Concentrations of 5 to 10 ,ug Cd/g soil are sufficiently high for a number of crop plants to accumulate excessive amounts of Cd (Tables 2 and 3). Hence the Cd uptake data for wheat are relevant to entry of Cd into the food chain. The Cd treatment rates for the above experiment with an acid soil ± lime were 0.1, 20, and 80 Ag Cd/g soil in combination with three concentrations each of Cu, Ni, and Zn. The grain Cd values for the unlimed soil experiment varied from 0.11 to 0.24 ,g Cd/g grain for the 0.1 ,ug Cd/g soil treatment, 9.3 to 16.4 ,ug Cd/g grain for the 20 ,ug Cd/g soil treatment, and from 21.3 to 37.0 Aug Cd/g grain for the 80 Ag Cd/g soil treatment. In general, liming reduced grain Cd concentrations 50%.
A quantitative relationship between the Cd content of wheat grain and addition rate of heavy metals was obtained through multiple regression analysis (Table 7). These equations predict accurately grain Cd levels over the entire range of addition rates of heavy metals. Essentially all of the variation in grain Cd data is accounted for by the addition rate of Cd; however, inclusion of other metals leads to equations with slightly higher coefficients of multiple determination (R2). The R2 values for the best-fit equations were 0.99 and 0.98 for the unlimed and limed soil experiments, both being unusually high (note that the equations are log1O transformed). These equations also indicate a significant negative effect of Zn applications on Cd uptake by wheat grain. The interested reader is referred to the paper by Bingham et al. (5) for further details of interactive effects of heavy metals on wheat.
pH Effects on Cd Availability
In the experiment with wheat described earlier in this paper, adding lime to the acid soil raised the pH from 5.2 to 6.7. The Cd content of grain from plants cultivated on the limed soil was approximately 50Wo of that from plants in the unlimed soil. This decrease in Cd content of grain is ascribed to a pH effect rather than to a Ca2+ per se. Additional details pertaining to availability of Cd as influenced by changes in soil pH stem from a recent investigation reported by Mahler, Bingham, and Page (6). The latter investigation consisted of amending four acid and four calcareous soils with sewage sludge (10 g sludge/kg soil) enriched with low to phytotoxic concentrations of CdSO,. The range of pH values for the acid soils was from 4.8 to 5.7; and for the calcareous soils, 7.4 to 7.8. These soils were cropped with romaine lettuce and swiss chard to have a biological measurement of soil Cd. In essence, the experiment was designed to compare Cd response Thus the evidence on hand shows Cd availability to decrease with increasing soil pH. An explanation of reduced availability of Cd existing in soils at identical soil solution concentrations upon increasing pH is lacking. Possibly root activity controlling Cd uptake is favored by acid pH conditions. Pre-liminary studies of the chemical species of Cd existing in acid versus calcareous soil systems have not led to a satisfactory explanation of the greater Cd uptake under acid soil conditions.
Conclusions
Plants differ greatly in tolerance to Cd as well as in Cd uptake and accumulation characteristics. Leafy plants such as spinach, lettuce, curlycress, and swiss chard are accumulators of Cd.
Based upon solution culture experiments, Cd concentrations as low as 0.05 Ag Cd/ml in soil solutions (or saturation extracts of soils) are sufficiently high for a number of crop species to accumulate Cd in levels rendering the plant unsafe for consumption. Likewise, Cd addition rates as low as 5 ,g Cd/g soil are excessive for such crops. Soil pH is a dominant factor influencing the availability of Cd added to soil. Liming an acid soil from pH 5.2 to 6.7 was associated with marked reduction of Cd uptake and accumulation by wheat. Comparison of Cd availability to lettuce and swiss chard grown on acid soils with that on calcareous soils also revealed less Cd uptake by plants under alkaline soil conditions. Speciation of Cd in soil solutions of the four acid and four calcareous soils examined indicates approximately 50% of the total Cd solubilized is free Cd2+; Cl, SO,, and fulvate-Cd species account for the remainder. Cd availability appears to be more closely related to the concentration of free Cd2+ in the soil solution than to that of Cd total or Cd complexes. | v3-fos |
2018-04-03T05:24:42.788Z | {
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} | s2 | High-pressure liquid chromatography analysis of antibiotic susceptibility disks.
The analysis of antibiotic susceptibility disks by high-pressure liquid chromatography (HPLC) was investigated. Methods are presented for the potency determination of mecillinam, ampicillin, carbenicillin, and cephalothin alone and in various combinations. Good agreement between HPLC and microbiological data is observed for potency determinations with recoveries of greater than 95%. Relative standard deviations of lower than 2% are recorded for each HPLC method. HPLC methods offer improved accuracy and greater precision when compared to the standard microbiological methods of analysis for susceptibility disks.
The use of antibiotics disks as in vitro diagnostic aids for the determination of the antibacterial activity of various compounds is an established practice and has been well documented.
The official method of potency analysis for those disks described in the Code of Federal Regulations (2) is a horizontal agar diffusion assay, using disks of known potency. This technique has the advantage of conveniently confirming the antibacterial activity of the compound during the assay; however, large experimental uncertainties due to difficulties in the preparation of standard disks in the microbiological assay often yield results which are imprecise and which may only approximate actual values. Furthermore, investigators who wish to work with nonstandard antibiotic concentrations must often develop individual sets of appropriate experimental parameters for each antibiotic level to be tested. The use of high-pressure liquid chromatography (HPLC) as a sensitive and specific method for the analysis of antibiotics, especially B-lactam compounds, is becoming more and more accepted. Recent literature contains an increasing number of papers devoted to the HPLC analysis of ,B-lactam antibiotics including specific assay methods for mecillinam (7), ampicillin (8,12), and cephalothin (9,11), to name a few.
Mecillinam is one of the new class of semisynthetic f8-amidino penicillins whose antibiotic properties have been described in detail (13). Mecilhinam shows biological activity against gram-negative organisms and, when tested in combination with acylamino penicilling, also exhibits a synergistic effect against those organisms in vitro (6,10). The principal,-lactam antibiotics chosen for synergy studies are ampi-cillin, carbenicillin, and cephalothin. As a supplemental in vitro diagnostic aid to physicians, a series of paper susceptibility disks have been formulated containing the antibiotics described above and in various combinations with mecillinam. Experimentally, a large variety of these disks has been prepared (Table 1) to determine the most clinically effective combinations and levels to be provided to researchers. Although microbiological methods are available for those single-component disks described in the Code of Federal Regulations, mecillinam is an experimental drug and no official methods of disk analysis have been published for its assay either aA a single component or in combination with other antibiotics. Microbiological methods have been developed for mecillinam alone; however the combination disks containing antibiotic mixtures do not lend themselves to simple microbiological assays for the components. To accomplish these assays, special organisms have to be employed, which are susceptible only to each of the antibiotics and are not affected by synergic combinations. This requires careful laboratory manipulation and considerably increases cost per assay and turn-around time. In an effort to develop an assay which could be used to determine antibiotic potencies for all of the disk samples, it was decided to investigate chemical methods which were both specific and sensitive enough to determine accurately the lowest level of each antibiotic that was present alone or in combination in these disks. The different chemical properties of each of these antibiotics led to the development of different analytical systems for each drug. These systems, however, are similar to each other and can be used for a wide range of drug levels, requiring only minor ad- Although it was necessary to develop a separate HPLC system for each compound, we elected to describe these different systems in one report because of their similarity and to demonstrate the special advantages HPLC has to offer in the quantitative analysis ofsusceptibility disks. Analysis by HPLC offers the advantages of combining both qualitative and quantitative determinations in one method, relatively quick analyses, and a more easily standardized control method for inter-laboratory comparison.
MATERIALS AND METHODS
Disks and reagents. The susceptibility disks used in these experiments were obtained from BBL Microbiology Systems (Cockeysville, Md.) and included mecillinam, ampicillin, cephalothin, and carbenicillin, alone and in combination. Standard potency disks for the microbiological assays were prepared in this laboratory by adding the appropriate weight of antibiotic(s) to plain paper disks and carefully air drying the samples.
The reference standard sample of mecillinam was obtained from Leo Pharmaceutical Products, Ltd.
(Copenhagen, Denmark). Ampicillin and cephalothin solutions and standards were prepared from certified U.S. Pharmacopeia reference standards, and carbenicillin reference standard was kindly provided by Pfizer Inc. (New York, N.Y.). All other chemicals were American Chemical Society reagent grade or equivalent except that chromatographic grade solvents were used for all chromatography and were filtered through a 0.5-,um filter before use.
Reference standard and sample preparation. Standard solutions were prepared;at appropriate concentrations depending on the expected drug level in the disk by dissolving and diluting the reference standards with filtered distilled water for mecillinam, ampicillin, and cephalothin, and with 1% phosphate buffer, pH 6.0, for carbenicilhin. Disk samples were prepared by extracting the contents of one disk with 1 ml of water or buffer and filtering the resulting solution through a 0.5-pm filter to remove insoluble material. The concentrations of working standards and sample solutions were approximately equal.
Chromatographic equipment. Chromatography was performed using a component chromatographic system composed of a Waters model 6000A solvent delivery system (Waters Associates, Milford, Mass.), a Rheodyne model 7120 injector fitted with a fixed 100-pl loop, and an L.D.C. model 1202 variable wavelength detector (Laboratory Data Control, Englewood Cliffs, N.J.). All separations were performed on Chromegabond C18 columns (4.6 mm by 30 cm) packed with 10-,um OX1.S. bonded phase (E.S. Industries, Mobile phase. The mobile phases consisted of wellshaken mixtures of acetonitrile and 0.01 M monosodium phosphate solution whose pH was adjusted to 5.0 with sodium hydroxide before mixing. Because of the variety of the antibiotics and combinations under test, the proportions of acetonitrile and buffer were adjusted for each sample to produce useful retention times and separations during analyses. Ratios of buffer and modifier ranged from 97:3 to 85:15 and are reported with appropriate retention data in Table 2 for each of the various compounds. Microbiological assay. The microbiological assays used in these studies for both single and combination disks were performed by the standard methods described in the Code of Federal Regulations for ampicillin and carbenicillin (2). Cephalothin at 2.5 pg VOL. 16, 1979 374 HAGEL, WAYSEK, AND CORT could not be assayed by the standard method using Klebsiella pneumoniae ATCC 10031 and was assayed microbiologically using Staphylococcus aureus ATCC 13150 similar to the ampicillin assay. Standard mecillinam disks at the 1-,ug level did not produce zones in the methods used for ampicillin, carbenicillin, and cephalothin, and at that level or below would not be expected to interfere. Mecillinam alone was assayed using Escherichia coli ATCC 10536 in a procedure similar to the ampicillin assay.
RESULTS AND DISCUSSION Separation of antibiotics. Suitable separations of the compounds of interest-mecillinam, ampicillin, cephalothin, and carbenicillinfrom each other and from blank susceptibility disk extracts were achieved using the chromatographic conditions described for each compound in Materials and Methods and in Table 2. Retention data for the antibiotics studied in the various mobile phases are also included in the table. A typical chromatogram demonstrating separation is shown in Fig. 1. The buffer-toacetonitrile ratio used for this sample was 85:15.
The use of several different buffer-acetonitrile ratios in the mobile phases was necessary to achieve complete separation of the disk components and to complete the analyses in reasonable time. The systems described here are designed to give the best results for mecillinam, ampicillin, cephalothin, or carbenicillin alone or in combination. The use of the solvent ratios described in Table 2 for ampicillin and cephalothin in the combination disks of these compounds permits simultaneous analysis of both components. Because of very different retention characteristics, it is necessary to analyze mecillinam and carbenicillin separately.
It was observed previously (7) that reducing the nonpolar component of the mobile phase generally increased the retention times of the types of compounds involved in this study and generally improved separation among all the compounds. When the acetonitrile in the mobile phase was decreased, corresponding increases in separation and retention times were observed for each of the drugs extracted from the disks and for several small peaks attributed to minor amounts of degradation of the antibiotics. These peaks are also observed in intentionally degraded standard solutions, and the presence of these compounds did not affect the microbiological performance of the disks. Plate assays performed at the time of the HPLC analyses yielded potency values which were in good agreement with the HPLC data and were well within the specifications of 67 to 150% of label claim established for disks (3). Linearity and precision of analysis. The linearity of the HPLC response for each antibiotic was calculated by a linear regression analysis of data for a series of standard weight injections. Data for the line y = m x + b for each drug is presented in Table 3, including the range of sample weights injected, parameters for the lines,-and correlation coefficients for the regression analyses. In each case, correlation coefficients of 0.999+ indicate that excellent linear response is observed over the working range of analytical sample weights used in the experiments. The second part of Table 3 contains ANTIMICROB. AGENTS CHEMOTHER. averages and standard deviations for a series of six replicate injections of a single disk extract of each compound. Relative standard deviations of less than 2% demonstrate acceptable precision for these methods. Quantitation was achieved by peak area or height measurement with equivalent results for mecillinam, ampicillin, and cephalothin. The two biologically active isomers of carbenicilhin produced two peaks in the chromatogram of preparations of this compound. A detailed discussion of these epimers by Butler et al. (1) indicates that they possess indistinguishable antibacterial activity. Therefore, it was necessary to sum the areas of the isomer peaks to calculate the correct potency figure for this drug.
Susceptibility disk assay. Susceptibility disks were assayed by extracting the contents of one disk into an appropriate volume of sample solvent, filtering the solution to remove particulate matter, and injecting the resulting solution. Water was used as the sample solvent rather than mobile phase to reduce analyst exposure to acetonitrile. To demonstrate the equivalence of both sample solve-nts, a group of mecillinam disks was divided and extracted with either water or mobile phase. The average potencies of these samples determined by HPLC were 0.795 ± 0.088 and 0.794 ± 0.075 jig per disk, respectively. No interfering peaks due to paper extracts are observed in any chromatogram at the various levels of antibiotic present. Similar results were obtained for both ampicilhin and cephalothin, and as a result water was selected as a suitable sample solvent for those compounds. Carbenicillin, however, was observed to degrade significantly in water in less than 1 h, leading to scattered potency values and additional peaks in the chromatogram. The recommended solvent for the assay of disodium carbenicillin diagnostic sensitivity powder is pH 6.0 phosphate buffer (4), and use of this buffer extended the sample solution stability to more than 4 h.
Initially, two different analytical procedures were investigated for disk analysis using mecil-linam as a model. The first involved the extraction of a sufficient number of disks into 4 ml of water to produce a working mecillinam concentration of approximately 5 ,ug/ml. The assay value obtained from the average of two replicate determinations was reported as the average potency of the sample. An alternative procedure and the one which eventually was adopted in this laboratory is a single disk assay procedure which calls for the extraction of the contents of 1 disk into 1 ml of water. Five replicate samples are prepared and analyzed, and the data are averaged to yield the appropriate potency value. The single disk assay is more time consuming, requiring five determinations versus two for the first method; however, using the second method the investigator is able to obtain a value for the standard deviation of the series of individual potencies which is an indication of the uniformity of the disk samples. This method provides data similar to that obtained from the microbiological diffusion assay which also calls for individual disk assays for uniformity. Comparative data for both HPLC procedures was generated for a sample of 1-,ug mecillinam disks. The average potency determined for 20 disks extracted with 4 ml of water was 1.2 jig per disk whereas the average result of a series of 6 single-disk assays was 1.3 + 0.08 ,ug per disk. This standard deviation is slightly larger than that observed for drug standard solutions and, to determine whether the observed varianee is due to experimental error or to disk-to-disk variation, a series of six individual disks from one representative lot of each antibiotic was analyzed. Table 4 contains data derived from peak area measurements from solutions prepared by extracting the contents of a single disk in 1 ml of sample solvent. The relative standard deviations are not excessively large compared to the standard single solution analyses described in Table 3 and do not charge the precision of analysis by very much. These data indicate that the content uniformity of disks is well within the range required VOL. 16, 1979 376 HAGEL, WAYSEK, AND CORT for the microbiological assay by the Code of Federal Regulations (3), which allows an absolute deviation of 2.5 mm for a zone of 25 mm and may represent as much as 50% deviation depending on the magnitude of the regression data.
To test the recovery of the extraction procedure, a batch of 1-,ug mecillinam disks was prepared carefully in this laboratory by the general Code of Federal Regulations method for preparation of disks (2). This sample contained 1.053 jug per disk and was analyzed versus an extemal mecillinam standard. The recovery for this sample was 1.02 ± 0.015 or 97.0 ± 1.5% of claim for five replicates.
Finally, a series of disks for which microbiological potency data were available was assayed by HPLC, and both results were compared with manufacturer's claims in Table 1. A typical chromatogram of a combination antibiotic disk sample, 1 ,ug of mecillinam and 10 ,ug of ampicillin, is presented in Fig. 2. Chromatograms of all the disks are essentially the same. No interferences with the peaks of interest are observed in chromatograms of control disk extracts or in chromatograms of combination disks. HPLC and microbiological potency figures generally are in good agreement with each other and with the claim. Occasionally, however, discrepancies between HPLC and microbiological values are observed. Carbenicillin 100-ag disks, for example, differed by as much as 28 JUg per disk (or approximately 30% of claimn). Similar differences betwreen chromatographic and microbiological methods have been reported by Diasio et al. (5) in the determination of 5-fluorocytosine by HPLC. These differences are a function of the precision of the microbiological methods and, in fact, contribute to the specification allowance of 67 to 150% of claim. | v3-fos |
2017-06-19T11:47:22.751Z | {
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} | s2 | Crossbreeding the Sardinian and East Friesian breeds in Sardinia
.Summary The genetic improvement of the milk production and prolificacy of dairy ewe populations can be obtained through the crossbreeding and upgrading of the ewes of the local breed with imported male lines. Until recently, the most interesting breed for this purpose in the Mediterranean Basin has been the East Friesian.
Introduction
The Sarda ewe, which gives very satisfactory results under the typical extensive Mediterranean husbandry conditions in Sardinia, has four major limitations for intensive husbandry systems: -a relatively low average level of milk production, -a relatively small body size, -a relatively low reproduction ability, -limited growth capacity of the lambs, with poor carcass conformation.
To correct these deficiencies and to obtain an animal better adapted to the more intensive husbandry conditions of the lowlands, it was decided in the early sixties, as in other Mediterranean countries, to introduce the East Friesian for crossbreeding purposes. According to the bibliography known at the time (ScH!INGRAB!R, 1933C REMER , 1934 ;MIJ HLBERG , 1934 ;Z EEB , 1934Z EEB , , 1941§ F', BB INGHAUS, 1949 ;BUITKAMP, 1952;GROENEVELD, 1953; I,ANTINGA, 1961; STEPHAN, Ig6I; BOYAZOGI,U, Ig63; DIJKSTRA, 1 9 66), it appeared to be the most prolific and productive of the dairy breeds, despite its difficulty in adapting to high temperature dryland environments in the Mediterranean and para-Mediterranean zone (DI M A KOPOULOS , 19 6 0 ; GooT, I g66; TAN!v et al., I g68;F L AMA N T and RICORDEAU, 1969).
The development of irrigation and intensive pastures in Sardinia called for a more intensive system of dairy sheep husbandry than had hitherto been practised in these regions (CAS U , I g7I; BATICI,!E, 1974 ). A major problem was to know whether the Sarda ewe could still be used successfully under these improved conditions or whether it would be better to replace her with a more productive kind of animal. A crossbreeding experiment was undertaken on the partly irrigated Bonassai experimental farm of the Istituto Zootecnico e Casea y io per la Sardegna, situated in the Sassari plain (North-west Sardinia).
For purposes of comparison, a small flock of 17 purebred Friesian ewes in lamb was introduced. As in other similar experiments elsewhere in the Mediterranean environment (FI, AM A N T and R I CO RD EAU, Ig6g; FI, AMANT, 1974), the adaptation of purebred East Friesian animals proved to be exceedingly poor. In this instance, half the ewes died during the first year; the remainder lambed and the mortality of the offspring in their first year was 8 0 p. I oo.
It is important to stress that all the available information from the! various Mediterranean experiments indicates that the Friesians initially show inadaptability, due to introductory stress in the new more arid environment. This could be bypassed, in some cases, by the importation of large populations, although it is doubtful whether this is economically valid. In this case, the so-called &dquo; routine or cruising mortality &dquo; would occur. The mortality level is linked to the local climate, poor resistance to diseases (pulmonary, piroplasmosis) and parasites, and nutritional and management stress. In some cases, the Friesian disappears within a generation of its introduction, while in others it adapts better and survives, although still with a relatively high rate of mortality (Z!ERVas et al., 1975 ;FI, AMAN T et al., 1975).
Material and methods
The possibility of improving the production potential of the Sa y da breed by crossbreeding to the East Friesian (Ostlriesisches Milchschal) was studied from as early as 19 66. The crossbreeding experiments were not limited to the first generation (F 1 ), and back-crosses were also carried out to obtain 25 p. 1 00 and 75 p. 100 Friesian females, the objective being to compare these three female genotypes with purebred Sarda contemporaries. In order to obtain the four genotypes (Table i), seven Friesian (Fr), nine Sarda (S) and four crossbred rams (5 0 p. 100 Fr) were used.
Because of the considerable climatic variability from one year to another in the Mediterranean region, a contemporary system of comparison was imperative. Thus, each of the groups of crossbred ewes ( 25 p. 100 Fr, 5 0 p. 100 Fr, 75 p. 100 Fr) was compared with a contemporary group of Sarda (S) animals, always originating from the same flock. Management conditions never varied during the experiment: intensive pasture (oats, lucerne, rye-grass, clover, etc.), with a maximum daily dose of 5 00 g of concentrates per ewe during the winter months, if necessary. These conditions are thus typical of the intensive irrigated regions of the Mediterranean Basin. Milking was carried out by machine without hand stripping ( 1 ). From the total available population (ewes baving completed four lactations), 25 8 ewes were chosen at random for this study. There was no planned selection programme, so as to allow free interplay of the normal genetic population factors.
Lambs were weighed at birth and then weekly until weaning at an average age of 35 days; thereafter, they were weighed monthly. Continuous observations on the reproduction of the ewes were carried out during the breeding season (individual mating). Milk yield and protein and fat content of the milk were measured twice monthly, starting after weaning. The first recording was made during the second month of lactation, but in most cases, 40 to 45 days after lambing. Both the total lactation yield and the standard reference lactation yield over i 5 o days of milking ( 12 o days for the first lactation) were calculated.
( 1 ) As defined at the International Symposium on the Machine Milking of Small Ruminants, Alghero, Sardinia, May 197 8.
For the present paper, only the results of four consecutive complete lactations were taken from the data obtained over the ten years of the study, except when otherwise indicated (e.g. the comparisons of wool quality). As far as more specific evaluation methods are concerned, the means and standard deviations were evaluated in all cases, while an attempt was made, where necessary, to express the averages (in percentage) of the different crossbred populations in relation to their contemporary purebred Sarda populations (taken as 100 p. 100 ) and to calculate the necessary standard errors. Figure I gives a complete explanatory reference of the body measurements, while the classification used for the udder conformation is presented in figure 4 . The live weights of the ewes were measured at lambing and shearing.
In 197 6, fleeces from a random sample of 15 adult ewes of each of the four genotypes were weighed at shearing and samples of the wool were analysed for staple length, crimping, percentage clean wool and fibre thickness at the wool laboratory of the S.A.A.D.S.R.I. ( 2 ) by the standard methods normally used.
Results
a. -Ewe weight and body measurements While there is generally little difference in weight between the 25 p. 100 Fr and the comparable Sarda ewes, the 50 p. 100 Fr animals are significantly heavier at first oestrus than the comparable Sarda purebreds (table 2 ).
There is also no real difference between the lambing and shearing weights of the 25 p. 100 Fr and the contemporary purebreds for all four lactations. Upgrading towards 50 p. 100 Fr and 75 p. 100 Fr is, however, always followed by a considerable increase in body size. This confirms the advantage of introducing heavier breeds to obtain females of better size and conformation than the ones traditionally found in the Mediterranean (table 3 ). The heavier adult weights of the upgraded crossbred animals are in line with the weights at first oestrus, the A representative number of one-year old ewes from the different genotypes were measured (fig. I ) and the various body measurements and live weights recorded as an indication of the conformation changes obtained through crossbreeding (table 4 ). b. -Precocity, gestation and lambing From tables 2 and 3 , the following values can be obtained when expressing the ewes' live weight at first cestrus as a percentage of their weight at fourth lambing (kg), which is considered as indicative of precocity: Thus, as previously shown by H AF E Z ( 1953 ) and Wnr,xav! et al. (1975), there is little variation between the purebreds (Sarda) and the 25 p. 100 and 5 0 p. I oo (Friesian) crossbreds, with a slightly better precocity for the F, as compared with the Sa y das (first oestrus at a lower weight as a percentage of the adult weight). The extremely high percentage ( 74 p. 100 ) obtained for the upgraded ( 75 p. 100 Fr) ewes, which could be interpreted as an indication of poor precocity, is contrary to all expectations. This could, however, be the result of the small numbers ( 22 ewes) involved.
A better indication of sexual precocity is probably the age at first oestrus, which gives very coherent average results: When compared with their contemporary Sardas, the crossbreds show a. pronounced tendency for an earlier first oestrus, from just over a 10 p. 100 difference for the 25 p. 100 Fr to i 3 p. 100 for the 5 o p. 100 Fr and over m p. 100 for the 75 p. 100 Fr. This is confirmed by an earlier average age at first lambing for all groups of crossbred females, when compared within years, to the contemporary purebred groups (table 2 ). An interesting feature is the highly significant difference in age at first lambing (table 3 ), to the advantage of the less upgraded crossbreds (25 p. 10 0 Fr and 5 0 p. 100 Fr).
The gestation period of the purebred Sarda is longer (in most cases significantly longer) than that of all crossbred groups (table 3 ). Our data confirms the results obtained in Greece by Zervas et al. (i 975 ), when comparing the Friesian (z 4 6 days) and its crosses ( 147 days) with the local Chios sheep (i5o days). The gestation period of the Sarda was, with few exceptions, between i 4 8-i 5 o days, while that of the crossbreds was rq 4 -r 4 6 days.
In Southern France, F LAMANT and J ACQUIN ( 197 8) obtained very similar results (purebreds): The purebred Sardas always lamb later in the season (also longer lactation) than the 2 p. 1 00 Fr and the 5 0 p. roo >! r.
The situation is reversed when comparing the 75 p. ioo Fr with its contemporary Sarda. This could naturally be confounded with a year effect. It can be considered as an indication of the lower adap-tability of the more upgraded ewes ( 75 p. ioo Fr) to local environmental conditions (despite good feeding and the particularly favourable year in which they were bred). As in most other Southern European countries, the lambing season extends from October to early April ( fig. 2 ). Contrary to the results obtained in Greece (ZE RVAS e1 al., 1975 ), there is no important difference between the ewes of the local breed and the crossbreds (not even the 75 p. 100 Fr). The adult crossbred ewes ( 2 nd, 3 rd and 4 th lactation), however, generally tend to lamb earlier on average than the purebred Sa y das. The results obtained by M AN rruTn. and C ASU ( 19 68) suggest that the Sarda is more seasonal in its breeding than other Mediterranean breeds.
c. -Numerical productivity and viability There was little difference between the purebred Sa y da ewes and the crossbreds in the percentage of ewes pregnant. However, there is apparently a slightly lower percentage of ewes actually lambing in the case of the crossbreds, particularly the 75 p. 100 Fr and 50 p. 100 Fr, when compared to their contemporary Sardas. The most interesting feature, though, is the high prolificacy of the crossbreds. The z5 p. 100 Fr are slightly superior to the Sarda ewes, but the 50 p. 100 Fr and 75 p. 100 Fr are significantly better (table 5 ).
It is Important to note the high percentage of early lamb mortality in the 75 p. 100 Fr group and the continued rise in total lamb mortality, up to one year of age, with the higher proportions of F y iesian blood (table 6), despite almost optimum intensive husbandry conditions. Data on the reasons for lamb mortality (table 7 ) show the main cause of death to be respiratory diseases. The limited available data on purebred Friesians on the farm is even more conclusive, as 77 , 7 8 p. 100 of all lambs born died with a year of birth (6 I , II p. 100 due to respiratory diseases). This information confirms our previous findings in Greece (Z ERVAS et al., 1975 ) and that of other authors (S HIMSHONI and L AVI , ig 7 2; F!.n- MAN T, 1974 One of the more positive results obtained through crossbreeding the Sarda to the East Friesian is that of body size, better growth rates and the obtention of crossbred male lambs more suitable for modern fattening techniques. The production of heavier lamb carcasses is naturally particularly interesting on the European continent where heavy lambs are more acceptable than on the island where the &dquo; agnello di latte &dquo; of 8-io kg remains the consumer's choice. An experimental attempt to evaluate the fattening potential of the crossbreds (SarrrrA and R U DA, 1 97 0; COSSE DU el al., 1972 ; CAS U et al., 197 6) confirmed the better growth potential and daily weight gains of these animals, when using both barley and maize rations; a very interesting feature was the better carcass conformation of the crossbred lambs.
e. -Milk production and culling An attempt was made to study the causes of culling of the different genotypes, taking into consideration the number of ewes of each group originally available and their performance to the end of the fourth lactation. There has to date been no conclusive indication between the different genotypes, although the most important reason for culling appears to be udder conformation and the related sanitary problems (table 9 ). Information on the milking period is presented in table 10 , while that of the standard lactations (i2o days of milking for the first lactation and 150 for the other three lactations) is given in table m.
As expected under the intensive rearing conditions of the experiment, the milked yield of the crossbreds is higher than that of the purebred Sarda, after a suckling period of nearly a month for the first lactation and over a month (::I: 35 days) for the adult lactations. Three interesting comments should be made at this stage: -There is no significant difference (not even at the first lactation) between the milk yield of the 25 p. 100 Fr and their contemporary Sardas.
-Strictly speaking, the best dairy ewe of the four genotypes appears to be the 50 p. 100 Fr; this confirms the information obtained from other similar Mediterranean projects (FI,A M A N T, 1974 ; KAi<AisSAKis ! al., Ig77; FLAMANT and C ASU , I g 77 ). The indication is that there is a drop in absolute and relative milk production when arriving at the 75 p. 100 Friesian blood level. This is clearer from the complete lactation data (table 12 ) than from the standard lactation data (table 13 ). It will eventually be interesting to compare this information with the data of the 8 7 , 5 p. I oo Fr genotype—which, unifortunately, is not yet available.
-Contrary to Greek findings (Kn!,mssnxrs et al., 1977 ), there is no significant difference between the different crossbred genotypes and their contemporary purebred groups in respect of milk quality (fat and protein content), although as expected, the milk of the purebreds is richer in fat content than that of the contemporary crossbreds (tables 10 and 11 ).
There is no significant difference between the lactation periods of the crossbreds and the purebreds, although the purebreds on average always show a relatively shorter lactation period than their contemporary crossbreds, which could be attributed to the slightly earlier average date of lambing of the crossbreds (fig. 2 ).
f. -Lactation curves An investigation within genotype and number of lactation of the average date on which the first milk records were effected, very seldom shows differences between the comparable genotypes (crossbreds and contemporary Sardas) from one year to another. Whenever a difference does occur, however, the average date of the first milk recording of the crossbreds—which is naturally linked to the date of lambing—is always the earlier of the two.
It is also interesting to note that the first milk recording (linked to the date of lambing) of the truly adult lactations ( 3 rd and 4 th) is always carried out in January and early February, within a period of one month. On an average, the first controls of the second lactation take place a month later (February and early March). The average duration of the milking period of the crossbreds is comparable to the milking periods for similar crossbreds in other parts of the Mediterranean. (table io). However, the initial yield by the 25 p. 100 Fr crossbreds at the beginning of lactation (after weaning) is higher and the final yield at the end of lactation (nth to y th control) is lower than that of their contemporary Sardas. This applies parti-cularly to the 2 nd-4 th lactations, whereas no noteworthy differences exist for the ist lactation.
-A comparison of the lactation curves between the 5 0 p. 100 Fr and their contemporary purebreds is always to the advantage of the crossbreds. The two lactation curves tend to be similar in evolution, but the individual milk controls of the crossbreds are consistently between 200 -5 00 gm higher than those of the purebreds. This confirms the value of F, ewes under intensive rearing conditions. -The results of the comparison between the 75 p. 100 Fr and the Sardas are far less striking, since the two lactation curves (S and 75 p. 100 Fr) are almost identical, particularly after the third lactation. The two groups of ewes in all cases start at a very comparable level of production, differing only towards the 7 th or 8th control effected at about I40 -I50 days from lambing.
A study of the maximum recordings (table 12 ) confirms the relatively late stage at which the Sarda ewe attains her maximum daily yield. It is interesting to note that some of the crossbreds also reach maximum yield rather late in comparison with results obtained with purebred Friesians elsewhere (B OYA zOGL U , ig6 3 ). K AIAISSAKI s et al. (ig!!) arrive at similar conclusions with their Friesian X Chios crossbreds, but at a much earlier stage after birth (adult lactations, 45 -6 0 days vs. 7 o-go days in our case; P I and 75 p. 100 Fr material). The fact that the maximum daily yield is attained at such a late stage could at least partially be ascribed to problems in adapting to machine milking.
With regard to the maximum recording data, two main remarks can be made: -The negligible difference between the purebred Sarda and the 25 p. 100 Fr can be confirmed. The volume of the maximum daily yield and the interval between birth and maximum yield are similar for these two genotypes.
-There are highly significant differences (PI, o.oi) between the volume of the maximum recording only in the case of the S / 5 o p. 100 Fr comparisons, while in the S / 75 p. 100 Fr comparisons, this is only true for the first lactation (also difference at PL 0 . 05 ewes in the third lactation).
Comparisons of the yield of the Prealpes and Friesian crossbreds ( 50 p. 100 and 75 p. 100 ) in the very short ascending period of the lactation curve (Iz Q uiERDO PRIMO et al.,ig6g) show that maximum recorded yield was lower for the 75 p. 100 .crossbreds than for the 5 0 p. 100 and that, contrary to our data, the crossbreds attain their maximum recorded yield at an earlier stage than the purebred Préalpes.
g. -Milking ability and machine stripping Machine milking is essential to the development of the dairy industry under intensive husbandry conditions. It is thus important to consider the udder conformation of breeds to be used under these conditions. A valuable presentation of the udder conformation of the Sardinian breed was made by Snrtrra and P ICINELU ( 1973 ) and for comparative purposes, an attempt was made here to classify the different genotypes in our experiment according to the type of udder ( fig. 4 ). Observations made at Bonassai with a random sample of contemporary Sardinian and crossbred ewes ( 50 p. 100 Fr and 75 p. 100 Fr) show that while only 4 p. 100 of the controlled purebred ewes had the &dquo; hard &dquo; type of udder, the comparative values for the 5 0 p. 100 Fr was 33 p. 100 and that for the 75 p. 100 Fr nearly 43 p. 1 0 0 . In the light of the observations of PA R T E A R xoYO and F!.A- MANT ( 197 8), this could be interpreted as indicating a better average milking ability of the purebreds, despite a less attractive udder conformation in general and some milk retained in the udder after each milking (table z 3 ).
The relation between the facility of machine milking, milk retention, etc., on the one hand, and the form of the udder, hardness, general conformation and position of the teats, on the other, is a very complex matter, approached by the farmer ( & d q u o ; éleveur &dquo; in French) and the milker ( & d q u o ; trayeur &dquo; in French) in different ways. In a recent publication, PARTS A RROYO and F L A M A N T ( 197 8) used three criteria to evaluate the milking ability of ewes of three dairy genotypes (Lacaune, Sarda and the F..S.L., the result of a Friesian, Sarda and Lacaune cross): milking characteristics, udder conformation and loss in milk production in relation to the reduction in the number of daily milkings. The milking characteristics of the Sarda ewes are apparently the least adequate, with the highest percentage of milk retained following udder massage. However, the average loss in production (one versus two milkings) is the least significant. Under simplified milking conditions, the F.S.L. ewes appear to have inherited the most favourable factors from their parental breed, the Sarda, at the same time maintaining the better udder conformation of the Lacaune breed.
According to C ASU and R UDA (r 973 ), machine stripping has little economic value under the local (even intensified) husbandry conditions. A study was consequently undertaken on the different genotypes to determine the quantity of milk obtained through stripping (table 14 ). The information thus obtained confirms their findings and indicates that in the case of the purebred Sardas and the 25 p. 100 Fr and 50 p. roo Fr crossbreds, the percentage of milk obtained through stripping is less than 5 p. 100 of the total milk yield; it is higher for the 75 p. 100 Fr group of ewes (6,8 p. 100 : ist lactation; 5, 3 p. 100 : 2 nd lactation; 5, 4 p. 100 : 3 rd lactation). Even when referring to the standard lactation period, the 75 p. 100 Fr group always shows the highest quantity and relative percentage of stripped milk.
h. -Wool characteristics Carpet wool production still plays an important role on the island where carpet weaving is traditional, particularly in the light of the recent re-activation of the family operated carpet industry. The development of tourism in recent years created a growing market for all handwoven textiles and the determined action of the local institute for popular arts (ISOLA) revived the interest in carpet weaving and local textile production.
The basic material for the traditional carpet industry has always been the wool of the longwool Sarda dairy breed, known for its high staple length, convenient fibre thickness and crimp value. Its quality and technical possibilities are amongst the best in the Mediterranean Basin (F ER xExo,i9!2). It thus appears important to have an indication of the effect of crossbreeding on wool quality and type.
The average of the data obtained is presented in figure 5 . It is evident that, despite the higher average quantity of shorn wool (table IS) from the crossbreds, the wool is less suitable for carpet weaving than that of the purebreds. It will undoubtedly be interesting to undertake a future study on the variability of the different measurements when a more representative number of animals will be available.
Discussion and conclusion z. The analysis and publication of the results obtained in Sardinia complete the crossbreeding experiments carried out over the past twenty years between the East Friesian breed and a number of the local Mediterranean breeds: the !4!'asM in Israel, the Chios in Greece and the Prialpes (Lacaune type) in France.
In general, these experiments were aimed at finding a genotype likely to improve the cheese industry and fully exploit the investments in the dairy sheep industry (machine milking, husbandry buildings and other facilities) undertaken in the richer agricultural areas (cereal planted plateaus and irrigated plains). By eliminating most of the constraints pertaining to the traditional animal husbandry systems, it is, in fact, possible to use more demanding animal material, however with all the productive qualities: milk yield, numerical productivity, high growth rate of the lambs, etc. It has been customary to use the East Friesiara breed in improved husbandry conditions to correct the shortcomings of the local breeds, such as: -low numerical productivity of the Awassi and Sarda breeds; -low growth capacities of the Awassi, Sarda and Chios breeds, and the fat-tail of the Awassi breed; -low milk yield (PréalPes-Lacaune), average milk yield (Sarda) or high milk yield, but lower than that of the purebred East Friesia1! (Chios).
The East Friesian undoubtedly possesses all the above qualities in its own environment, but experiments as a whole reveal certain qualities specific to the local breeds and which might deteriorate by crossbreeding with the Friesian breed. These qualities essentially concern adaptability in difficult environments, which better husbandry conditions have not or cannot completely correct: resistance to heat due to strong thermic variations (F!,AMarr2 and RiCOxD!nu, i 9 6 9 ), possibility of out-of-season sexual activity whereby the natural growth. of the grass in autumn and spring can be used (GooT, r 9 66; Z!RVns et al., i 975 ). It can also be assumed that the high nutritional requirements of the East Friesian or its crossbreds are not always satisfied, even under improved husbandry conditions. Frequent sanitary problems are found in the purebred Friesian or the crossbred 75 p. 100 Friesian ewes.
The experiments with the Sarda breed confirm, however, that under certain improved and controlled conditions, the defects of the East Friesian can be reduced. Thus, sexual seasoning observed elsewhere with an increased proportion of Friesian blood (Z ERVAS et al., 1975 ) was not found in the Sardinian experiments and the sanitary problems appear less frequently with the crossbreds than indicated, for example, by GooT ( 19 66), R ICORDEAU and F LAMANT (ig6g) and Z!RVns et al. (1975). The observations of S HIMSHONI and L AVI ( 1972 ) in Israel, ZE RVAS et al. (1975) in Greece and Fr,nMnrrT and C ASU ( 1977 ) in the Roquefort region (France) confirm our own observations; through various methods of flock management a farmer can attain a certain amount of success. In general, the rearing of ewes with over 50 p. roo of Friesian blood cannot be recommended without risking setbacks.
The lower productivity of crossbred Friesian ewes beyond the first generation (F 1 ) is due to the influence of unfavourable factors at several levels (K AI , AISSAKIS et al., 1977) : -on the reproduction efficacy, resulting in a decrease in the birth rate of lambs and the milk yield of ewes; -on the viability of the crossbred lambs and adults; -on the level of milk yield per lactation, either through a direct effect on the lactation curve or its level at different stages (I ZQUIERDO P RIMO et al., ig6g), or through an indirect effect: the reproductive ability and viability as an intermediary, determining the number of reproductive females available for renewal and consequently a possible rate of selection.
2 . In comparison with the other Mediterranean sheep breeds, the Sarda has other particularities: milking ability and interesting fleece characteristics.
Observations made on the morphology of the udder and the milk fractions collected during milking were not really conclusive with regard to the milking ability of the various crossbred genotypes in comparison with the Sarda ewes. They thus had to be submitted to the same experimental schemes, whereby stripping was suppressed or a single daily milking was carried out, thus revealing the particularities of the Sarda ewes in comparison with other breeds (Cnsu et al., 1978).
With regard to the fleece characteristics, the East Friesian crossing undeniably lowers the qualities required for carpet making. Crossbreeding thus has to be accompanied by the regional planning of the area concerned. It can, for example, be considered limiting the rearing of crossbred ewes in the irrigated plains, however maintaining the rearing of Sarda ewes in the extensive and mountainous regions covering most of the island (L E I,nvrrou, 1941 ). The combination of farming with the local breed and a homecraft industry, technologically linked to it and likely to bring about an important increase in value, probably constitutes one of the conditions for the perpetuation of rural society in this region. On the contrary, farms in the irrigated plains can undertake specialised dairy and meat production (CAS U , 1971 ). 3 . Through these results different crossbreeding strategies can be foreseen for farmers having undertaken a degree of improvement. It is thus possible to compare the milk yield of the following flocks, taking a 20 p. 100 renewal rate of the flock into account (table 1 6): a) purebred Sarda, b) purebred Sarda -E-production of the F, ewe with a second meat crossing, c) purebred Sarda j -production of the F l ewe + production of the -!5 p. 100 Friesian ewes, d) idem c, but with 75 p. 100 Friesian ewes.
In spite of a ± 34 p. 100 higher milk yield for F, ewes in comparison with the Sarda ewes, the global superiority of a flock which would assure the renewal of purebred Sarda females and the production of F, females is only 20 p. 100 . On the other hand, an additional 6 9 p. 100 of lambs can be sold, which amongst others, have a higher growth rate than the purebred Sarda lambs. It can also be observed that despite a fall in production of the 75 p. l oo Fr ewes, in comparison with F 1 ewes, the producer centres practising this type of crossbreeding can hopefully use the better milk potential of the East Friesian breed. It must, however, be considered whether tl.e slow progress aimed at is worth the risk engendered by the higher frailty of the genotypes produced, since the production of lambs is generalised in comparison with flocks which are only limited to first generation crossbreeding.
The effective productivity of the flock where the F, ewes are fully used, could be increased by guaranteeing the renewal of the Sarda females through outside purchases or in a satellite flock (under more difficult husbandry conditions) in the mountainous areas. It could also be envisaged to supplement the flocks in the plains with F, females produced directly in the satellite flock (genetic and geographical stratification of breeding systems). A system of this nature, however, comes up against definite difficulties which, other than organisatory problems, essentially reside in the extremely poor adaptation of East Friesian rams to Mediterranean husbandry conditions and notably with regard to reproduction. This handicap probably also exists under plain conditions and would limit the theoretic effectiveness of the schemes that could be established. In order to solve the adaptation problem of the males under extensive pasture conditions, East Friesian rams were used in Greece mainly for artificial insemination over a period of approximately twenty years (ZE RVAS and BO Y AzOGLU, 1 977 ). Supposing that one even succeeded in mastering the crossbreeding technique and limiting it to the first generation, which was never the case, the global effectiveness of the operation would appear doubtful, since it was not possible to maintain a sufficient number of purebred Friesian flocks and since there was a high rate of mortality amongst the rams in the production centres, necessitating frequent imports.
The crossbreeding experiments with the East Friesian breed undertaken over the past twenty-five years in Israel, France, Greece and Sardinia have thus proved that the use of rams of this breed on a large scale is not to be recommended. It would appear preferable to exploit investments in the Mediterranean region by the creation of synthetic lines where the East Friesian breed can be used (FiAMaN2 et al., 1975 : F.S.L. in France, Assaf in Israel), or perhaps even better, if the local breeds possess relatively high productive qualities (Chios and Kyme-Sho!elos in Greece) or are characteristically linked to the local economy (Sarda in Italy, Lacaune in France), to establish a purebreed selection programme of these breeds ( fig. 6). | v3-fos |
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} | s2 | Cadmium from soil amended with sewage sludge: effects and residues in swine.
Liquid digested sewage sludge from a Chicago waste treatment plant was applied to experimental corn plots starting in 1968. The treatment plant received a high proportion of industrial effluent and the sludge averaged about 200 ppm Cd (dry weight). Corn grain harvested from the plots in 1974 was fed to growing swine for 56 days, and other swine were permitted to forage on the plots during the winters of 1975-76 and 1976-77. The sludge-fertilized corn contained higher concentrations of nutrient and toxic elements, but did not interfere with swine performance. Minor changes in hepatic microsomal oxidases and red blood cells accompanied significant increases in renal Cd and decreases in hepatic Fe. Swine foraging on these plots ingested considerable amounts of sludge soil and accumulated significantly higher concentrations of renal Cd. At lower rates of sludge application the swine outperformed those foraging both on control plots and those receiving heavy sludge applications in terms of weight gain, in-utero piglet survival, blood hemoglobin, and tissue Fe concentrations.
Introduction
Municipal sewage sludge, the product of secondary and tertiary wastewater treatment, presents an increasingly burdensome disposal problem to sanitary districts of all sizes. Surface land disposal is an attractive alternative, but there are some potential hazards. Accumulation of toxic trace elements is the current primary concern and cadmium is the trace element receiving the most attention. Trace element composition of digested sewage sludges is highly dependent on the city of origin and on the metal processing activities within the recipient sanitary district (1). Heavy sewage sludge application to agricultural soils for several years results in significantly increased concentrations of nutrient elements, increased organic matter and moisture holding capacity, and does not adversely affect microbial action (2). Increased productivity is possible, especially on devastated soils or when suboptimum levels of commercial fertilizers are used (3). Sewage sludge applications also result in the ac-cumulation of significant amounts of trace elements in the soils (2)(3)(4).
The effects of sludge amendment on crops include a frequent enhancement of production and increases in nutrient elements and nonessential trace elements in plant tissues (4)(5)(6). Plant leaves accumulate higher metal concentrations than do grains. Incorporation of previously applied sludge into the soil immediately prior to planting seems to make the metals more available than with an even heavier soil load from long term cumulative applications.
Feeding plant material grown on soils heavily fertilized with sewage sludge to animals has not elicited any definitive toxicity syndrome; however, there have been accumulations of potentially toxic trace elements in animal tissues, especially in kidney but rarely in muscle (6)(7)(8). The presence and accumulation of potentially toxic elements makes it desirable to conduct more intensive investigations for possible effects. We have generated residue data not available when a previous swine feeding study was published (8), and this has necessitated reevaluation of some conclusions. In addition, a longer-term swine reproduction study involving more intensive exposure has recently been completed and some of these data will also be evaluated.
Clinical and Analytical
Complete blood cell counts, serum enzymes and electrolytes, histopathology, electroencephalography, and electrocardiography were conducted using standard techniques. Metal concentrations were determined by atomic absorption spectroscopy after wet and/or dry ashing the samples. Hepatic microsomes were isolated from perfused liver sections by differential centrifugation and assayed as previously described (9, 10).
Grain Feeding Study
Nine weanling crossbred (Hampshire/Yorkshire) gilts were randomly assigned to one of three slotfloored pens (1.2 x 4.7 m) equipped with automatic waterers. After a 2-week acclimation period on full feed, the gilts were limit-fed a standard growing ration according to one of the following regimens: "'control" received a ration formulated with control corn for 56 days; "'14-day" received a ration formulated with sewage sludge-fertilized (SF) corn for 14 days and the control ration for 42 days; "'56-day" received the SF ration for the full 56 days. The SF corn was pooled from experimental plots and incorporated as 79.4% of the ration (8). Weekly weight and total feed consumption were recorded and various clinical parameters were monitored periodically. The swine were sacrificed at the end of the 56 day feeding period.
Winter Foraging
Sixteen weanling purebred Berkshire gilts were assigned to experimental corn plots in a random fashion, except that no siblings were permitted within the same dosage group. Each 6.1 x 12.2 m plot had received fractions (0, ¼4, 1/2, or 1) of the maximum amount of sludge which could be applied, dependent on weather, during each of eight growing seasons. Additional sludge applications occurred between winters. The sludges applied to these plots were from the Chicago Southwest plant which received a high proportion of industrial effluent and the Cd content averaged about 200 ppm dry weight.
The plots were divided between the two buffer rows and individual shelters, feeders and waterers were provided. The gilts were introduced to the plots November 4, 1975 andremoved March 20, 1976. They were bred by one of two Berkshire boars that spring, farrowed in October and returned to the same plots as sows December 2, 1976. They were again bred in January while on the plots, removed from the plots March 15,1977 and farrowed in spring 1977. The sows were killed for examination after weaning the second litter.
Results and Discussion
Grain Feeding Table 1 presents selected metal concentrations in the control and sludge-fertilized (SF) corn. The SF corn also contained 1.14 times more Kjeldahl nitrogen than did the control (NPK-fertilized) corn resulting in calculated protein concentrations of 16.5% for the control diet and 17.7% for the SF diet (8). The swine fed SF corn for the full 56 days performed slightly better than the other two groups, possibly due to the limit-feeding and slightly higher nutritive elements in the SF diets (8). Relative organ weights, microscopic pathology, clinical chemistry, cardiac function, and central nervous function showed no dose related differences.
The swine fed SF corn diets accumulated significantly more Cd in the liver and kidney than did controls, but hepatic Fe and renal Mn were lower than controls (Table 2). There were no significant changes in Cd, Cu, Fe, Mn, or Zn content in spleen, muscle, brain, or bone. The Fe decrease, also seen as a trend in the kidney, may be related to the Cd interference with Fe absorption described by Freeland and Cousins (11), since the SF corn contained a higher Fe concentration (Table 1).
A possible correlation may exist between the lower tissue Fe and some minor, but significant, differences in red blood cells (RBC). At 56 days, the pigs which had received SF corn had slightly lower packed cell volumes (PCV) and hemoglobin (Hb) values ( Table 3). The red cell counts were lower for the SF fed pigs than for the control animals resulting in a significant increase of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). The 56 days of slightly decreased Fe absorption would probably not be adequate for the pigs to develop a significant iron deficiency anemia, as the life span of porcine erythrocytes is 85 + 11 days. At higher Cd concentrations, however, Cousins et al. (12) produced pigs with decreased packed cell volumes in 42 days. At the low level of "biological" Cd fed in our study, packed cell volumes were not depressed because of an apparent increase in the volume of erythrocytes (MCV). This slight change in the MCV may be attributed to a release of reticulocytes in response to minimal anemia. However, the fact that the mean corpuscular hemoglobin in concentration (MCHC) for all groups was essentially the same, indicates the possibility that the enumeration of erythrocytes (the most variable of the measurements) was in error. A more intensive survey of the effects of the higher metal content of sludge-fertilized grains on hematology is necessary.
The pigs receiving SF corn for 56 days had decreased hepatic microsomal O-dealkylation activity, another possible consequence of interference with iron metabolism. It was previously erroneously reported that this activity was enhanced (8). The swine fed SF corn for only 14 days did not differ from the controls, but the 56-day feeding re-sulted in higher microsomal protein concentrations but lower and more variable activity ( Table 4). The 56-day group has a lower affinity for low substrate concentrations and a lower maximum reaction velocity V for high substrate concentrations (Table 5 and Fig. 1).
Although this type of biphasic plot is expected when a wide range of substrate concentrations are used (9, 13) the shape of the plot for swine liver microsomal metabolism of p-nitrophenetole is unusual (10). The low-substrate portion of the plot extrapolates to a higher Vma, and is not as sensitive to CO inhibition as the high-substrate (low affinity) "'enzyme." The factor(s) in SF corn which depress the microsomal activity maintain the general pattern even though slopes and intercepts are somewhat different.
Direct feeding of large amounts of sewage sludge (50% of diet) to male rats resulted in enhanced pentobarbital metabolism and liver enlargement (14). High levels of dietary cadmium have been shown to enhance microsomal enzymes and to potentiate microsomal inducing agents (15), while peritoneal administration of cadmium depresses drug metabolism (16). Furthermore, several metals, including Cd, Cu, Fe, Ni, and Zn, induce microsomal heme oxygenase which degrades free heme as well as cytochrome P-450 (17). It is unlikely that the low concentration of Cd in the SF corn acted directly to depress the microsomal mixed function oxidase system. It is probable that an interaction between depressed Fe levels, increased Cd and/or increased dietary protein resulted in the increased synthesis of microsomal protein not related to drug metabolism and/or increased degradation of heme decreasing the metabolic potential of the protein present; thus, the increase in microsomal protein would not be accompanied by an increase in microsomal O-dealkylation activity. The question of Cd influence on microsomal drug metabolism presents some interesting paradoxes and should be a fertile area for future investigation.
Winter Foraging
Subjective evaluation of holes and wallows indicated that the swine on the maximum treated plots tended to root less than the other groups. In order to more precisely evaluate exposure through inges- Table 6. Mean metal residues in soils amended for 8 years with sewage sludge and in the feces and kidneys of sows winter-foraging on these soils. hSignificantly lower than 1/4 max at p <0.003. '*Significantly lower than 1/4 max at p = 0.0005. Table 9. Liveborn and total apparent conceptions in two litters from swine winter-foraging on sewage sludge-amended soils (mean + S D tion, a series of fecal samples were collected and analyzed for heavy metals. Samples collected in February 1977, while the ground was frozen and moderately snow-covered, indicated that there was a dose related increase in fecal metal residues, but this was not necessarily directly related to propensity to root. The relationship between soil, fecal and renal residues of selected metals is shown in Table 6. Cadmium was the only metal which accumulated significantly in the kidney tissue. Soil ingestion increased as spring approached and the ground thawed, but residues of metals in the feces declined rapidly once the swine were removed from the plots ( Table 7). Complete data for residues and biological effects are not yet available. Pigs started out uniform and gained weight at a reasonably uniform rate up to maturity (Table 8); however, after weaning the first litters a trend started in which the animals on maximum treated plots seemed to suffer slightly from the heavy sludge application rates. The swine foraging on plots amended with 1/4 the maximum application rate responded more favorably to the stress of reproduction and ultimately gained significantly more weight than any of the other groups ( Table 8).
Environmental Health Perspectives
The first year's farrowing records cannot be taken too literally. Some gilts had unusually traumatic first parturitions and, in fact, one from the maximum treated group died from massive hemorrhages suffered during delivery. To make the comparisons more precise, only sows which farrowed both times are included in the data: the sow that died in the maximum treated group is not included, but had 6 liveborn pigs; the replacement for this sow is not included (11 liveborn piglets and 4 fetal deaths); one sow each from the control and 1/4 maximum groups did not conceive the second time in spite of multiple attempts at breeding.
The swine in the control and maximum treated groups seemed to be slightly more fertile than the two intermediate groups, both as gilts and as sows ( Table 9). The group foraging on plots amended with 1/2 the maximum possible sewage sludge application for two winters generally conceived fewer pigs, but all of these were born alive and fully de-veloped (Table 10). These data must be tempered with an appreciation for the low experimental numbers and the high degree of variability. The number of stillborn piglets is exaggerated, for example, in the first-litter 1/4 maximum group and the secondlitter maximum group by single sows, which had breech birth complications resulting in 5 and 6 stillborn piglets, respectively. The number of piglets not fully developed is more closely related to total litter size than to treatment group; all but one of the 17 fetal deaths occurred in litters totaling 12 pigs or more. Thus, it is equally as likely that the excellent in-utero survival rate of 1/2 maximum piglets is due to lower fertility as to a positive influence of nutrient factor(s) in the sludge. On the other hand, the fact that gilt-to-sow performance improved and there were no piglet deaths indicates that this rate of sewage sludge application does not render these soils toxic to foraging swine.
Although there is a direct relationship between treatment level and Cd residues, there were no significant differences in the iron content of treated and untreated soils. This is not surprising, since the iron content was near 2% in the soil and the sludge contribution would be minimal. However, acid extractable (bioavailable) Fe is significantly increased in sewage sludge amended soils and the pH of the soil is decreased (Table 11). Even though there is a direct relationship between treatment level and available soil Fe, tissue Fe does not increase linearly (Fig. 2). Thus, swine store the increased available Fe at lower sludge application rates, but as the rate of sludge application increases the Fe stores and blood hemoglobin decline again (Fig. 2). This may be related to the increased Cd which interferes with Fe absorption and/or to some of the many other factors in the sewage sludge. It appears that soil concentrations of 4 or 5 ppm Cd would not interfere with Fe absorption in swine ingesting the soils, but at higher Cd concentrations, it is more difficult for the swine to take advantage of the in-creased available Fe. Again, it would be desirable to investigate these relationships in more detail. In summary, the swine foraging on moderately heavily amended plots (1/4 and 1/2 maximum) frequently outperformed the animals on control or maximum treated plots. This pattern persisted for weight gain, in-utero piglet survival, blood hemoglobin, and tissue iron concentrations, even though the diet was standard and nutritionally adequate. At the lower Cd intake of the grain-feeding study there was also a modest performance advantage of the SF corn, but this could have been due to higher nutritive value, since these pigs were limit-fed. Only at very high sewage sludge application rates did there appear to be signs of toxicity, and these were generally significant only when compared to intermediate application rates, essentially the same as controls. Nevertheless, the modest performance advantage gained must be balanced against the increased Cd residues even though these increases are not observed in muscle tissue. Since the swine foraging study probably represents maximum animal exposure to land applied sewage sludge, it suggests that moderate agricultural use should present little direct toxicity hazard; however, the data are scant and present some apparent anomalies. Reliable riskbenefit analysis is impossible at this time with only these data. | v3-fos |
2018-04-03T01:54:35.611Z | {
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} | s2 | Partial purification and properties of proteases from defatted soybean flour.
Four chromatographically different proteases were partially purified from defatted soybean flour, and their pH optima were around 5.0 to 5.6 using casein as the substrate. These soybean proteases were designated S1, S2, S3 and S4 according to their order of elution from a DEAE-cellulose column. Each gave a single peak of caseinolytic activity on a Sephadex G-200 column chromatogram, and corresponded to the molecular weights of about 50,000(S1), 35,000(S2), 60,000(S3) and 200,000(S4). The proteases could hydrolyze casein and poly-Glu. alpha-Casein was more rapidly hydrolyzed than beta-casein, but the esters or dipeptide could not be hydrolyzed. Aliquots of 10(-3) M Hg2+, Cu2+ and Zn2+ inhibited the caseinolytic activities by 70% to 90%, while other cations, Mn2+, Mg2+, Ca2+ and Ni2+, at the same concentration did not. SPI (10(-5) M) inhibited 80--90% of their activities, and EPNP (10(-5) M) inhibited their activities 30--60%, but DFP (10(-3) M), SSI (10(-3) M), PCMB (10(-4) M), NEM (10(-3) M) and EDTA (10(-3) M) were not inhibitory. The above results indicate that proteases S1, S2, S3 and S4 from defatted soybean flour can be classified as acid proteases.
Since LAUFER et al. (1) reported proteolytic activity in soybean seeds, investigators have described the purification and the properties of the enzyme in the seed. OFELT et al.
(2) reported the presence of a protease most active at pH 5.5 in soybean flour. The partial purification and some properties hove been Abbreviations: DFP, diisopropylfluorophosphate; PCMB, p-chloromercuribenzoate; NEM, N ethylmaleimide; EPNP, 1,2-epoxy-3 (p-nitrophenoxy) propane; SSI, subtilisin inhibitor; SPI, pepsin inhibitor; ATEE, acetyltyrosineethylester; BAEE, benzoylarginineethylester; TAME, tosylarginine methylester; poly-Glu, poly-glutamic acid. 333 reported by WELL et al. (3), who separated six active proteolytic fractions from dormant soybean cotyledons. They showed that these enzymes had optimal pH values between pH 5.0 and 5.4 by using casein as the substrate, and different Michaelis constants on poly-Glu. FUKAZAWA et al. investigated the proteases in germinating and dormant soybean seeds (4) and found four protease fractions in the dormant seeds on DEAE-sephadex column, although the activities were weak.
In this paper, the authors present the results of the partial purification of the proteases from defatted soybean flour and their enzymatic properties .
Purlcation of proteases
Step 1. Preparation of crude extract: The defatted flour was suspended in 10 volumes of extraction medium and stirred for 15 min in an ice cold bath. The suspension was centrifuged at 15,000rpm for 30 min. The effect of medium on extraction is shown in Table 1. As the best result was obtained with regard to both total activity and specific activity with 50 mM phosphate buffer it was used in the following purification. For the purification of proteases, 200g of the soybean defatted flour was suspended in 2,000ml of medium and stirred for 15min. A clear supernatant was obtained after centrifugation of the suspension at 15,000rpm for 30min. Step. 2. Preparation of the pH 5.5 soluble protein fraction: As a preliminary experiment, a portion of the clear supernatant (step 1) was divided into several fractions and in each the pH was adjusted to between 7.0 and 3.3 by acetic acid. The precipitates were collected by centrifugation and dissolved in medium. An aliquot of the solution was used for proteolytic activity and protein content. At pH 5.5, the ratio of non-protease protein to proteolytic activity was greatest in the precipitate. Thus, supernatant at pH 5.5 was gathered by centrifugation at 10,000 rpm for 15 min. In this step, protease was purified about 2.4-fold (Table 2).
Step 3. Ammonium sulfate fractionation: To the pH 5 .5 soluble fraction, solid (NH4) 2SO4 was added gradually to 65% saturation, then the suspension was kept for over one hour in an ice bath. The precipitate was collected by centrifugation at 10,000rpm for 15min, dissolved in a minimum volume of medium and stored frozen state until use. Before using, it was dialyzed against medium. Step 4. Sephadex G-150 column chromatography: The preparation from Step 3 was subjected to Sephadex G-150 column , equilibrated with medium, and two peaks of proteolytic activity were obtained (Fig . 1). The fractions, 25-35, and 51-60, were combined respectively , concentrated with ammonium sulfate (60%), and kept frozen until the next step.
Step 5. DEAE-cellulose column chromatography: The concentrated frac tion (fraction numbers 25-35) in Step 4 were dissolved in medium and dialyzed
DISCUSSION
In the present experiment, the four proteases that we separated from defatted soybean flour had pH optima between 5.0-5.5. In other dormant seeds, cotton (14), barley (15) and hempseed (16), the presence of acid proteases having pH optima of 2-4 have beeen described, but proteases in sunflower seeds had pH optima at 5.2 and were classified as acid proteases (17). The application of specific protease inhibitors of microbial origin have facilitated the characteri zation of proteases in biological materials. Thus, we were able to demonstrate that the soybean proteases S1, S2, S3 and S4 are acid proteases from the effects of inhibitors, SSI or SPI (Table 3).
Although we studied the enzymatic properties of these proteases, there was little difference in the optimum pH, effects of inhibitors, effects of metal ions or specificities for substrates; however, the molecular weights differed markedly. Furthermore, the enzyme activities, which had been inhibited by heavy metal ions (10-3M), were restored by more than 100% by the addition of SH reagents. However, the enzymes are not inhibited by SH-blocking reagents such as PCMB or NEM. Thus, it is suggested that free SH groups on the enzymes are participating in the reactivation, but do not contribute to the active sites. Detailed characterization of the proteases in soybean seeds must await further investigation. M | v3-fos |
2016-05-17T04:13:01.210Z | {
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} | s2 | Experience during two generations of within lines boar performance testing, using 5α-androst-16-ene-3-one (5α-androstenone) and an olfactory judgement of boar taint
Summary The Danish work undertaken within the B.A.A.P. Commission oj Pig Production working group on the production and the utilization of meat from entire male animals is presented from the Danish breeding experiment station « Tylstrup » on thelbasis of 8 entire male pigs of the Dconish Landrace breed during the generations 1974 and 197From the year of herd establishment, 1974are given the means, the frequencies and the variation of a panel judgment of boar odour intensity (in boar fat) together with those of two steroid criteria, 5 stenone (in boar fat and blood plasma) and testosterone (in blood plasma). For the steroids, the repeatabilities (intra-blood sample) and the correlations between morning and afternoon samples are given. The purpose of this presentation is to forward estimates of genetic parameters for the potential selection objective boar taint and of its probable genetic relation to male reproductive performance and growth. The highest estimate of p. 100 additive gene action (his found for a linear biological expression, adding the level of the steroid 5and the olfactory panel score:
Introduction
In 1973 , The European Association of Animal Production (E.A.A.P.) Commission of Pig Production, took the initiative to establish a working group on ce Production and Utilization of Meat from Entire Male Animals » (ig 7 q.). The present report is updating the relevant Danish work undertaken.
Materials and Methods
Samples were collected from 8 45 performance tested young boars at a live weight of 90 -!-3 kg. Correction for age effect was not undertaken because of the insignificance of regression in this limited live weight interval of maximum 6 kg.
The samples of fatty tissue were collected from the live boars by use of a biopsy needle (LUNDSTRØM et al. 1973 ) within the generations -2 (year r974-75)! zero (year 197 6-77 ) and one (year 1977 -7 8) of the selection experiment herd of Danish Landrace pigs (Table i), which was established in the winter 1973 -74 . Samples of blood were collected from vena cava cranialis (generations -2 and zero). The level of 5 «-androstenone in fat, taken in the generations o and 1 ( 197 6-7 8), and in peripheral plasma, taken in the generation -2 ( tg 74 -75 ), were evaluated by radio-immunoassay according to A NDR ESE N ( 1974, 1975, and the peripheral plasma levels of testosterone as described by Sarrwar, et al. ( 1974 ). All samples were analyzed in duplicate.
The samples of fat were judged for the occurrence of boar taint by 4 women and 4 men. A 10 class scale was used for the material from the generation -2 (1 974 -75), and a 4 class scale was then used henceforward, as suggested by the E.A.A.P. working group mentioned above for reasons of uniformity in scaling between member countries.
The foundation generation in the breeding experiment station cc Tylstrup » was established by means of a stratified sampling procedure by systematically intermating boars with gilts from exactly 100 litters sampled, each litter bought from one of 100 elite herds which made up the elite breed nucleus of the recognition year 1973 -74 of the Danish Landrace pig, i.e. 100 litters out of the total of 8 6 71 litters produced that year within the elite nucleus, or i.i5 p. 100 of litters sampled.
The successive generations are partitioned into seasonal blocks of 8 0 to 100 contemporary probands for selection. Heritabilities and genetic correlations are estimated from the within-lines sire components of variance and of covariance.
Results and Discussion
ig!4-75: The year of herd establishment The results of the organoleptic judgement for boar taint in the foundation generation is given in Table 2 . As the difference between gilts and young boars proved to be highly significant (P G o.ooi), the gilts were not tested for sex odour intensity in the following generations. The difference between the two sexes is demonstrated in the frequency distributions, the minimum and the maximum points, the standard deviations, and the coefficients of variation in the two sexes, all given in Table 2 .
The level of 5 a-androstenone and testosterone in boar peripheral plasma in the foundation generation is given in Table 3 . From the table section A it is seen that the mean level and the variation, both absolute and relative, in the 5a-androstenone level are slightly lower than those three estimates for testosterone.
The sufficient accuracy in the biochemical analysis is proved by the intraclass correlation between the first and the second duplicates within the same boar within the same plasma as shown in the Table section 3 , B.
The levels of 5«-androstenone and testosterone in peripheral plasma of boars are not constant during a 24 hours cycle (A NDR E S E N 1975 b, C LAUS andGI MÉ N EZ , 1977 ). In order to reduce the effect of this variation, from each boar one sample of peripheral plasma was collected in the morning and another 7 hours later. In Table 3 , section C, is seen that the correlation between the morning plasma and the afternoon plasma levels of the steroid 5«-androstenone was r = o.81, whereas that between the morning plasma and the afternoon plasma levels of the steroid testosterone was only r = o.61. It is apparent that this morning and afternoon sampling of blood with 7 hrs distance seemed sufficient to reduce the diurnal variation of 5 «-androstenone considerably, but no that of testosterone. The difference between the two correlation coefficients, o.81 vrs. o.6I, being transformed to their specific z-values, proved to be significant at the probability level of P G o.ool. The values of the two periods were then pooled, and the phenotypic correlation between those mean values of testosterone and 5«-androstenone was rp = + 0 . 7 8 (within boar within diurnal time). This correlation is given in the section E of Table 3 . In the following calculations, the mean steroid level of the two samples collected 7 hrs if apart has been used.
Of interest is the intra-litter correlation between two full-brothers in Table 3 , > section D ( 93 degrees of freedom) of 0 . 17 and of 0 . 25 for the two steroid levels, respectively. When doubling these, a rough heritability estimate is obtained for each steroid, which may be compared with the corresponding estimates in Table 5 .
1976-1978
The mean level of the three selection criteria in question in these two years, together with the daily weight gain, were as given in Table 4 .
The phenotypic correlation between 5 x-androstenone and odour intensity, both in boar fat, was found to be rp =_ !-0 . 5 . This estimate was in agreement with MAI, M FO R S and ANDRESEN (1975). The reasoning behind the linear combination of the boar odour panel score and the level of 5 «-androstenone was to combine an expression of the consumer's response (housewife's criterion) with the steroid response of the animal itself, the level of 5 «-androstenone. Before combining these two variates, each of them was of course expressed relative to its own standard deviation, and these two variates were then added, constituting the combined expression of boar 'taint. Table 5 demonstrates clearly the improvement in the estimation of genetic action against boar taint in the consumer's product when using this combined expression. It is promising that this a biological » criterion yields the highest estimation of relative gene action.
In the sequence of their selection project against and for boar taint, using only the level of 5 «-androstenone and using the selection response evaluation technique given by HILL (rg72), WI LL EC K E et al. ( 1979 ) obtained, combining their first three generations of selection, realized heritability estimates of similar magnitude as those given in Table 5 .
In their Danish Landrace breed nucleus performance test on elite herds investigation, J ON SSO N and WISMER-P!DI;RSEN ( 1974 ) found a heritability estimate of h 2 = 0.54 ! 0 . 32 ( 4 2 d.f. for sires, 0.05 ! P G 0 . 10 ), which is comparable to the last estimate given in Table 5 , but not with respect to its standard error of estimate.
The heritability estimate of the testosterone level was found to be in the same range as for 5 «-androstenone, and in Table 6 it is demonstrated that a significant genetic covariation exists between 5 «-androstenone and testosterone. At the breeding experiment station, therefore, an upward and a downward selection line were started in the autumn of 197 8 to demonstrate as quickly as possible the correlated response on the level of testosterone when selecting solely on the combination of the two boar taint criteria, as discussed above.
The response to libido, reproduction and weight gain will also be followed very carefully. A control line is maintained alongside, contemporarily. Therefore, the testosterone level is determined in the plasma of each boar pig reaching go kg live weight in that experimental herd.
The relationship between each of the two steroid levels to daily weight gain is only negligible and not significant (see Table 6). These relationships have, however, to be carefully followed along with libido and other reproduction criteria in order to study the selection response upon these criteria.
From 197 8, the determination of 5a-androstenone in boar fat has been performed by a simplified method, giving relative concentrations of the steroid in counts per minute (C.P.M.) (ANDx!s!rr, ig 7 g). The selection procedure practised from the same year is mass selection on the basis of performance test within and across selection blocks of contemporary animals of similar age. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | A new autosomal translocation in "Alpine grey cattle"
In the cytogenetical research experiments at present carried out in Italian cattle breds which are in way of numerical decrease, we have taken into consideration a sample number of 5o bulls of the « Grigia Alpina breed n, chosen out of a group coming from an A.I. Centre and from those which were selected for reproduction purposes at the Pure Breed Show. Five bulls resulted to be carriers of a new autosomal translocation involving two of the smallest chromosomes. On the basis of the results obtained by need of measurements and the application of band- ing, we are of the opinion the translocation concerns the chromosomes of the pairs 25 and 27. Two of the translocation carriers, kept at an A.I. Centre have never given semen suitable for freezing purposes.
Introduction
Starting from the famous translocation i / 2 g discovered by G US T AVSSON and R OCKBORN in 19 6 4 in the Swedish Red and White breed, a considerable number of other chromosomal anomalies have been discovered since then in various parts of the world and in several different breeds of cattle. The translocation 1 / 29 seems to be the most widespread (Pop!sCU, ig 77 ), and even in our country cattle breeds seem to be particularly affected. It is to be found in the Romagnola breed at the level of heterozygous state equivalent to 28.8 p. 100 (DE G IOVANNI and M OL T ENI , 197 6). It is also met with other breeds such as Chianina, Marchigiana and Modicana (S UCCI and DE G I O VANNI , 197 6;MOLTENI et al., 1977). However other anomalies have been discovered and identified either by means of banding methods or by measuring the relative length of the chromosomes in question. P OPESCU ' S scientific paper ( 1977 ) explains in great depth all the anomalies discovered up till now in the Bos taurus I,. To these abnormalities one must add the translocation 14 ) 20 which was more recently identified by L OGUE and H ARV E Y (1978).
VVe present a new type of Robertsonian translocation in which the chromosomes involved are identified by measurements and banding studies.
Materials and methods
In the research program of the Italian National Research Comacil (C.N.R.) &dquo; Defense of animal genetic resources &dquo; meant to identify the chromosomal complement of various Italian cattle breeds in way of exstinction, we have examined 50 bulls, belonging to the Alpine Grey (Grigia Alpina) breed&dquo;. Out of the group some were chosen by the Zootechnic Commission of the Herd Book to be destined for the Progeny test and the rest were selected at the Bolzano Pure Breed Show as Prime specimens of their breed. The latter group were set aside in various breeding stations in Alto Adige for reproduction purposes.
The microscopic specimens were obtained by culture of leucocytes from the peripheral blood according to the method of M OORHEAD et al. ( I9 6 0 ) slightly modified.
The chromosomes involved in the translocation were identified by the Rbanding method using BUDR, and Orange Acridine staining (PoPEscu, 1975 ). The densitometric profiles were obtained with a 1 /E iTZ MPV II microphotometer.
The idiogram was constructed on the basis of 10 cells from all the heterozygous animals.
The C-bands were obtained by S UMMER ' S method ( 1972 ) modified by Poruscu (1974).
Results
The karotype of 5 animals evidenced the normal number of chromosomes reduced to 59 , due to the presence of a small metacentric chromosome, whose centrometric index is 47 . 44 ± 2 . 57 , obtained by fusing 2 chromosomes of small dimensions ( fig. i).
Moreover in one of the carriers, probably co-twin of a free-martin, were found only female cells. Four of the 5 carrier bulls were related to each other (they had a 3 rd generation ancestor in common), whereas the 5 th had no close connection with the others.
From the measurement idiogram it may be seen that the long arm is within the range of the pairs 21 -2 8 and the short one within the range of the pairs 25 -2 8 ( fig. 2 ).
After R-band labelling, the long arm of the abnormal chromosome showed the same pattern as the free chromosome z 5 and the short one as the free chromosome 27 , according to Gus2evssorr and IIaG!!,TORrr ( 197 6) ( fig. 3).
With the use of the I, E iTZ NIPV II microphotometer a sketch was made of the banded chromosomal profiles. In spite of the small number of metaphases we were able to examine, due to the impossibility of being connected with a suitable computer, non-the-less, we were able to confirm that the chromosomes involved in the translocation are those belonging to the pairs 25 and 27 (fig. 4 ).
The fused chromosome in cells treated by C-banding method, appears as carrying a single constitutive heterochromatin block on its long arm (fig. 5).
Up till now we have only been able to study the sexual behaviour and semen picture of 2 out of the 5 mentioned bulls which were brought to an Artificial Insemination Centre.
Apart from the 2 bulls'poor &dquo; libido & d q u o ; , the few ejaculations presented, seemed to be of bad quality and insufficient and not suitable for freezing purposes. On the contrary all the bulls belonging to the same bred that were present in the same Centre have always supplied spermatic material of good quality and quantity and suitable for freezing purposes. In the light of these first comments, we intend to carry out the study of this new translocation. We intend to study the meiosis of male translocation heterozygotes with the aim of pointing out the presence of the trivalent and, if that is the case, of cells with unbalanced karyotypes in the second meiotic metaphases.
We intend also to go into the study of fertility in greater depth by keeping a greater number of the translocation carriers under control. | v3-fos |
2018-04-03T01:40:06.605Z | {
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} | s2 | Vitamin B2 activity of 7,8-dimethyl-10-(2,3,4-trihydroxy-4-formylbutyl)isoalloxazine in Lactobacillus casei.
The microbial activities of vitamin B2-aldehyde and vitamin B2-acid, produced by Schizophyllum commune, a Basidiomycete, were studied. Lactobacillus casei ATCC No. 7469 was used as a test microorganism. B2-aldehyde exhibited a good response curve in the growth of L. casei. B2-acid had neither a stimulatory nor an inhibitory effect on the growth. When B2-aldehyde was incubated with the homogenate of L. casei, it was converted to riboflavin. The flavin formed from B2-aldehyde by the homogenate not only exhibited an equivalent response curve to authentic riboflavin in the growth of L. casei, but also showed the same Rf value as authentic riboflavin in any paper chromatogram, as far as tested. Hence, the microbial activity of B2-aldehyde for L. casei seems to be ascribable to riboflavin which is a reduction product of B2-aldehyde.
Microbial activity of B2-acid
The B2-aldehyde preparation contained B2-acid as an impurity. The microbial activity of B2-acid for L. casei was studied and B2-acid was isolated and crystallized according to a previous paper (3,4). The result is shown in curve 3 of Fig. 1. Although the concentration of B2-acid was increased to 53.5ng/ml, growth was not observed. The effect of adding B2-acid together with B2 was also tested. As shown in curve 4 of Fig. 1, the response of L, casei was dependent on the concentration only of B2; therefore, B2-acid did not seem to have any effect on the growth of L . casei. This result suggested that the observed activity of B2-aldehyde was not influenced by any contaminating B2-acid. 3 4. Microbial activity of the B2 produced from B2-aldehyde with the homogenate of L. casei Some isomers of B2 have been studied with respect to their biological activities. For example, it was reported that araboflavin had some activity for L. casei (9,10). We studied the microbial activity of B2 produced by the homogenate (the sample B2), comparing it with authentic B2 in order to obtain information as to whether the sample B2 was a true D-riboflavin. If the product from B2-aldehyde with the homogenate is D-riboflavin, its activity should be equivalent to that of B2.
The sample B2 in the homogenate reaction mixture was separated by PPC using ethyl acetate-pyridine-water as a developer and then was eluted from the paper with deionized water. The eluate was used as the sample B2 without further purification.
Authentic B2 was charged on the same filter paper, and treated by PPC in the same way as the sample B2 as described above, so that the influence of impurities during the preparation of the sample B2 might be compensated.
As shown in Fig. 4, the sample B2 showed the same response curve in the growth of L. casei as the authentic B2, D-riboflavin. Therefore, it is almost conclusive that the product from B2-aldehyde is D-riboflavin.
From the results of these studies it could be determined that B2-aldehyde possessed the stimulatory activity for growth of B2-requiring L. casei, and that the microbial activity of B2-aldehyde seems attributable to the normal metabolism of B2 which was produced by an enzymatic reduction of B2-aldehyde in L. casei. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Sewage sludge as a source of cadmium in soil-plant-animal systems.
The objective of this presentation is to relate the abundance and mobility of Cd in components of terrestrial ecosystems with implications for land utilization of sewage sludge. The uptake of Cd by crop plants is a function of the quantity of the element in the soil plus other soil factors affecting the Cd ion activity or electrochemical potential at the plant root surface. The natural abundance of Cd in soils has been reported as 0.5 μg/g which is higher than the background level of 0.2 μg/g found in soils studied in Pennsylvania. Experimental results indicate that the plant availability of Cd increases with each soil addition. While the plant availability of Cd is decreased by liming to increase soil pH, it has not been possible to add Cd salts or sewage sludge Cd without significantly increasing plant uptake. Field studies have shown that land application of sewage sludge can be expected to increase the Cd concentration of corn leaves from a range of 0.05–0.1 μg/g to 1–3 μg/g. Two years after the last application of sludge which added up to 10 ppm Cd to the surface soil, corn grain, sorghum grain, wheat grain, and potatoes showed a 10- to 15-fold increase in Cd over background levels. Studies were conducted with chicks, laying hens, and meadow voles (Microtus Pennsylvanias) to assess the impact of this increase in plant Cd upon the food chain. Corn and sorghum plants were grown on soils with either inorganic or sludge fertilizer for the purpose of producing herbage for use in feeding trials with meadow voles. Eight diets and a synthetic control diet were formulated to study the effect of source (plant vs. inorganic) of Cd on tissue accumulation. Significant accumulation of Cd occurred in kidney and liver, but not muscle, of voles fed diets containing sludge fertilized corn (1.09 μg/g) or sludge fertilized sorghum (2.76 μg/g). The source of Cd had little influence on tissue accumulation. In studies with broiler chicks and laying hens, natural diets containing 0.2 ppm Cd were supplemented with 3 ppm of this element. As with the meadow voles, Cd readily accumulated in liver and kidney. Although the results were not statistically significant, 3 ppm dietary Cd doubled muscle Cd content. There was no transfer of Cd to egg in a long term (12 month) experiment with laying hens. Soil management programs have been developed to maintain animal dietary levels of Cd at less than 1.0 μg/g from the use of sewage sludge on land in Pennsylvania. However, it is concluded that this level over time may cause a significant accumulation of Cd in animal tissues. Interpretation of these results in relation to those for human intake of Cd and the long range health effects of Cd is required for the proper monitoring of sewage sludge applications on land used for production of crops which enter the food chain.
Introduction
The accumulation of Cd and other ions by plants from soil is affected by inherent differences among species and varieties within species, soil abundance of the element, ionic interactions in soils, and soilplant interactions. Interpretations of experimental results relating soil testing, soil chemical equilibria, plant analysis, and plant physiological processes have been subjects of several reviews (/-9). The review on trace element cycling by Allaway in 1968 * Departments of Agronomy and Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802. (10) provides an excellent discussion of principles. Bingham et al. (11) reported that additions of 4 to 640 ppm Cd to soil resulted in a 25% decrement in plant yield, diagnostic leaf concentrations of 3 to 160 ppm Cd, and concentrations within the edible portions of plants ranging from 2.0 to 80 ppm Cd, depending upon plant species. The relatively high concentrations of Cd found toxic to plants when compared to concentrations of Cd considered harmful to animals and man make results of plant toxicity studies seem irrelevant to the problem of Cd in components of terrestrial ecosystems. The objective of this presentation is to review experimental results from several investigations conducted at Penn State University that have led to a February 1979 chemical monitoring program designed to prevent excessive food chain levels of Cd from the utilization of sewage sludge as fertilizer on land used to produce food chain crops. The suitability of sludge for use on cropland is determined by the Cd content and the rate of sludge applied to supply the required N and P.
Soils and Soil-Plant Relationship
From results summarized by Baker and Chesnin (2), the abundance of Cd increases from an average of 0.13 ppm in igneous rocks to 0.18 ppm for the average in the lithosphere to 0.5 ppm as an average for soils. The range of Cd in virgin soils has been reported as 0.01 to 0.7 ppm. The field soil used for the Penn State studies was on a land boundary between Hagerstown silt loam and Murrill silt loam. The total Cd in the soil, sampled from different areas of the field, ranged from 0.2 to 0.3 ppm. Thus, the plants were established on plots with a relatively low background level of Cd. The details of the experimental methods and results for 1974 and 1975 have been presented by Baker, Amacher, and Doty (12). Variable treatments included check plots with row or starter fertilizer only, plots receiving row fertilizer plus other commercial fertilizer in 1974 and 1975, plots treated with row fertilizer and dairy cattle manure at 5 dry tons per acre in 1974 and 1975, and plots treated with row fertilizer plus 5, 10, and 20 dry tons of a sewage sludge containing 257 ppm Cd on a dry matter basis. Since 1975, all plots have received only row fertilizer for the production of various food chain crops in order to study the residual effect of sewage sludge on crop yields and Cd accumulation in plant parts in relation to soil test levels of Cd.
It has been shown that chemical analysis of corn leaves removed from the node from which the ear emerges at the silking stage of maturity will reflect the soil availability of several elements (13). The results presented in Table 1 for corn leaves from plots receiving no sludge Cd are in close agreement with the range of 0.05 to 0.2 ppm reported as the normal range for plant leaves by Melsted in 1973 (7). His suggested maximum of 3 ppm Cd in plant leaves was reduced to I ppm Cd by Baker and Chesnin in 1975 (2) on the basis of the relationship between plant leaf Cd and the level expected in the food chain. While the upper limit of I ppm in corn leaves has no official validity, its use as a maximum level to be attained in corn ear leaves provides for interesting observations from the data summarized in Table 1. The lower values reported for plots receiving no sludge Cd in 1975 compared with those reported previously (12) resulted from an improved flameless atomic absorption technique which is capable of more accuracy when the level of Cd in plant tissue is below 0.1 ppm. The 5 dry ton rate of sludge supplied approximately 2.6 ppm Cd to the surface soil over the two-year period. The corn ear leaf Cd decreased from 1.09 ppm in 1974 after the first addition of 5 tons dry sludge to 0.4 ppm in 1976 and 1977. At all rates of sludge, the uptake of Cd from the second application was not greatly different from that observed for the initial application. This trend has been observed by other investigators and suggests that the availability is determined initially by the Cd binding properties of the sludge and not by the soil. However, over time as the sludge colloidal material is decomposed, the availability of Cd will be more a function of its chemistry in the soil. At the 10 and 20 dry ton rates of sludge the uptake of Cd was proportionately less than at the 5 ton rate. These results indicate that higher rates of Cd on fewer acres could result in less Cd entering the food chain. In the CAST Report No. 64 (14), it was reported that "repeated annual applications of sludge to soil cropped to corn show that the amounts applied in a given year influenced the Cd contents in leaves to a greater extent than did the total cumulative amounts of Cd applied." From our data as well as data reported by others (14,15), it is concluded that the plant accumulation of Cd from a given soil is a function of the total Cd loading of the soil, the properties of the sludge added, and the soil chemistry in relation to Cd binding once the sludge applications have been discontinued. However, once the soil abundance of Cd was increased to levels above the desired minimum, the plant concentration of Cd has attained levels in corn leaves above I ppm. Since it has not been possible to apply substantial quantities of Cd without increasing plant uptake of Cd, food chain buildup of Cd may be prevented only by preventing Cd additions to soil or by limiting the applications of Cd contaminated sludge to land dedicated to the production of non-food chain crops.
Environmental Health Perspectives 3.66 a Assumes an acre furrow slice to weigh 2,000,000 lb. b Method of Lindsay and Norvell (9). c Method of Baker (12). Sludge-10 11.9 11.9 12.0 11.8 Sludge-20 11.0 11.6 11.6 11.5 a A test level of 12.0 or greater is recommended for soils.
The amounts of Cd added by the various treatments and the quantities extracted by various testing methods may be compared from results presented in Table 2. Since the acre furrow slice treated with sludge was probably less than 2 million pounds, the results for O.IN HCI suggest that this extractant removed all of the Cd added to the soil, while the Baker method, like the corn plants, removed proportionally less Cd as the soil level increased. The results by the Baker method for pH, Cd, Fe, Zn, Cu, Mn, and Ni are related to DTPA chemistry by a computer program to calculate the negative logarithm of the activity of each metal in each sample. The soil test results and the calculated pCd results are presented in Tables 3 and 4. From the results for the sludge-treated plots it does not appear that the test values are decreasing with time. When means for corn leaf Cd were correlated with soil test Cd and pCd over the four years, the re-spective coefficients were 0.82 and -0.84. Since the soils within the field were not extremely variable and the pH was not altered substantially by the treatments, a close correlation between soil test Cd and pCd (r = -0.86) was expected. However, where soil type and pH are expected to vary, the calculated pCd is used to obtain a theoretically superior measure of Cd availability. Both measurements are needed to predict the long term effect of sludge on Cd abundance as reflected by the amount removed with the testing solution and the current availability as reflected by pCd. 0.025b a Mean in column not followed by a common letter, differ significantly (p < 0.05). All results on a wet basis. 4.75 ± 1.98b 9.27 ± 2.25c 0.15 ± 0.02b a Means ± standard deviation dry fat-free basis. Means not followed by the same letter are significantly different (p < 0.05).
Accumulation of Cd in the Food Chain
The concentrations of Cd in cereal grains and potatoes for different field treatments are presented in Table 5. Except for corn grain, the crops accumulated substantial quantities of Cd. Two years after the last application of sludge the corn grain contained about 10 times the background level for check plots; the wheat contained about 15 times the background levels; and potatoes contained 10 to 12 times the background levels. Unless sludge containing Cd is used exclusively for the production of corn for grain which is relatively low in Cd, the effect on the food chain can be expected to be about equal for all these crops.
Cadmium in Animal Tissues
In order to assess the impact of increased plant Cd content on the food chain, several animal feeding trials have been conducted for the purpose of determining the relationship between dietary and tissue Cd content. In one study, meadow voles (Microtus Pennsylvanias) were fed herbage grown on sludge fertilized plots (16). Diets containing the sludge fertilized corn herbage had a Cd content of 1.09 ppm while the diets containing sludge fertilized sorghum contained 2.76 ppm Cd. For the purpose of determining the relative availability of Cd, the synthetic control diet and diets containing herbage grown on check plots were supplemented with Cd salts to give comparable levels of dietary Cd. The results are presented in Table 6. All diets containing supplemental Cd resulted in significant increases in liver and kidney Cd content. Muscle showed a greatly reduced ability to accumulate Cd. Although cadmium supplementation resulted in an increase in muscle Cd content, these changes did not prove to be statistically significant. The availability of Cd in herbage from sludge amended plots was equal to or greater than Cd added as soluble Cd to the herbage from check plots.
Feeding trials have been conducted with broiler chicks and laying hens (17). Normal diets which contained 0.20 ppm Cd were supplemented with 3 ppm Cd in order to approximate the levels observed with sludge treated herbage. The data from these experiments are presented in Tables 7 and 8. The results were very similar to those obtained with the meadow voles. Cadmium readily accumulated in liver and kidney while muscle was not as sensitive to dietary Cd content. Although the results are not statistically significant, 3 ppm dietary Cd consistently doubled muscle cadmium content. There was no transfer of cadmium to the egg in this experiment. These results are consistent with observations of Sell (18), who studied the transfer of Cd109 to the egg, as well as the results of Mills and Delgarno (19), who reported poor placental transfer of cadmium in sheep.
The results of these studies show that the amounts of Cd found in plants grown on sludge amended soils can increase the Cd content of tissues such as liver and kidney. Although there was not a statistically significant increase in muscle Cd content, these amounts of dietary Cd (1-3 ppm) consistently doubled muscle cadmium content. Interpretation of these results in relation to human intake of cadmium and long range health effects of Cd is required for the proper monitoring of sewage sludge application on land used for production of crops which enter the food chain. | v3-fos |
2018-04-03T01:54:55.052Z | {
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} | s2 | HAEMATOLOGICAL VALUES IN VITAMIN B12 RESPONSIVE CALVES
observed as early as 18 hours after inoculation of virus. These foci could be seen in unstained cultures, but were most obvious in infected cells that had been grown on cover slips and stained with haematoxylin and eosin. The virus suspension was found to directly haemagglutinate mouse, rat, chicken and turkey erythrocytes at room temperature, but not goose, duck, guinea pig, rabbit, sheep, horse, cow, pig or human type 0 erythrocytes. Haemagglutinating encephalomyelitis virus is at present classified as a coronavirus (Greig e t a / 1971; Phillip e ta1 1971). Supernatant fluid from the 9th passage of the virus in LLCPKI cells was mixed with equal volumes of serial tenfold dilutions of pig serum collected from gilts that had HEV affected litters. The mixtures were allowed to react at 4°C overnight centrifuged at 10,000 g for 60 minutes and the supernatant discarded. The deposit was resuspended in 3 drops of sterile distilled water, the preparation negatively stained with potassium phosphotungstate at pH 6.4 and examined in a Hitachi H300 electron microscope. Aggregates of virus particles morphologically resembling coronavirus were observed at serum dilutions to 100. The isolation of haemagglutinating agent with the morphological features of a coronavirus from the brains of pigs exhibiting a nervous disease suggested the virus was HEV of pigs. The virus was referred to the Central Veterinary Laboratory, Weybridge, Surrey, England for confirmatory tests, as no reference HEV antiserum was available in Australia. The virus isolated was identical, or closely related, to the Weybridge strain of HEV when tested by an indirect fluorescent antibody test (S.F. Cartwright, personal communication). A serological survey of pig herds in the State of Victoria using a haemagglutination-inhibition (HI) test was subsequently undertaken. The HI test was performed in V-shaped microtitre trays using 8 haemagglutinating units of virus, serial twofold dilutions of serum inactivated at 56OC for 30 minutes and 0.75% suspension of chicken erythrocytes. All serums were treated with a 25% suspension of acid washed kaolin in borate saline at pH 9.0 and adsorbed against chicken erythrocytes prior to testing. The virus-serum mixtures were allowed to react for 60 minutes and the test was read at room temperature 45 minutes after the addition of erythrocytes. An HI titre of 1 in 16 and greater was considered to be indicative of HEV specific antibody. A total of 364 serums from 24 herds (a minimum of 10 serums per herd) were examined. Ten of the 24 herds were found to have specific antibody, with the HI titre of most serums from infected herds being 1:64 to 1:128. Retrospective examination of stored serum revealed the presence of specific antibody in serum collected from herds of pigs in 1972, 1973 and 1974. These herds had experienced outbreaks of a syndrome resembling Vomiting and Wasting Disease as described by Cartwright ec al(1969) but the virus had not been isolated. The results indicate that HEV is endemic in pigs in Victoria, and that i t has been present since at least 1972. Clinical outbreaks of the disease, however, appear to be uncommon. Grateful acknowledgements are made to the Director and Dr. S. Cartwright, Central Veterinary Laboratory, Ministry of Agriculture, Fisheries and Food, Weybridge, Surrey, England for assisting with the identification of the virus. We are also indebted to Dr I . G . W. Parish, Leitchville, for submitting the diseased pigs. A. J . FORMAN. c . J . HALE; R. T . JONES, I. D. CONNAUGHTON. Regional Veterinary Laboratory, Department of Agriculture, Midlands Highway, Bendigo, Victoria, 3550
HAEMATOLOGICAL VALUES IN VITAMIN B,, RESPONSIVE CALVES
We have recently investigated haematological values in calves responding to vitamin B,, supplementation. The investigation was carried out near Robe, South Australia with Hereford calves depastured on calcareous sands supporting largely strawberry clover and lucerne. I n May-June 1978 the calves, aged between 1 and 12 weeks, were stratified by bodyweight within sex and allocated at random to treatment groups. One group of calves the 'BIZ' group, received subcutaneous injections of vitamin BIZ* at approximately 6weekly intervals. In a second group of calves, the 'COP' group each calf received by mouth two log cobalt pellets t when introduced to the trial and a third group, the 'Nil' group, were not treated. All calves grazed as one herd and were weighed ~ * 2 mg hydroxocobalamin/50 k g hodyweighi: 'Cohalex', V R Lahorarories, t ICI Ausrralia Lid, Melbourne. Vicioria. Wales. and given anthelmintic $ at 6-weekly intervals. Subcutaneous injections of copper b were given to all calves when introduced into the trial and then at 6, 13 and 32 weeks after the trial had commenced: serum copper of the calves, monitored at 6weekly intervals, was maintained in the normal range of 8 to 22 unmol/l.
Sydney. New South
Blood samples for haematology were taken into EDTA and values determined using a Coulter S blood cell counterll. The haematocrit values obtained by this method were checked manually using a micro-haematocrit centrifuge. When the values did not agree, the manual value was substituted to establish the true red blood cell count. This procedure was undertaken because the more microcytic cells may have fallen Ausrralian Veterinary Journal, Vol. 5 5 , October, 1979 below the threshold for the red cell count and be missed, thus resulting in a falsely low red cell count and haematocrit value. The Coulter cell size estimations may be considered valid since there are obtained by pulse height analysis independent o f red cell count or blood dilution.
Bodyweights of the calves in each experimental group were similar until the 26th week of the experiment when the 'Nil' group calves were significantly less (P<0.05) than the calves in the 'Bi,' and 'COP' groups. At the 32nd week of the trial, the bodyweights of the 'BI2' calves were heavier than the bodyweights of the 'COP' calves which in turn were heavier than the 'Nil'calves. 'Nil' calves were then given subcutaneous injection of vitamin B , , to prevent bodyweight losses and at the 38th week of the trial the 'Nil' calves were similar in bodyweight to the 'COP' calves but both groups were less than the bodyweight of 'Bi,' calves (Table 1).
Differences in bodyweights of calves were associated with differences in haematological values (Table 1). At 32 weeks the 'Nil' calves had a greater number of red cells but of smaller size, than calves from the 'B,2' group. A reduction in red cell numbers concomitant with an increase in cell size was observed in the 'Nil' calves by the 38th week of the trial, about 6 weeks after giving the 'Nil' calves single subcutaneous injections of vitamin B,? (5 mg hydroxocobalamin/calf). No response in haemoglobin concentration in red blood cells was observed with vitamin B,, supplementation. These findings appear to be at variance to those of Filmer (1933) and Neal and Ahmann (1937) who reported that cobalt-deficiency in cattle was associated with microcytic hypochromic anaemia.
Red cell size and numbers of red cells in the 'COP' calves were similar to those observed in the 'Nil' calves (Table 1) suggesting that the cobalt pellets in young calves may not be com-Ausrralian Vererinary Journal, Vol. 55, October, 1979 pletely effective in preventing the onset of vitamin B,, deficiency. It is of interest to note however, that the haematological values of the 'COP' calves approximated to those of the 'Bi2' calves at the 38th week of the trial. The altered haematological values may be related to an improvement in the vitamin B,, status of the 'COP' calves over the last 6 weeks of the trial although the effect of other factors such as age cannot be excluded. Schalm et a/ (1975) in summarising haematological values of cattle reported that red blood cells initially decrease in size over the first 3 to 4 months of life of the calf and then gradually increase in parallel with a gradual decrease in red cell numbers. | v3-fos |
2018-12-14T03:56:34.152Z | {
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} | s2 | The Effects of Ethephon on Cattleya aurantiaca (Orchidaceae) Seedlings
In Cattleya aurantiaca seedlings, ethephon (2-chloroethylphosphonic acid, also known as Ethrel), slightly accelerated leaf development at concentrations between 2.5 and 20 ppm but suppressed it at 50 ppm. It inhibited leaf length at concentrations of 2.5, 5, and 50 ppm but enhanced it at 10 and 20 ppm. Root formation was inhibited by concentrations higher than 2.5 ppm. Chlorophyll content of seedlings was highest on a medium containing 5 ppm ethephon.
Intro duct ion
Knowledge regarding the contribution of mycorrhizae to germinating orchid seeds and developing seedlings has increased in recent years, but uncertainties still exist, especially with regard to the functions of plant hormones. The available evidence suggests that germinating seeds and developing seedlings do not require an exogenous supply of auxins but may temporarily benefit from naphthaleneacetic acid (STRAUSS and REISINGER 1976). Effects of cytokinins or gibberellins are unclear (M; ITHNER 1959ITHNER , 1974ARDITTI 1967a, in press;STOUTAMIRE 1974), and there are no reports on the influence of ethylene on orchid seed germination and seedling development.
Ethylene promotes germination of rape, Brassica napus (TAKAYANAGI and HARRINGTON 197 1 ), but this by itself is not enough to suggest that it may have a similar effect on orchid seeds and seedlings because they differ from those of other flowering plants. However, a report that mycorrhizal fungi of orchids produce ethylene (HANKE and DOLLWET 1976) suggests that this hormone may affect seed germination and seedling development.
Ethylene, a gas which diffuses easily, is diicult to incorporate in culture media. Therefore, compollnds which gradually evolve ethrlene are better suited for in vitro experiments. Ethephon or Ethrel (2-chloroethylphosphonic acid) is such a compound (ANONYMOUS 1967;WARNER and LEOPOLD 1969 ;YANG 1969). It can elicit ethylene-like effects such as flowering of pineapple, abscission, and suppression of asexual embryogenesis in vitro (ANONYMOUS 1967;COOKE and RANDAL 1968;TISSERAT and MURASHIGE 1977). We used ethephon as a nutrient medium component in a study of ethylene effects on seedling development in CattZeya aurantiaca. . 1), and reduced the percentage of those with both (table 1). At concentrations of 5, 10, and 20 ppm, ethephon stimulated the appearance of numerous root hairs on circa 6'Wo of the seedlings.
Material and methods
LEAF DEVELOPMENT AND LENGTH. Concentrations of 2.5, 5, 10, and 20 ppm slightly enhanced leaf development, as indicated by the decrease in the number of stage 4 seedlings coupled with an increase of stage 5 plantlets (table 1). Leaf development was suppressed by 50 ppm, and as a consequence the number of stage 5 seedlings increased (table 1). Ethephon enhanced first and second leaf elongation at 20 ppm ( fig. 3) 5 ppm (fig. 3).
but, surprisingly, caused a reduction in length at 2.5 and
CHLOROPHYLL CONTENT. Chlorophyll a, chlorophyll b, and total chlorophyll content all followed the same trend ( fig. 4). At all concentrations of ethephon, chlorophyll content increased relative to the KC and the KC plus 2-ml ethanol controls. The greatest increase occurred at 5 ppm ( fig. 4).
ADDITIONAL OBSERVATIONS. Some excessively swollen seedlings, occurring singly or in aggregates, were observed on the KC plus 2-ml ethanol control and at 20 and 50 ppm ethephon but not at the lower concentrations. Many seedlings died after reaching growth stage 5 ( fig. 1) on a medium containing 50 ppm ethephon.
Discussion
Cumulative release of ethylene from ethephon is proportional to its concentration in the medium (TISSERAT and MURASHIGE 1977). During 4 wk a total of 60 ,ul/liter were released from a medium containing 3 ppm ethephon and "... 225 pl/l for 10 mg/l and 450 ,ul/l for 30 mg/l" (TISSERAT and MURASHIGE 1977). Comparisons with or extrapolation and interpolation from these data indicate that the amounts of ethylene released in our media were 55 ,ul/liter (in the presence of 2.5 ppm ethephon), 110 pl/liter (5 ppm), 225 ,ul/liter (10 ppm), 340 ,ul/liter (20 ppm), and 600 ,ul/liter (50 ppm). Therefore, it is reasonable to assume that the effects we observed w ere brought about by differences in ethylene levels. The large number of excessively swollen seedlings we noted on 20 and 50 ppm ethephon, more than on the ethanol control, also argues for a specific ethylene effect since this enlargement is similar to the typical swelling brought about by the gas (BURG and BURG 1966;CHADWICK and BURG 1967). This effect may be due to disruption of normal polar cell expansion and increases in fresh weight as in pea internodes (EISINGER and BURG 197 2) . However, it is possible that phosphonate produced by the decomposition of Ethrel may have also had an effect, as in asexual embryogenesis in carrot callus cultures (TISSERAT and @tURASHIGE 1977). A comparison between our observations and the effects of naphthaleneacetic acid on seedlings of the same species (STRAUSS and REISINGER 1976) indicates that the influence of auxins and ethylene on orchid seedlings is not related.
The reduced or inhibited development of Cattleya aurantiaca seedlings brought about by ethephon is reminiscent of its inhibitory effects on Algerian ivy tissue cultures (STOUTEMYER and BRITT 1970) and asexual embryogenesis as well as the development of advanced embryonic stages in carrot tissue cultures (WOCHOK and WETHERELL 1971; TISSERAT and MURASHIGE 197 7 ) .
Concentrations of 5 and 50 ppm ethephon severely inhibited foliar elongation; and 2.5 ppm, although inhibitory, had a more moderate effect (fig. 3). The highest concentration, 50 ppm, also inhibited leaf development. This agrees with reports that growth of tomato and marigold leaves was inhibited by low concentrations of ethylene (ABELES 1973). Inhibition by 50 ppm is undoubtedly due to supraoptimal concentrations, as indicated by the death of seedlings past growth stage 5 on this medium. The other iBLE 1
BOTANICAL GAZETTE effects of low ethylene concentrations (ca. 20 ppm) on pea seedlings (SMITH and RUSSELL 1969).
Increases in chlorophyll content ( fig. 4) are not an expected response to ethylene. In fact, this hormone induces senescence and, therefore, often brings about reductions in chlorophyll content (ABELES 1973). One possible explanation of our findings is based on the observation that the ethephon concentrations which increase chlorophyll levels ( fig. 4) reduce leaf length. It is conceivable, therefore, that these concentrations may reduce the size of cells but not the amount of chlorophyll per cell and, consequently, per leaf and/or seedling. Hence, unchanged chlorophyll content in smaller leaves results in higher pigment levels per unit of weight ( fig. 4). differences in leaf elongation are those between suboptimal (10 ppm) and optimal (20 ppm) concentrations.
A comparison of the percentages of plantlets at stages 4, 5, and 6 in the ethephon-containing cultures, the controls, and the 6-mo-old seedlings (table 1) indicates that low levels of ethylene can enhance leaf development somewhat, even if the same concentrations may inhibit subsequent expansion.
Root formation distinguishes between stages 5 and 6 ( fig. 1). A comparison of the percentages of seedlings at these stages (table 1) is an indication of the effects of ethephon on root production. It shows that the percentage of stage 6 seedlings (i.e., those with roots) is higher on 2.5 ppm than on any of the other ethephon-containing media. This is in line with reports that Ethrel can enhance rooting in apples, blueberries, mung beans, and tomatoes (ABELES 1973). However, in orchids it appears that concentrations above 2.5 ppm are supraoptimal for root formation. The stimulation of root hair formation by 5, 10, and 20 ppm ethephon is similar to the | v3-fos |
2018-04-03T04:48:15.680Z | {
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} | s2 | Comparative susceptibility of starch granules of double- and triple-mutants containing amylose-extender, waxy, sugary-1, sugary-2 and dull genes of maize inbred OH43 (Zea mays L.) to amylase.
susceptibility fungal
This paper presents the relative susceptibility and extent of enzyme degradation in yet other endosperm mutant combinations which have been synthesized in the maize inbred Oh43, but not reported, and confirms the results of a few genetic effects and interactions by these combinations on the relative susceptibility and extent of enzyme degradation. We prepared starch granules from kernels of near isogenic conversions of 14 double-and 26 triple-mutants containing ae, 14 double-and 18 triple-mutants containing wx, 15 double-and 20 triple-mutants containing su1, 13 double-and 23 triple-mutants containing su2 and 14 double-and 19 triple-mutants containing the dull (du) gene and compared the relative susceptibility of these starch granules to fungal glucoamylase.
Starch granules. Starch granules were prepared by a method reported previously (2).
Sources of enzymes. Sources of Rhizopus glucoamylase, glucose oxidase and peroxidase were described earlier (2).
Analytical methods. Susceptibility of starch granules to glucoamylase was studied by a method reported previously (2). Percent degradation shown in Figs respectively. The data (Table 1) were expressed on the basis of the relative percentage of the commercial normal control enzyme degradation determined as a percentage glucose equivalent. Preparation of starch granules attacked by glucoamylase, specimen mounting and scanning electron microscopy. Starch granules were attacked by glucoamylase under the conditions reported previously (2). Procedures for specimen mounting and scanning electron microscopic observations were followed as described earlier (5) except that starch granules were coated with 100-200 A of gold layers by ion spattering by the use of an Ion Coater IB-3 of Eiko Engineering Co., Ltd.
RESULTS
Starch granule susceptibility to glucoamylase of double-and triple-mutants containing ae gene of the Oh43 maize Figure 1 and Table 1 show that among the starch granules of 14 doublemutants containing ae, those of ae sh2 and ae wx tended to be digested faster than normal, while those granules of the remaining mutant combinations were digested at a similar rate to, or slower than normal.
Starch granules of seven triple-mutants containing ae, which were digested faster than normal, showed that ae du wx and ae su2 sh2 were fastest followed by ae wx sh2, ae du su1, ae su1 wx, ae su1 sh2 and ae O2 sh2 ( Fig. 2 and Table 1). Granules of the ae du wx and ae su2 sh2 mutants were digested 2.7 and 2.6 times faster, respectively, than normal. Starch granules of 19 triple-mutants containing ae were digested at a similar rate to, or slower than normal. Table 1). The wx su2 mutant granules were 3 to 4 times more susceptible than normal.
Starch granule susceptibility to glucoamylase of double-and triple-mutants containing su1 of the Oh43 Figure 4 and Table 1 show that starch granules of 10 of the double-and 10 of the triple-mutants containing su1 were digested 3.0 to 7.9 times faster than normal. Those starch granules of su1 su2 sh1, su1 su2 sh2 and su1 h were digested nearly eight times more rapidly than normal.
Starch granule susceptibility to glucoamylase of double-and triple-mutants containing su2 of the Oh43 Starch granules of 11 double-and 16 triple-mutants containing su2 were digested 2.8 to 8.4 times faster than normal (Fig. 5 and Table 1). Those granules of the su2 wx sh2, su2 wx sh1, su1 su2 sh1 and su1 su2 she triple-mutants were digested the most rapidly, while su2 bt2 and sue she were the most rapidly digested of the su2 double-mutants.
Starch granule susceptibility to glucoamylase of double-and triple-mutants containing du of the Oh43 Figure 6 and Table 1 show that starch granules of six double-and 13 triple -mutants containing du were digested faster than normal. Granules of du su2 wx mutant were digested 5.2 times faster than normal and the du su1 double-mutant was digested 3.2 times faster than normal. Granules of seven double-and one triple -mutant containing du were digested at a similar rate to those of normal maize. SEM observations of starch granules attacked by glucoamylase SEM observation of starch granules of the double-and triple-mutants containing wx and du with the exception of ae wx, ae du, ae wx fl2, ae du sh1, ae su1 wx, ae du su1, du su1, du su1 fl2, du su2, du su2 o2, du sue sh1, du su2 sh2, su2 wx o2, su2 wx sh1 and su2 wx sh2 showed, in general, numerous pinholes on the surface layers after enzymatic reaction and pores that penetrated into the inner layers of the granules during enzymatic attack (Figs. 7b-7g). On the other hand, starch granules of the double-and triple-mutants containing ae, which had a lower susceptibility to amylase, showed shapes and surfaces similar to those of the native undegraded granules after enzymatic reaction (Fig. 7h). However, starch granules of the doubleand triple-mutants containing ae, which had a similar or greater susceptibility to
DISCUSSION
As stated by SMITH and LINEBACK (7), it is apparent that the extents and patterns of degradation of native starch granules are functions of the source of the starch and type of amylase (1,2,8). This is true for starch granules of endosperm mutants of maize (2,5,6).
Among single endosperm mutants, starch granules of the su1 and sue mutants were digested by amylases two to three times faster than normal. Starch granules of the wx, sh2, bt1 and bt2 mutants tended to be digested faster than normal (2)(3)(4)(5)(6).
Starch granules of the double-and triple-mutants containing su1 and su2 were digested two to eight times faster than normal (Figs. 5 and 6 and Table 1). The su2, bt1 and h and combinations of sh1 fl2, su2 fl2, su2 o2, su2 sh1, su2 sh2, wx fl2 and o2 sh2 further enhanced the starch granule susceptibility of the double-and triple-mutants containing su1. The bt2 and sh2 and combinations of wx o2, wx sh1, wx sh2 and sh1 fl2 enhanced the effect of sue. However, the ae gene (2) seems to be epistatic to su1 and sue, which resulted in starch granules that were less susceptible to glucoamylase in the double-mutants, ae su1 and ae su2 (Fig. 1) and the triple-mutants, ae su2 fl2, ae su2 o2 and ae du su2 (Fig. 2).
Starch granules of the double-and triple-mutants containing wx were digested about two times faster than normal ( Fig. 3 and Table 1). Recently, YAMAHA and TAKI (9) and BOYER et al. (10) reported that ae wx starch consists of nearly 100% amylopectin with an anomalous fine structure. In this connection, elucidation of the relationship between the relative susceptibility to glucoamylase of starch granules of the ae du wx, ae wx sh2, ae su1 wx, ae wx, ae wx sh1, ae wx o2, ae su2 wx and ae wx fl2 mutants (Table 1) and the structural characteristics of these starches is needed. Further studies are in progress.
Starch granules of the double-and triple-mutants containing sh2 were digested 1.2 to 8 times faster than normal ( Table 1). The interactions of sh2 with wx, su2, su1 and du genes enhanced the effect of sh2 on starch granule susceptibility of the double-and triple-mutants containing sh2.
Starch granules of double-mutant combinations with o2 showed digestion properties that were comparable to their respective nonopaque single-mutant counterpart (2,5,6). The same general situation was observed for triple-mutant combinations with o2 and their respective nonopaque double-mutant counterparts (Table 1). | v3-fos |
2018-04-03T03:40:39.146Z | {
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} | 0 | [] | 1979-05-25T00:00:00.000Z | 7817397 | {
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} | s2 | Isolation and characterization of proteoglycans from porcine lungs.
Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.
Proteoglycans
were extracted from porcine lungs with 4 M guanidinium chloride.
The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through Ad from the bottom to the top of the gradient, were obtained. 'Ihe pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled AIDI through AID4 from the bottom to the top of the gradient.
These fractions were analyzed for their protein, uranic acid, glucosamine, galactosamine, hexose, and sialic acid content.
The fraction AIDI with the highest buoyant density had the highest content of uranic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient.
As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uranic acid and galactosamine content decreased.
The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leutine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.
Proteoglycans, like collagen and elastic fibers, are a major component of all connective tissues. In recent years, proteoglycans of cartilage from various sources have been isolated and characterized (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Results of these studies indicate that the cartilage proteoglycans are large aggregates composed of up to 140 proteoglycan subunits linked noncovalently to a linear molecule of hyaluronic acid in association with two link proteins. According to the current structural model, the proteoglycan monomer consists of approximately 110 chondroitin sulfate and 50 keratan sulfate side chains attached covalently to a linear core protein . The average molecular weights of the core protein, chondroitin sulfate, and keratan sulfate are about 200,000,20,000 and 5,000, respectively. The average molecular weight of the proteoglycan monomer is approximately 2 to 3 x 10" (3,13,14). The proteoglycan monomer can be divided into three distinct portions: a smaller nonvariable hyaluronic acid-binding region with a few or no polysaccharide side chains at one end, and a much larger variable segment containing chondroitin sulfates on the other end, with a keratan sulfate-rich region in the middle of the molecule. The variation in the size of the proteoglycan molecule is mainly due to the variation in the size of the chondroitin sulfate-enriched region (12,15,16).
In terms of the overall composition by weight, the
AND DISCUSSION
The lung tissue used in the present study was free from bronchi, large blood vessels, and trachea. Light micrographs of this tissue taken from random samples show the smooth muscle around arterioles, veins, and terminal bronchi and demonstrate the absence of cartilage ( Figs. 1 and 2). Proteoglycans were extracted from the lung tissue in 4 M guanidinium chloride, pH 5.8, which solubilized approximately 60 to 70% of the total glycosaminoglycans as measured by uranic acid. The major glycosaminoglycans extracted by this dissociative solvent were hyaluronic acid, chondroitin sulfate, and heparin sulfate (Fig. 3). When this proteoglycan extract was subjected to the density gradient centrifugation under associative conditions (20), four equal fractions labeled A, through Aq from the bottom to the top of the gradient were obtained. The distribution of uranic acid and protein in these fractions is shown in Fig. 4. The pools of Fractions Al through Aq contained approximately 75, 14, 9, and 2% of the total uranic acid, respectively.
The material recovered as a surface gel at the top of the associative gradient accounted for approximately 2 to 3% of the applied sample, based on the dry weight, and contained less than 1% of the total uranic acid. To ensure removal of extraneous proteins, the A, samples were recycled through a second associative density gradient, and the bottom one-half of these samples were pooled. These recycled A, samples containing proteoglycan aggregates (5) were subjected to the dissociative density gradient centrifugation (20), and four equal fractions labeled A,D, through AID4 from the bottom to the top of the gradient were obtained. The relative amounts and composition of these four fractions are given in Table I. Approximately 4 to 6 mg of proteoglycans were obtained from 1 g of wet tissue. The proteoglycans recovered at the highest density had the lowest protein and highest uranic acid and galactosamine content. Chondroitin sulfates were the only detectable glycosaminoglycans present in this fraction (Fig. 3). Their identification was confirmed by the disappearance of the band corresponding to chondroitin sulfates on the cellulose acetate electrophoresis after digestion with chondroitinase AC and chondroitinase ABC. With the decrease in the density of the dissociative gradient the protein, hexoses, as well as the sialic acid content increased, whereas uranic acid and galactosamine decreased. AID2 and AlDs fractions contained mostly hyaluronic acid and small amounts of heparin sulfate (Fig. 3). After digestion with Streptomyces hyaluronidase the major band corresponding to hyaluronic acid on the cellulose acetate paper disappeared, but a minor band corresponding to heparin sulfate could be detected. Heparin sulfate was further confirmed by its susceptibility to deamination cleavage (30). The AID4 fraction was rich in protein, glucosamine, hexoses, and sialic acid. This fraction was found to contain heparin sulfate (Fig. 3) which could be degraded by the action of nitrous acid. The SDS polyacrylamide gel electrophoresis of AID.+ fraction showed two protein bands with apparent molecular weights of 50,009 and 65,000 (Fig. 5). These two proteins may be compared with the two hyaluronic acid-binding proteins of apparent molecular weights 40,000 and 65,006 isolated by Hascall and Heinegard (6) (4), dermatan sulfate (5), keratan sulfate (S), and heparin (7). dalton protein present in the AID4 fraction (Fig. 5) represents the hyaluronic acid-binding region of the proteoglycan monomer as described by Hascall and Heinegard (6) is not known. Gel chromatography (Fig. 6) was used to assess the size of the proteoglycans as well as the effect of combining samples from the top and middle of the dissociative gradient on the proteoglycan monomers. Most of the proteoglycan aggregates, AI, were excluded from the Bio-Gel A-50m column, whereas most of the monomers, AIDI, were included in the column. However, a small fraction of AI was included in the column, and a small portion of AIDI was excluded from the column. The fractions from the middle of the dissociative gradient increased the size of the proteoglycan monomers (Fig. 6, f proteoglycans very little by itself (Fig. 6h), although in the presence of the middle fractions, AID2 and AIDS, it did increase the size of the proteoglycan monomers (Fig. 6, i and j) . It, therefore, appears that an active component was present in the middle fractions, which contain hyaluronic acid. The amino acid composition of four fractions, AlDl through A1D4, are somewhat similar but not identical (Table II). The major constituent amino acids are aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine. No hydroxyproline or hydroxylysine was detected in any of these fractions. The lung AIDl fraction differs in its amino acid composition from that of the cartilage AIDI fraction (5) especially in its higher content of aspartic acid and alanine and lower content of serine and proline. The amino acid composition of the AID4 fraction is quite different from those of collagen and elastin. It is similar to the amino acid composition of the "structural glycoproteins" present in the connective tissue matrix (41). Our results agree with the previously published reports that the content of aspartic acid increases and that of glycine decreases with the decrease in buoyant density of the proteoglycan fractions (7,42,43). The proteoglycans isolated from the lung tissue are comparable to those of aorta (44). The 4 M guanidinium chloride extracts of bovine aorta contained chondroitin sulfates, hyaluronic acid, and heparin sulfate. The proteoglycans of aorta interacted with hyaluronic acid and cardiovascular connective tissue proteins to form aggregates.
The results described in this report clearly demonstrate that cartilage-free lung tissue contains cartilage-like proteo-4266 Lung Proteoglycans | v3-fos |
2017-07-29T04:25:16.416Z | {
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} | s2 | A review of literature on the syndrome of arthrogryposis and palatoschisis (S.A.P.) in Charolais cattle 1976–1979
vous system at the level of the motor end plaque, followed by a disorganisation of muscle tissue. The anomaly presents similarities with a hereditary condition in the mouse known as musculav dysgenesis (mdg). The frequency of the syndrome in purebred Chavolais in France as well as in Canada has been reported at 0.5 p. 100 among newborn calves. The gene frequency is q’= 0.2o and penetrance in homozygotes is 0.15 and 0.8 in males and females respectively. It has been demonstrated that penetrance may appear to be complete as a result of differences in the probabilities of affected calves being reported depending on the numbers occuring in individual herds or sire groups. It is also pointed out that breeding experiments using animals already identified as carriers could result in elevated estimates of penetrance. Two methods of detecting carrier bulls have been applied: using a test herd of carrier females as in Canada or in the normal course of progeny performance testing pure bred beef cattle as in France. In each case only a small number of bulls can be tested compared with the number required to meet the needs of the breed. Total eradication of calf losses by 5.A.P. is not only theoretically impossible but could even be undesirable, at least from an economic point of view since heterozygous females may be sufficiently more prolific to more than compensate for the low losses resulting from the presence of the gene in the population. Nevertheless, national associations of Chavolais breeders have other concerns than those associated with the genetics of calf losses, namely commercial considerations.
Summary
Twenty or so papers concerning S.A.P. have been published since 197 6 and can be added to the q 9 already included in a descriptive bibliography published in 197 6. Important information on the syndrome has been added: the age of the embryo at first appearance has been determined (about 3 . 5 months). An identifiable anomaly affects the nervous system at the level of the motor end plaque, followed by a disorganisation of muscle tissue. The anomaly presents similarities with a hereditary condition in the mouse known as musculav dysgenesis (mdg).
The frequency of the syndrome in purebred Chavolais in France as well as in Canada has been reported at 0 . 5 p. 100 among newborn calves. The gene frequency is q'= 0 . 2 o and penetrance in homozygotes is 0 . 15 and 0 . 0 8 in males and females respectively.
It has been demonstrated that penetrance may appear to be complete as a result of differences in the probabilities of affected calves being reported depending on the numbers occuring in individual herds or sire groups. It is also pointed out that breeding experiments using animals already identified as carriers could result in elevated estimates of penetrance.
Two methods of detecting carrier bulls have been applied: using a test herd of carrier females as in Canada or in the normal course of progeny performance testing pure bred beef cattle as in France. In each case only a small number of bulls can be tested compared with the number required to meet the needs of the breed.
Total eradication of calf losses by 5.A.P. is not only theoretically impossible but could even be undesirable, at least from an economic point of view since heterozygous females may be sufficiently more prolific to more than compensate for the low losses resulting from the presence of the gene in the population. Nevertheless, national associations of Chavolais breeders have other concerns than those associated with the genetics of calf losses, namely commercial considerations.
Introduction
The first scientific publications of the Arthrogryposis and Cleft Palate Syndrome (S.A.P.) appeared in 19 67 (I,A UV B R GrNB et BLIN).
A bibliography prepared by I aAUV B R G N B and F AU C ON ( 197 6) contained 49 papers published during the period 19 6 7 -75 . Since then some 2 o additional articles have been published on this subject which are reviewed in this paper. Source of the Reports A!pearing Since 197 5 There is a series of articles describing the investigations conducted at the University of Saskatchewan, Saskatoon, Canada supported financially by the Canadian Charolais Association (C.C.A.) and the International Charolais Federation (F.I.A.E.R.B.C.): Q UANTZ ( 197 6), R USSEL ( 197 6a), RussEL ( I g 7 6b), ANONYMOUS ( 1977 ), NI!I,SFN et al. ( 1977 ), RUSSEI, ( 1977 ), ANONYMOUS (1978), RUSSEI, (1979 (HARTr,!Y and WANNER, 1974 , overlooked in the listing by I, AUVER G NB and F AUCON , 197 6) have recorded cases of S.A.P. appearing in Charolais in these countries.
Included also is a report on the Canadian situation requested by F.LA.E.R.B.C. (I,AUVERGNE ,197 6), on the prospects of eradication (I,AUV!RGN! and How!r.r., 1977 ) and on the situation in the United States of America (L AU - VERGNE, 1979).
Description of the syndrome (S.A.P.) These last years have seen marked progress in an understanding of the histopathology of S.A.P. beginning in I g 7 6 with work by RussEL on which he reported at the Monter y ey Convention (Russ!r., 1977 ) and described in detail in his Ph. D. thesis of 1979.
Using a part of the C.C.A.'s experimental herd assembled at Goodale near Saskatoon, RussEL obtained embryos displaying various stages in the development of the syndrome. In addition he studied S.A.P. calves at birth. From this study if was possible to determine the prenatal age of the embryo at which the syndrome could first be detected ( 3 . 5 months) and the kind of abnormalities present at this age: retraction of axons from the post-synaptic membranes.
It appears that a change in the differentiation of neurons could be the cause of muscle hypotonia and the accompanying leg deformities observed in S.A.P.
calves.
In the muscle of newborn S.A.P. calves, structural abnormalities are evident at the level of the neural plaques accompanied by faulty differentiation of 111ts::le fibers.
The characteristics of nerve-striated muscle interaction in the S.A.P. calf have been specified by Rr!G!R ( 1979 ) and RWG!R et al. ( 19 8 0 ). A detailed parallel study in a neuromuscular mutant in the Mouse (« Muscular dysgenesis !· mdg) has shown similarities in the morphological expression of the neuropathologic impairment: the atrophic muscular fibers present a disorganized myofibrilar structure (Z stria), the innervation networks are deeply desorganized as shown by tests of metallic impregnation of nerves, with a lot of contacts with an immature aspect and an abnormal distribution of the innervation (R IEGER and PINÇON-RAYMOND, 1980).
There are also defects of biochemical nature: in the Mouse the biosynthesis of molecular forms of acetylcholinesterase is quite incomplete (as N AWRO T, I g 7 6; NAwxoT et al., 1979 have also pointed out). In the S.A.P. calf the mechanisms of localisation and restriction of the specific molecular forms of innervation are abnormal. The functional receptor of acetylcholine (neurotransmitter of the neuromuscular synapse) shows an aberrant distribution in the mdg mouse as well as in the S.A.P. calf.
The irregular character of the affected muscles has been stressed by R IEGER ( 1979 ). The choice of abnormals examined by R USSELL ( 1979 ) (all cases has a cleft palate) must not detract from the fact that there are frequent cases where the animal has analogous muscular and tendinous abnormalities but lacks the cleft palate and can be classed as true S.A.P. (I,W P or,D et al., 19 69, GIROUD, 1975, LAUVE R G N E, 1975. Behaviour and frequency of the S.A.P. gene Studies already cited by L AUVERÜNE and F AUCON ( 197 6) such as that of LA UV E R G N E ( 1975 ), established that the S.A.P. condition was hereditary, monofactorial, and caused by an autosomal recessive gene. Although in France the gene seems to have incomplete penetrance, it may be complete in certain cases, at least in Canada.
In France this result has been confirmed beyond doubt from an analysis of beef cattle test data obtained from purebred animals (I,!xoRT et al., ig 77 ); estimations of penetrance were o.i5 for males and 0 . 09 for females and the gene frequency was calculated to be 0 . 20 . Similar frequencies of the abnormality were revealed by a survey among Charolais breeders in Saskatchewan (H OWELL and I,nuv!RGN!), 197 8) after allowance was made for percentage of Charolais breeding in the crossbreds. This would indicate that the penetrance of the gene is not influenced significantly by crossing with other breeds.
The first explanation of the almost total penetrance estimated from data obtained from cattle breeders such as reported by N AWROT ( 1973 ) in Canada or HArrs!'r et al. ( 197 8) in Belgium derives from the fact that inclusion of the proband (the first observation) strongly biassed the date because of its variable probability of observation: escaping inclusion when only one abnormal appears but certain to be included when several abnormal calves appear in a short time span within a herd. This explanation could nevertheless hardly be applied to account for the mendelian segregation compatible with complete penetrance of the recessive homozyzote observed in the experimental crosses conducted at Kinsella (BERG and G OONEVARDENE , 1974 ) Two comments can be made: -it is possible that some normal appearing breeding animals may be homozygous recessive as demonstrated by BERG and Goorr!wnxD!N! (rg76), -the use of female carriers of the S.A.P. gene, detected as such because they have already produced an abnormal calf may well lead to an increased penetrance. That being the case, it may be wise to exert some selection against the tendency of certain carrier females to increase the penetrance of the S.A.P. gene when their offspring are homozygous recessive.
The problem of eradication At Harrogate, a C.C.A. report (ANONYMOUS, zg 7 8) emphasized that the histopathological studies in Canada do not allow distinction to be made between the heterozygous carrier and the homozygous normal animals by a simple examination of the living animals.
But it does appear that the works of R U SSE L ( 1979 ) and Ri!GEx et al. (1979) could offer orientations for the search of some biochemical or morphological means of detection. The only method for the detection of normal appearing carriers (hetero or homozygous) remains a breeding test. Detection and removal of the carriers from the breeding population has so far been undertaken only in two countries and by different methods.
-In Canada, the C.C.A.. set up an experimental herd of normal appearing carriers. As already stated, a part of this herd was used by R USSEL ( 197 6) to produce the abnormal embryos used in his studies. Sires to be tested were mated to these presumed heterozygous cows.
Apparently penetrance is very nearly complete (refer to the foregoing remarks concerning augmentation of penetrance in descendants of cows having aleardy produced a defective calf), so it would only be necessary to perform a few matings to identify the genotype of a bull with reasonable probability of not having made an error (QunrrTZ, 1976).
-In France, for pure bred bull testing, the frequency of the gene in the entire female propulation is used (q = 0 . 20 ) which is high enough but it doesn't have as high a penetrance as in the experimental herd in Canada. Also, the number of calves required for equal precision is much higher (about ten times). According to L EFORT et al. (ig 77 ), the probability of nondetection of a heterozygous carrier with i5o progeny is o.16.
The advantage of the French method over the Canadian is the very low cost since the detection of abnormals is made in the ordinary routine of progeny performance testing. However, these two methods permit the testing and detection of only a small number of males each year, and in populations where artificial insemination is not used to any extent, at least in seed stock herd:, they are not effective for eradication of the gene.
In fact, as concluded by L AUV E R G N E and HO W E LL ( 1977 ) in their report to F.I.A.E.R.B.C. it can be asked whether eradication is possible or even desirable.
To the first question, the reply is in the negative. Neither of the two methods examined above is capable of detecting all the carrier males in a breed. What's more, if the French method was rigidly applied it would become less and less effective because, as the gene frequency decreased in the female population, there would be required larger and larger numbers of progeny from a bull to eventually declare him a non carrier with the same high level of probability. In the event of there being a selective advantage for the heterozygote, eradication would be theoretically impossible (I,!xoxT and LAUVERGNE, 1974).
The second question can also be answered in the negative. For one thing the economic loss by S.A.P. is very low ( 0 . 5 p. ioo of newborn calves compared to total losses from all causes currently about 10 p. 100 ), and is probably less than the cost of an intensive eradication program. Secondly if the observations of BERG and G OON E WARD E N E ( 1974 ) are accepted (that heterozygous females were more prolific than homozygous normal females), the losses caused by the abnormals would be less than the gains resulting from the heterosis phenomenon ( 3 . 3 p. 100 additional newborn calves at a gene frequency of q = 0 . 20 ). However, there are other reasons for undertaking an eradication program. Commercial considerations may dictate it, especially in North America where the competition among beef breeds is very keen. This partially explains the program undertaken by the C.C.A. to prove that nothing is neglected in breed improvement. Reasoning from the same position of commercial competition, authorities in the American Charolais Association (A.I.C.A.) have on the other hand chosen to systematically ignore the very existence of the anomaly, an attitude in line with the conclusions of a genetic study by I,nuv!RGN! (zg79).
Conclusions
Even if it is recommended to the various Charolais associations that the anomaly (S.A.P.) be ignored in their testing and selection programs, it would be desirable for them to routinely monitor cases of S.A.P. if only to be assured that it is truly S.A.P. and not an anomaly caused by a different gene or even a virus.
In any case, the study of S.A.P. in all its manifestations at the histological and histochemical levels, is far from complete and must be pursued logically.
The fundamental neurobiologic studies of neuromuscular system in the S.A.P. calf could be developed with profit using the experience from the study of mutants in the Mouse (the already quoted mdg; R IEGER , 1979 ), but also of variants in Man such as « !4!A!'og'r!'OMS multiplex congenita o studied by B ANKER et al., 1957 . This type of studies could not only bring new views on the neuro-muscle contact and its stabilization but also new contributions to the knowledge of biochemical parameters in these pathological cases. It could even be possible in the future to detect normal overlap carriers by checking light abnomalities of some of these parameters in the living population of Charolais calves (RI!G!x et al., ig8o).
It would be equally desirable to continue on-farm studies of the frequency of the syndrome in the pure breed and its crosses to confirm the values already reported in France and Canada.
Chronological bibliography of S. A.P. papers 197 6-1979 The numeration of the papers comes in the succession of the numeration of the 19 6 7 -1975 article given by I,AUV!RGN! and F AU C O rr ( 197 6 He gave also the basic principle for the law of eradication of gene in the case we are interested in.
Without him the difficult statistical situations we faced in the study of S.A.P. as well in France as in Canada could certainly not have been so quickly solved. | v3-fos |
2017-06-19T09:33:17.763Z | {
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} | s2 | Inheritance of body weight and breast length at 8 weeks of age in meat type strains of chickens
Two pure strains of meat type chickens were used, White Pbymouth Rock and Light Sussex. They were involved in a cross breeding programme. Heterotic effect on body weight was present and confirmed the theory of homogenetic heterosis. Performances and heritabilities of body weight at 8 weeks of age and breast length at the same age were estimated. Results showed a high magnitude of heritabilities for these two characters. A high positive phenotypic and genetic correlation between the two characters gives an expected response of indirect selection and suggest an advantage of establishing a selection index including breast length to improve body weight.
Introduction
The present investigation used both, White Plymouth Rock and Light Sussex breeds in a crossbreeding programme. The aim of this study was to report estimates of heritabilities of body weight and breast length at 8 weeeks of age in the two strains and their reciprocal crosses. Phenotypic and Genetic correlations between the two previous characters were reported. Many reported estimates of such parameters were available in the literature but relatively few of the reported estimates have been based on closed flock populations and crosses. The two used strains in this experiment were imported since about twenty five years ago, and kept closed in the experimental farm belonging to the Ministry of Agriculture, U.A.R.
Estimates of such genetic parameters based on random and control strains were reported by M ERRITT ( 19 66). Estimates based on unselected populations were reported by KING (ig6i) and estimates based on selected population have been more numerous and summerized by SWG!!, ( 19 6 2 ). estimates of heritability of body weight at 8 weeks of age were summarized by K INNEY (ig6g) and were of high magnitude.
Heritability estimates of breast length were reported by many investigators, G ODFREY & GoonMarr ( 195 6) found estimates of heritabilities of breast length at 9 weeks of age using the various components methods as o. 4 . The same authors obtained phenotypic and genetic correlations between breast length and body weight, estimates were 0 . 3 and o.5 respectively.
CoCK et al. ( 195 6) found heritabilities of body weight and breast length at 10 weeks of age, average about o. 5 o and 0 . 30 respectively. They also reported the phenotypic correlation between the two characters as o.2g. BxuNSON et al. ( 195 6) estimated the mean phenotypic correlation between 10 week body weight and breast angle to be o.ig for males and 0 . 13 for females. Finally these estimates, in addition to characterizing the population itself, can be useful in selecting either for one character or to be involved in selection index programme to obtain maximum progress in both characters.
Material and methods
This experiment was carried out during 1975 and rg 7 6 at El Kanater-Khairia, Research Station belonging to the Agricultural Research Centre, Ministry of Agriculture, U.A.R.
Two breeds were used in this experiment, namely, White Ply7nouth Rock (PP) and Light Sussex (SS). The two pure breeds and their reciprocal crosses were obtained.
Sixteen pedigree pens were available, eight of them were headed by sires from PP and others were headed by sires from SS strain. In respect to females, equal number of dams from each strain were distributed randomly into each pedigree pen.
Body weight and length of breast were recorded at 8 weeks of age. Regarding the first year, females of SP crosses were the heaviest, followed by PS crosses, PP strain and finally SS strain showed the lowest body weight.
In the second season PP strain was the heaviest followed by PS, SP and finally SS. The intermediate parental body weight in the first season was 510 g and the mean body weights of crosses were 5 8 2 and 5 90 g. for SP and PS respectively. In the second year the intermediate parental body weight was q. 7 q. grams and the mean body weights of the crosses were 5 21 and 5 19 g. for PS and SP respectively.
As for males in the first year PS crosses were the heaviest followed by SP, PP and SS strain. In the second season PP strain was the heaviest followed by PS and SP crosses and latest SS strain. These results showed the same trend as for females in both years indicating that the two reciprocal crosses execeded the mean body weights of the two pure breeds in the first year and the mean parental body weight in the second year.
Thus, the role of dominance and over dominance effect is important in the inheritance of body weight at 8 weeks of age in the studied genetic groups. Table 2 shows a tendancy for females to have a greater coefficient of variation than males. Crosses generally showed a lower coefficient of variation than the pure breeds which agrees with available references, stating a greater sensitivity of pure breeds to uncontrolled environmental conditions than for crosses. However the variances did not show great differences according to sex and genotypes.
2 . -Heterotic effect for each sex The differences between sexes were in general greater in the crosses than the two pure breeds, being 8 2 .8, 70 , 0 , 10 8. 3 and 8g.5 g. for PP, SS, PS, and SP strain in the first year respectively, while they were in the same order 104 .5, 6q..5, Io5. I and 99 . 3 grams in the second year. Table 3 shows the herotic percentage of the two crosses over the parental body weight. In all crosses males gained more from crosses than females.
It could be observed that heterotic effects were greater in 1975 than 197 6. Table 4 shows that there was a highly significant difference between years and systems of mating. The year effect may be due to uncontrolled enviromental factors such as temperature, humidity, etc... Interaction between system of mating and year was significant for both sexes. The results suggest different response to these uncontrolled factors according to genetic makenup as show-in table 2 . It may be also stated that the heterotic effect is expected to be greater in better conditions. These results were also tested by analysis of variance with two controlled factors, sex and system of mating. This analysis of variances was listed in table 5 . Results showed a significant effect of sex and system of mating on body weight at 8 weeks of age at i p. 100 level, while the variances of interaction were significant at 5 p. ioo level.
These results may confirm the term of homoganetic heterosis suggested by S TONAK E R ( 19 6 3 ). It should be mentioned that the two strains were kept closed since more than 25 years.
3 . -Breast length at 8 weeks of age Table 6 shows the mean, standard deviation and coefficient of variation of breast length for both sexes in the two years at 8 weeks of age.
Generally the Ligth Sussex females showed the shortest breast length while the pure White Plymouth Rock and the two crosses showed approximately the same length. In respect to males, they showed a greater breast length than females. The Light Sussex was the shorter all over the experiment while the two crosses showed superior length than the pure Plymouth in the first year, and nearly the same, in the second year. These results in general may confirm the importance of dominance and overdominance effect on the inheritance of this character. The coefficient of variation results did not show a considerable difference among the studied genetic groups.
4 . -Heritability estimates of body weight at 8 weeks of age Table 7 presents the estimates of heritability as obtained by variance components. Estimates when obtained by dams components of variance were higher than those based on sire components of variance, in 25 cases out of 30 . The relative importances of sire components of variance were slightly greater for males than females. These findings may suggest that sex linked genetic variance was not of much importance in the inheritance of body weight in the studied groups. M ERRITT ( 19 66) stated that in birds, the sire components would contain one quarter and one half of the additive sex linked genetic variance, while the dam component would contain one half and none when the analyses are based on males and females respectively. Moreover, the dominance variance due to sex linked genes would not exist for females. Estimates of heritabilities based on pooled data were for females 0 . 30 , 0 .5 3 and o.q.2 when obtained by paternal, maternal and average component respectively. They were in the same order o.q.o, 0 . 70 and 0 . 45 for females. These values were within the estimates listed in the literatures e.g. K INNEY (ig6g). Heritability estimates did not show different magnitude as obtained for pure strains or crosses.
5 . -Heritability estimates of breast length at 8 weeks of age These estimates were within the available estimates of many references. Some of these estimates were negative or higher than unit, that may be due to sampling error or scaling effect (FALCONER, ig6o).
6. -Phenotypic correlation between body weight and breast length Table 9 presents the phenotypic correlation between the two characters. All estimates ranged from 0 . 3 6 to 0 . 92 . These results suggest a close positive correlation between the two characters in both sexes.
7 . -Genetic correlation Table 10 shows that in general there was a high genetic correlations between the two characters. These results indicated that selection programme carried out for body weight will lead to a considerable increase in breast length, and improve conformation.
On the other hand, breast length may be of some advantage when included in selection indexes to improve body weight regarding its rather high heritability and positive genetic and phenotypic correlation with body weight. | v3-fos |
2019-03-19T13:09:03.171Z | {
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} | s2 | Growth factors and management technique in relation to the developmental rhythm yield formation pattern of a seeding year lucerne stand
The investigation of seeding year lucerne stand development and yield formation pattern was carried out at the University of Helsinki in Viikki in 1973—74. The management techniques studied were the number of cuttings and the density of stand establishment. The variety used was Danish Isis-Daenfcldt. The maximum DM yield was obtained from a seeding rate of 20 kg/ha. The maximum DM yield produced under the favourable growing conditions in 1973 was 5. 7 tons/ha and under the less favourable conditions in 1974 3.3 tons/ha. No more than two cuts in the year of seeding are recommended. The radiation used by lucerne was 0.3 0.5 %of the total radiation reaching the surface. A 10 % decrease in radiation in 1974 resulted in a 50 % decrease in photosynthctic activity mainly due to differences in temperature and overabundant precipitation. The optimum LAI was obtained from scedingratcs 5 kg/ha. From seeding to emergence a temperature sum of 120 °C is needed and from seeding to flowering a sum of 800°C. From cutting to the next flowering a sum of 600— 700°C is needed. Three cuts provided forage 6.3 % units more digestible than two cuts. Root carbohydrate levels suggest that the last cut should not be made later than mid-August.
I Introduction
Lucerne is generally accepted as one of the most important forage species for domestic animals in those areas where it may be successfully cultivated. The actual range where lucerne is cultivated extends between 30 and 60 degrees latitude in the northern and 20 and 40 degrees latitude in the southern hemisphere (SMITH 1962).
Lucerne is well adapted to different temperature regimes as its wide growing area illustrates. Lucerne thrives best in an area where the growing season's average temperature is 18-20°C. Germination begins already at 4-6°C, but its optimum temperature is 3 1-37°C. The optimum temperature for lucerne nitrogen fixing bacteria is 1 s-3o°C (BOLTON 1962). A substantial reduction in bacteria activity is observed in temperature ranges of 10-12°C and 35-40°C because the nitrogen fixing capability is retarded (BOLTON 1962).
After wintering the specie's spring development begins when temperatures reach B-lo°C. Hardened lucerne can withstand freezing temperatures of up to -2 5°C on bare ground. After the hardening is lost in the spring the root meristematic area may suffer damage if the temperature falls to just ±0°C (SMITH 1962). Young lucerne shoots are considerably more succeptible to cold spring nights than those of red clover. Nevertheless, the adaptability of lucerne is best illustrated by the fact that it is successfully cultivated in areas where winter temperatures exceed -4O°C.
In areas such as Hungary, where the average annual temperature is 11°C, five harvests of lucerne are possible. Where the average annual temperature ranges from 7.5 to 9°C, as in Germany, 3-4 harvests are common. These average annual temperatures represent temperature sums of 4500-3000°C for growing seasons covering April to October (BOLTON 1962).
For flowering (BECKER-DILLINGER 1929) lucerne needs a temperature sum of 850-900°C and the same amount of heat for each of the following cuts. As the average temperature sum in southern Finland is 1900-2100°C, this means that at most two lucerne harvests are obtainable during the growing season.
Because of its deep growing (6-9 m) main root lucerne can withstand drought better than any other forage plant (SCHANTZ and PIEMESEL 1927). On the otherhand abundant ground and surface water are detrimental and one of the major obstacles to successful establishment of lucerne. According to SCHANTZ and PIEMESEL (1927) the evaporation coefficient for lucerne is equivalent to about 180 mm of precipitation per ton of dry matter. For areas where 2-3 cuts are made the precipitation needed ranges from 450-600 mm and in areas where more cuts are possible from 900-1200 mm. Investigations and practical cultivation have shown that 850-1000 mm of precipitation is the upper limit lucerne can tolerate without serious problems.
Because of its deeprootedness lucerne requires a growing medium where the subsoil is permeable. Lucerne is known to thrive also where the lime content of a permeable subsoil is high (MULTAMÄKI 1965). It requires a soil where the pH is >6.5.
In the plough layer lime should be present in a concentration of 12 t/ha. A lucerne harvest of 6 t/ha removes from the soil about 30 kg P/ha, 210 kg K/ha and 150 CaO/ha. At the same time it leaves 5-7 t/ha of organic matter and 300-400 kg N/ha. Lucerne obtains its nitrogen from nutrients in the soil or via its root bacteria which fixes it from the air. The nitrogen fixing capacity of lucerne is 40 and 84 % more efficient than for red clover and peas respectively. Cultivation possibilities for lucerne in Finland have been investigated by HEIKINHEIMO (195 5), TEITTINEN (195 5), RAVANTTI (195 5,I 960), MULTAMÄKI (1962MULTAMÄKI ( , 1965MULTAMÄKI ( , 1969MULTAMÄKI ( , 1970, BÖCKELMAN (1967), RAININKO (1970) and PULLI (1973PULLI ( , 1977. All have shown that in areas where lucerne thrives its yield production has surpassed that of red clover and fescue. The factors which most seriously hinder lucerne growth are the low reaction of the soil, low nutrient content of the soil, too much groundwater, surface water, a poorly suited variety, short growing season, low temperatures and various factors resulting in poor wintering.
The purpose of this study was to determine the relationship between growth and growth factors in the early development of lucerne, in respect to the intraspecific compentition and other cultivation intensities. The study was conducted during 1973-74.
Field arrangements
The field trials were established on the University Farm in Viikki in 1973. The plots were organized in the following ways; The emergence measurements were carried out June 6 in both years.
Leaf area index (LAI) measurements were conducted weekly throughout the entire growing season in both tcst years. The LAI sample size per plot was 0.045 m 2 . The overall height of the crop was measured daily in 1973. and twice weekly in 1974. The root samples for carbohydrate analysis and forage for in vitro digcstibility tests were collected once a week throughout the entire growing season. Stand density and root yields were measured on Nov. 5 in 1973 and Nov. 21 in 1974. Metcorologic data was obtained from the weather station at nearby Seutula airport.
Laboratory analyses
Leaf area was measured with an optical leaf planimeter (Model Kl) designed and built by the Technical University of Helsinki.
Forage in vitro degestibility was determined according to the procedure of TILLEY and TERRY (1963). The reducing sugars of lucerne roots were measured with the calorimetric methods of NELSON (1944) and SOMOGYE (19 52) as applied by SMITH (1969). The dry matter energy content of forage was determined with a PHILLIPSON oxygen microbomb calorimeter according to PHILLIPSON'S (1964) procedure.
Weather conditions
The weather conditions of the 1973 and 1974 growing seasons differed considerably from each other. Summer 1973 was warm and dry while summer 1974 was chilly and rainy (Tables 1,2, 3). When comparing the temperature conditions in degree days (Table 4), the temperature sum of the 1974 growing season on Aug. 8 was one month late compared to that of 1973. Also radiation conditions were more favourable in 1973 than in 1974. Because of the weather conditions prevailing in 1973 several irrigation treatments were needed in order to meet the plant's water requirements.
4. Results and discussion 4.1. Yields and stand characteristics Yields The seeding-year yields (Table 5) during the favourable 1973 growing season varied in the 2-cut system at different seeding rates from 1.1 -5.7 tons DM/ha and in the 3-cut system between 1.0 and 5.7 tons DM/ha. In 1974, which was a less favourable season for lucerne, the 1-cut system yielded 1.2 tons DM/ha and the 2-cut system 3.3 tons DM/ha. The yields of 5.7 and 3.3 tons DM/ha represent 70 and 30 % respectively of the average lucerne yield level in Finland (MULTAMÄKI 1969). According to TESAR and JACKOBS (1972), in the northern United States seeding-year lucerne yields average 4-5.8 tons DM/ha and represent 40-60 % of the yields from older stands. Under unfavourable conditions the seeding-year yield may be even less.
Of the seeding rates tested 20 kg/ha proved to be sufficient to obtain the maximum yield ( Fig. 1,2). These results represent a 5 10 kg less performance level than MULTAMÄKI's (1965) results but considerably higher seeding-year results than those reported by BOLTON (1962), TESAR (1972) and PULLI (1973) in the United States.
The tests showed that an above normal seeding rate did not increase the yield significantly but because of the increased interplant competition the roots remained smaller and wintering ability weakened. It was also found that in sparse stands the competition between lucerne and weeds reduced lucerne's growth vigor particularly when a low cutting frequency was used. On the basis of this finding two cuts is the optimum cutting frequency under favourable growing conditions. Stand characteristics: In both of the growing seasons (Table 6) lucerne required a temperature sum of about 120°C from seeding to emergence. The average temperature sum needed from seeding to flowering was 860°C. The sum required from the first cutting to flowering and the second cutting to flowering was about 610-650°C. These require- BECKER-DILLINGER (1929), first in regard to variety development and second as to whether the basis for observation is the onset of flowering or full flowering.
The population development of lucerne was studied very closely in 1973. According to the results of BROWN and STAFFORD (1970) the seedling development could be considered excellent.
Thinning of the stand which occurred by the autumn of the seeding-year was greatest at high seeding rates as shown earlier by PULLI (1973). The substantial thinning which occurred at the lowest seeding rate (1 kg/ha) was due to the severe competition with weeds in the spring.
The shooting of the stand (Table 7) at different seeding densities was clearly similar to results obtained in earlier studies, for example CLEMENTS et al. (1929) and COWETT and SPRAGUE (1962). Shoot formation on an individual basis was 3.9 9.3, depending on the seeding rate and cutting frequency. The primary development of the yield was strongest at the high seeding rates and weakest in the sparse stands, despite the fact that shooting increased at the same time, as also DAVIDSON and DONALD (1958) have shown. No LAI differences (Fig. 4) were observed in the final autumn cuttings of stands having seed amounts of 5 kg/ha or more and the yield differences were minor (Table 5). This supports PULLI's observations (1973) in the USA.
Photosynthetic efficiency
Lucerne used an average of 0.5 % and 0.3 % of the total radiation in 1973 and 1974 respectively for yield formation (Fig. 3). There were few differences between the cutting systems. The energy value of the yield ranged from 3.9-4.7 keal/g in individual cuts at different seeding rates (Table 8), but differed very little for complete cutting systems. These results were very similar to those PULLI (1980) obtained with a mixed clover-grass stand.
The total radiation for 1974 was about 10 % less than in 1973 (Table 3), whereas stand radiation utilization in 1974 was almost 50 % less than in 1973 (Fig. 3). This is an indication that at Finnish latitudes radiation is not a limiting factor for lucerne photosynthesis, but that temperature and excess precipitation are, as CHANG (1968) reported.
LAI and Plant Height Relationships Yields and LAI
The large temperature differences between 1973 and 1974 resulted in noticeably slower lucerne development in 1974. September of 1974, however, was exceptionally warm. The largest LAI value measured in 1973 was 4.3 and in 1974 5.2 (Fig. 4,5). At high seeding amounts maximum LAI was reached faster than in hinner stands. This tendency weakened with the arrival of autumn and in the last autumn cut at the end of the growing season all stands with seeding rates > 5 kg/ha had developed nearly equal LAIs. This phenomenon was even more pronounced the more often the stands were cut (Fig. 4). The dry matter yields developed in very much the same way as LAI. This result differed from that of FUESS and TESAR (1968) where LAI reached its peak rather quickly after which the old leaves dropped off but the dry matter yield still increased. In Viikki lucerne's growth potential was obviously less and its growth rhythm slower and the maximum LAI values were Fig. 3. Photosynthctic efficiency of a lucerne stand with different seeding rates cut 1-3 times in 1973-74 Table 8. Energy-value of lucerne stand keal/g DM in invidual cuts at different seeding rates in 1973-74. Plant height relationships: The daily increase in plant height (Fig. 6), average temperature and the radiation amount followed each other well. Temperature alone accounted for 70-7 5 % of the increase as the following indicate; x = avg. daily temp (°C), y = daily height increment (cm): According to the calculated linear relationships the border temperatures for spring, summer and autumn growth are + 10°C, +l3°C and +B°C respectively. Of these the summer temperature value can be questioned. The high activation value most likely is a result of the abundant irrigation and relatively high daily temperatures in 1973. Strong midsummer height growth can also be seen in Table 9. Under favourable conditions height growth in the 2-cut system is steady throughout the entire growing season. From Fig. 7 it can be seen that temperature exerted a strong influence into the autumn despite the fact that illumination had somewhat weakened. These results support those reported by PULLI (1980) for meadow fescue and a mixed clovergrass stand. As part of this investigation the in vitro digestibility of lucerne was monitored weekly during the period 25.6.-4.10. The reduction in digestibility occurred evenly in the 2-cut system (Table 10), being less than 0.5 % in each cut. In the 3-cut system as the development progressed toward the autumn so too did the rate of change in digestibility, as the values of 0.20, 0.30 and 0.75 % for 1973 indicate. These digestibility changes agree with those observed by REID et al. (1959) andPULLI (1973) for lucerne and by countless others in studies of forage, for example HUOKUNA (1973), RAININKO (1973), SYRJÄLÄ et al. (1978. The large changes in digestibility during the early autumn were influenced by the strong growth potential of the stand combined with the favourable growing conditions. The 3-cut system produced fodder about 6.3% units more digestible than the 2cut system (Table 11). Because the final cut of the 3-cut system was made on Oct. 4, abundant shoot development in September increased the digestibility of this final cut significantly. At the same time there was a loss of terminal shoot dominance.
4J. Carbohydrate reserves in lucerne roots GRANFIELD (1935) found that a substantial reserve nutrient level is a prerequisite for the successful wintering of lucerne in northern cultivation areas. In lucerne the carbohydrates available for growth should be about 35 % of the dry weight of the roots in order to assure satisfactory wintering (SMITH 1962). Carbohydrate reserves vary with the cuttings (GRABER et al. 1927) and too many cuttings during the growing season may result in a too low reserve nutrient level in the autumn and winter (SMITH 1962).
In the lucerne study at Viikki the reserve nutrient level of lucerne roots dropped by about 10 % of the total dry matter content following cutting (Fig-8) in the favourable 1973 season, but then rose to a level exceeding 30 %. The rate of increase was slower in the 3-cut system than the 2-cut system. During the rainy and cool growing season of 1974 carbohydrate reserves were not used much for yield formation Table 10. Changes in in vitro digestibility of lucerne stand % per day when cut two or three times during the growing season.
Cutting
Changes in in vitro digestibility % per day system 25/6-9/7 25/6-23/7 23/7-10/8 6/8-3/9 20/8-3/9 2-cut -0.45 -0.48 3-cut -0.21 -0.30 -0.75 and as a result the reserve level remained high throughout the entire growing season (Fig. 9). The carbohydrate level in lucerne roots in the Viikki test should have ensured good wintering. As satisfactory wintering did not occur the reasons are many. Good wintering always requires favourable growing conditions. In addition, making the last cut at the latest by mid-August appears to assure good wintering of lucerne. Having the last cut in mid-August means making only two cuts during the growing season.
Summary and conclusions
At the university farm in Viikki a test series was conducted during 1973-74 on the growth and development of lucerne during the seeding-ycar. The variety used was the Danish Isis-Daenfeldt lucerne. On the basis of the results the following conelusions can be made: 1.) The maximum dry matter yield was obtained from a seeding rate of 20 kg/ha. By the autumn of the seeding year the stand density was 60 % of the seeding density. 2.) For good wintering lucerne may be cut at most twice during the growing season. The last cut should be made by mid-August in order that 4 weeks of growth following the cut may be reached before the first frost. 3.) The radiation used by lucerne was 0.3-0.5 %of the total radiation reaching the surface. Differences in the utilization amounts of radiation are due to temperature differences.
4.) In both study years the optimum LAI was obtained from seeding rates > 5 kg-/ha. The greatest measured LAI values were 4.3 and 5.2 in 1973 and 1974 respectively. LAI and the yield as well as LAI and height growth were closely correlated to each other.
5.) From seeding to emergence a temperature sum of 120°C is needed, and from seeding to flowering a sum of 800°C. From cutting to the next flowering a sum of 600-700°C is needed.
6.) Three cuts provided forage 6.3 % units more digestible than two cuts. 7.) Satisfactory wintering of lucerne requires high reserves of carbohydrates in the roots as well as good root development. | v3-fos |
2018-04-03T02:43:13.935Z | {
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} | s2 | Patients and spiders.
Once, while in country practice, I was asked to see an inexperienced farm worker who had lacerated a leg by the over-zealous use of a scythe; the telephone message stated that the wound was bleeding badly. On arrival at the farm, however, I found no panic and, on inspecting the deep wound, I found very little blood. The laceration had been packed with cobwebs, which had been pulled hastily from corners of the cowshed; the intricate mesh of multiple, fine silken threads had produced excellent haemostasis. Of course, this procedure was a long-established country method of firct-aid. Cobwebs have been used as applications to bleeding wounds for many centuries in rural areas; no doubt battle casualties, in the Middle Ages, for example, were often treated in this fashion. The patient I saw at the farm made a good recovery, following initial suturing. Tetanus did not supervene and no local sepsis was observed. Yet, it is obvious that bacteriological investigation of stable and pigsty spiders' webs is not going to yield results to satisfy the modern microbiologist. Doubtless it is simpler, and certainly far safer, in a case of extensive capillary haemorrhage, to apply pressure to the wound by means of the usual sterile gauze pad. But there might still be the odd occasion when spiders' webs are the only suitable dressing material available in an emergency. Cobwebs are not welcomed in many households today, even though they may be tolerated in the stable. On the discovery of a spider's web the housewife usually sweeps it away, to be deposited in the dustbin. Sometimes the dislike of webs is extended to the spider itself; many people flee from spiders and some become intent on killing them. Such persons maintain that the quick and unexpected movement of these eight-legged creatures is really upsetting to them; usually it is women who hold these views. Clearly, Miss Muffet's behaviour and influence continues at the present day. Some time back I interviewed an intelligent woman who told me that she had been accused of shoplifting. She explained that she had been scared that day by a big, black spider which had run suddenly across the floor of a shed; apparently, she had been so upset by the incident that on going into a store that afternoon she had not been responsible for her actions. Having told me the story, she said that the expression on …
Patients and Spiders
Philip Radford General Practitioner, Westbury-on-Trym, Bristol Once, while in country practice, I was asked to see an inexperienced farm worker who had lacerated a leg by the over-zealous use of a scythe; the telephone message stated that the wound was bleeding badly. On arrival at the farm, however, I found no panic and, on inspecting the deep wound, I found very little blood. The laceration had been packed with cobwebs, which had been pulled hastily from corners of the cowshed; the intricate mesh of multiple, fine silken threads had produced excellent haemostasis. Of course, this procedure was a longestablished country method of firct-aid. Cobwebs have been used as applications to bleeding wounds for many centuries in rural areas; no doubt battle casualties, in the Middle Ages, for example, were often treated in this fashion.
The patient I saw at the farm made a good recovery, following initial suturing. Tetanus did not supervene and no local sepsis was observed. Yet, it is obvious that bacteriological investigation of stable and pigsty spiders' webs is not going to yield results to satisfy the modern microbiologist.
Doubtless it is simpler, and certainly far safer, in a case of extensive capillary haemorrhage, to apply pressure to the wound by means of the usual sterile gauze pad. But there might still be the odd occasion when spiders' webs are the only suitable dressing material available in an emergency.
Cobwebs are not welcomed in many households today, even though they may be tolerated in the stable. On the discovery of a spider's web the housewife usually sweeps it away, to be deposited in the dust-bin. Sometimes the dislike of webs is extended to the spider itself; many people flee from spiders and some become intent on killing them. Such persons maintain that the quick and unexpected movement of these eight-legged creatures is really upsetting to them; usually it is women who hold these views. Clearly, Miss Muffet's behaviour and influence continues at the present day. Some time back I interviewed an intelligent woman who told me that she had been accused of shoplifting. She explained that she had been scared that day by a big, black spider which had run suddenly across the floor of a shed; apparently, she had been so upset by the incident that on going into a store that afternoon she had not been responsible for her actions. Having told me the story, she said that the expression on my face had shown that I had not believed her and, because of my reaction, she felt that she could not possibly give such an explanation before a court of law. In her opinion, doctors just did not understand how terrified some people were of spiders and she seemed to think that magistrates had a similar lack of sympathy. Rightly or wrongly, I let the matter rest there.
Women are not alone in being afraid of spiders. I recall a male patient who flatly refused to visit Burma on the grounds of his intense spider-phobia although, to be fair, he also had a fear of snakes and scorpions. Perhaps I should have delved deeply into psychological events of his childhood to see if some relief could be given but, as life in Britain was satisfactory for him, I did not initiate any investigation of his mental state. Happily, he was reassured that none of Britain's numerous spider species could inflict physical harm on man.
Even so, I understand that he continued to flee from the presence of spiders.
Whatever may be our attitude to moving spiders, or spiders suddenly located in some cranny, most of us are prepared to admire the beauty of spiders' webs. Some autumn scenes are made lovely by gossamer, composed of the numerous webs of so many small spiders; an October afternoon may well reveal such a multitude, borne on the herbage or floating in the air if sufficiently dry. A fine description of a fall of gossamer was given by the naturalist Gilbert White in the 18th Century; in one of his letters in The Natural History of Selborne he tells how a shower of cobwebs continued for the whole of one day in late September and, had anyone wished, whole baskets of cobwebs could easily have been gathered.
For small spiders to produce such an amount of fine, silk fibre is surely a remarkable and specialised process. The silk threads are prepared by the actions of the two or three pairs of spinning organs situated at the back of the abdomen of the spider; these spinnerets have multiple spinning tubes from which the silk, coming from the silk glands, is extruded.
It is not surprising that many birds make use of cobweb, building this light and filmy material into their nest structures, thereby adding to the insulation and giving decoration and camouflage.
Probably the best-known cobweb architect is the long-tailed tit Aegithalos caudatus; with this bird webs are interwoven with lichens and wool to make a cosy, ovoid nest which, with a lining of hundreds of feathers, must give as much comfort and warmth to the nestlings as is possible in so small a space.
Another common bird to seek spiders' webs for nest construction is the chaffinch Fringilla coelebs.
In this case, web material is twined with moss, grasses and lichens; the resulting inconspicuous nest blends beautifully with a lichened tree branch. Then goldfinches Carduelis carduelis, building on branches of orchard trees, mix rootlets with the wool of sheep and spiders' webs; hence the young find a well-insulated and strong nest-base when hatched from their eggs. Yet another bird skilful in incorporating spiders' webs into the wall of its nest is the goldcrest Regulus regulus. Here, the suspended, bag-type nest is normally composed of mosses and webs.
We have no information as to whether Miss Muffet approved of the design of spiders' webs even though she disliked their builders. Nevertheless, many persons enjoy the sight of webs outlined by frozen dew on a December morning and admire the geometric pattern of the concentrically placed and radial silk fibres; such webs look particularly attractive when stretched between the dead, frosted stems and the seedheads of umbelliferous plants such as hogweed.
We admire also the agility of the spider in grasping the fine, silk lines of its web and note the speed with which it rushes to deal with an entrapped fly. But perhaps most of all we delight in watching a spider descend to the ground by its ever-lengthening, thin silken thread, prepared as it travels, and wonder at the mechanism of its production. With such an activity before one, it is surely difficult to follow the cult and example of Miss Muffet. While many British bird species utilise spiders' webs regularly in the building of their nests, others search for spiders as a normal article of diet. The tiny wren Troglodytes troglodytes, for instance, ingests a remarkable number of spiders in the course of a day, often including some remarkably large specimens. Goldcrests, the smallest of British birds, behave in the same way, looking diligently in bark crevices for spiders which form a high proportion of their food intake. One can but speculate whether any such birds behave like Miss Muffet and, if so, do any of them survive a truly hard winter?
Just as many birds eat spiders, so there are some large, tropical spider species which swallow small birds. Birds living in the vicinity of these spiders obviously have to be vigilant and, to survive, it behoves them to follow the behaviour of Miss Muffet. Vulnerable birds could well be excused for developing a spider-phobia and, indeed, their continued existance depends on its development and constant maintenance. Yet why should certain humans, who have nothing to fear from spiders and who have the mental ability to comprehend their skilled and characteristic activities, fear them so strongly? It is a pity that Miss Muffet does not live today, so that we might have a full psychiatric report on her background and present symptoms. Perhaps an effective treatment could even be considered for her. | v3-fos |
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} | s2 | Botrytis porri Buchw . on leek as an important storage fungus in Finland
Botrytis porri Buchw. was found to be a common and economically significant pathogenic fungus on leeks in storage. The increase in the number of fungi caused a linear decrease in the number of marketable leeks when stored at 0.5° C. B. porri was found to spoil leeks even at —o.s° C. Spraying with benorayl and thiophanatemethyl one or two weeks before harvesting significantly decreased the numbers of the fungus and the amount of damage caused during storage. Botrytis allii Munn and Fusarium avenaceum Sacc. rarely caused spoilage of leeks.
Introduction
Leeks come second to onions as important Allium plants in Finland. The area under cultivation has increased from about 30 ha to 80 ha during the 1970'5. The value of the crop is high since the price of leeks is about three times that of onions in Finland. Leeks are stored in the field in more southerly countries, but owing to the severe winter in Finland they have to be moved to temporary stores or refrigerated stores. Although rather extensive studies have been carried out in Finland (Suhonen 1970) on storage techniques and conditions, diseases occurring during storage have not been studied at all. In practice, however, the most serious risk factor in the storage of leeks has proved to be storage diseases. Rather little research has been carried out abroad on the storage diseases of leek presumably as a result of the specialised cultivation technique required. In Norway, the most important fungal pathogen of leeks in storage has been found to be Botrytis porri Buchw. (Roed 1952). Pathogens are considered to be a serious problem in the storage of leeks in Norway ( Hoetun 1978 a).
The aim of this study was to determine the fungal pathogens of leeks in storage, their significance and possible ways of controlling them by means of fungicide sprays before harvesting.
Material and methods
Storage experiments were carried out in 1975/76, 1976/77, 1977/78 and 1978/79. The stored leeks, variety »Copenhagen marget», were grown each year on the same site in Viikki which had not earlier been used for growing leeks. The seedlings were grown in the greenhouse, planted during the period 15.-25. 5. and the crop harvested during the first or second week in October. The field, which was of good fertility, was given a basic fertilization each year of 1 200-1 400 kg/ha of chloride-free compound fertilizer (N : P a 0 6 : K 2 »J = 7:24 : 14) and nitrogen was added 2-3 times during the growing season as calcium nitrate to give a total annual nitrogen dosage of 250 kg N/ha. The leeks were placed in a refrigerated store at 0-(-1°C and a relative humidity of 96-98 %. From 10 to 15 normal-sized leeks per replication were placed in open, perforated (0 1 cm, at intervals of 15 x 15 cm) plastic bags.
Before storage, the roots of the leeks were trimmed to a length of about 1 cm and the leaves shortened by about 1/3. At the end of the storage period, the fungi on each leek were determined and the degree of fungal infection estimated using the scale 0-5. The leeks were then prepared for marketing in the normal way. The fungus determinations were carried out using a stereomicroscope. Whenever necessary, plant samples were placed on moist filter paper in petri dishes for more precise determinations and for preparing pure cultures.
Spraying was carried out on the field one or two weeks before harvesting using different types of fungicide. The growth of Botrytis porri at different temperatures was studied in 1977/78.
A piece of B. porri agar was placed on the upper part of a leek which had been cut to a length of about 10 cm. The leeks were stored on damp filter paper in containers covered by plastic film. Eight infected leeks were incubated at each temperature: -0.5°± 0.5°, +l°± 0.5°, +4°± 0.5°and +B°± 1°C.
A similar experiment using leek discs was carried out the previous year. The results of this test will not be discussed here as they were in complete agreement with those obtained in 1977/78.
Leek samples obtained during the course of the experiments from leek stores situated in different parts of Finland were studied in order to determine the types of fungi which affect leeks in storage.
Botrytis porri Buchw. on stored leeks
Botrytis porri (syn.: Botryotinia porri (van Beyma) Whetz., Sclerotinia porri van Beyma) produced 9-ll pt (11.7 pi) thick, initially light coloured, later grey mycelia. Conidiophores and conidia (Fig. 1) were formed on the host plants, but conidia were not formed at all on potato dextrose (PDA, Difco) agar. The conidia were 11 -lB pi (14.5 pi) x 9-12 pi (10.8 pi) in size. The fungus produced 3-lo mm, irregular-shaped sclerotia on the PDA medium (Fig. 2) and also on leeks (Fig. 3). The sclerotia were initially light grey in colour, but as they aged turned almost completely black. When leeks were stored in the normal way, the plants lying closely packed together, sclerotia were rarely formed. The fungus produced large numbers of sclerotia in the autumn in the field and in the temperature experiments where the leeks were kept separate from each other. The fungus was found in all parts of the leek plants. B. porri grew extensively throughout the plant tissues and also grew out between the leaves. The infected plant tissue became soft and watery (Fig. 4). Leeks which were packed tightly together became very slimy and appeared at first glance to have been spoilt by bacteria.
B. porri was found in all the areas studied: the Helsinki, Turku and Jyväskylä areas and the Aland Islands. On the basis of discussions carried out with farmers and consultants, it is apparent that the fungus is also to be found in other areas where leeks are cultivated. B. porri was usually found only on leeks which had been stored, but in 1977 and 1978 the disease was already apparent in October on the upper parts of leek plants growing in the field. The fungus did not cause any damage in the field.
At Viikki, where the disease was followed for four years in succession, the fungus occurred in less than 1 % of the leeks during the first year, in about 10 % ot the leeks during the second, and in 80-90 % of the leeks in the next two years. The increase in the amount of B. porri in stored leeks brought about a linear (y --0.56 x 73.8) decrease in the marketable proportion of leeks at 0.5°C (Fig. 5). After four months storage, a B. porri content of 80 -9O % produced almost complete spoilage. B. porri was still able to grow on the leeks at a temperature of -o,s°C and caused a serious amount of spoilage in 4 months (Fig. 6). Raising the temperature to 8°C speeded up spoilage.
Control of Botrytis porri
Spraying with benomyl and thiophanate methyl before harvesting significantly reduced the B. porri content and also the storage losses (Tables 1 and 2). In 1977/78, when the fungus was artificially spread on the field, two sprayings gave better results than one only. In the last year of the experiment, spraying carried out two weeks before harvesting was as effective as two sprayings, but better than spraying carried out one week before harvesting. The same result was obtained with both preparations. The best treatment reduced the fungus content from about 90 % to about 20 %.
Non-systemic control chemicals did not reduce the B. porri content of the stored leeks (Table 1).
At harvesting, the amount of fungicide residues present in leeks sprayed two weeks before harvesting with benomyl and thiophanate methyl was always less than 0.4 ppm. No residues were found in samples taken at the end of the storage period. Other fungi on stored leeks Bolrytis allii Munn was rarely found in the stored leeks. This fungus causes spoilage of leeks in a similar way to B. porri, but owing to its rare occurrence it was of no importance as a storage fungus.
Fusarium avenaceum Sacc. was found on a very few occasions in spoiled leeks. Spoilage always started from the lower part of infected leeks. Infected tissue was red-brown in colour.
Discussion
In this study, grey mold of leek has been called Bolrytis porri Buchw., although in some studies (Cronshey 1947, Röed 1952, Cook 1976) the conidial stage of Botryotinia porri (Van Beyma) Whetz. is considered to be identical to Bolrytis hyssoidea Walker. B. porri forms large sclerotia on PDA medium (cf. Röed 1952) but B. hyssoidea does not form sclerotia at all on PDA medium (Walker 1925). Buchwald (1949), Ellis (1971) and Jarvis (1977) consider the conidial stage of Botryotinia porri to be different from B. hyssoidea. B. porri was now found for the first time in Finland. As the fungus is an extremely common and damaging storage pathogen of leeks, it has undoubtedly been present in Finland for a long time. It has possibly been confused with B. cinerea Pers., as has occurred elsewhere (Röed 1952). It may have been introduced to Finland along with imported seed as it is also a seed-borne pathogen (Neergaard 1945). According to the observations made in this study, infection of the crop in the field takes place during the end of summer and autumn before harvesting. The fungus may be already visible, but in most cases it is hidden (cf. Hoftun 1978 b). Continuous cultivation of leeks appears to strongly increase the incidence of the disease.
B. porri can grow at temperatures below 0°C. According to Hoftun (1978 b), it can even take place below -2°C. Control of the disease by lowering the storage temperature is therefore not possible. Adding carbon dioxide to the air in the store reduces or inhibits the growth of B. porri (Hoftun 1978 b), but the construction of storage space in Finland where the air mixture can be controlled, has not become common obviously as a result of the high costs.
Control of B. porri should therefore be done in the field before harvesting. | v3-fos |
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} | s2 | The analysis of sire buying policies
An expression is derived for the net present value of returns obtained through increased productivity of descendants of a purchased sire. This expression is useful for evaluating a given sire purchase, but is not so helpful in determining a sire buying policy. For this purpose, an analysis is made of the value of sire purchase from a breeding nucleus which is making constant genetic gains when the lag of the commercial population behind the nucleus has stabilised under a fixed policy. This leads to a criterion for deciding how long a sire should be used, which depends
Introduction
Most economic assessments of animal breeding programs, such as those of P OUTOUS and V IS S A C ( 19 6 2 ), HILL ( 1971 ), JAMES ( 197 2) and !r,s!N and M O C QUO T ( I974 ) have dealt with decisions made in the breeding nucleus. However, genetic gains made in the nucleus are mostly realised by gene flow into commercial populations and, therefore, the benefit of genetic gain is dependent on decisions made by owners of commercial stock to buy breeding animals, normally sires, from the breeding nucleus. Thus genetic gains made in the nucleus will have a significant impact on production to the extent that purchase of sires from the nucleus is seen as a profitable use of resources by owners of production stock. N APIER and Jorr!s ( 197 6) have discussed the value of Australian merino rams by calculating the discounted value of returns from the progeny of a purchased ram. However, they ignored contributions to later generations, and considered evaluation of a particular sire, rather than a sire buying policy. Their approach may thus be more relevant to buying a terminal sire than to purchase of a sire for purebreeding. In this paper, the analysis of sire buying policies for purebred populations will be developed in relation to the rate of genetic gain being achieved in the nucleus, and to the relative breeding value and price of available sires.
Evaluation of a purchased sire
We first develop an expression for the value of a purchased sire, given all the conditions of use. This will be expressed as present value of discounted returns, comparing the purchased sire with a randomly chosen male from the commercial population or base. The notation to be used in this paper is listed with definitions in the Appendix.
N sires are used altogether at any one time, a fraction W of them surviving to the next time unit. For simplicity, survivals rate is assumed independent of age, and time units are referred to as periods. The maximum number of times a sire will be used is T, provided he survives long enough. If n replacement sires are bought each period : Thus, if C is the purchase cost per sire, the expense of sire buying each period is NC( I -W) 1( 1 -W T ) or NO /T if there is no wastage.
If h j = 1 , 2 , .... F, are the genetic contributions of females of age j to the progeny crop, where ¿,fj = 0 . 5 , and each sire contributes an equal number of i offspring in any period, it can be shown that the genetic contribution of a siie to the progeny crop at time t from its initial use is e' F t-I e 12N, where Using a discount rate d, the contribution at time t is multiplied by r to bring it to present value, where r = i /(i -!-d), and summing these discounted values over all values of t gives rf' (Iy!)w e / 2 N. Letting J denote e' (Iy F)-1 e . it can be shown that : &dquo; &dquo; &dquo; &dquo; The sire will make a similar contribution at each subsequent mating in which he is used, and these can be combined after discounting by a further factor of r for each time unit. Allowing for wastage, the expected total genetic contribution of a sire is : If it is expected that NP progeny will be produced each period, the number of discounted progeny genotypes expected from a sire is : We now suppose returns from progeny are realised at an age Y units in both sexes.
There is no real loss of generality in this assumption since, if returns occur over several periods, as in wool sheep, these can all be discounted back to the same period and summed (H OPKINS and JAMES, 1979 ). If each unit of breeding value is worth B economic units per animal, the value of a unit of breeding value in the sire is : This remains true when W = i. Since the sire cost is C, if he has a breeding value R relative to the mean of the base, his net present value is : It is obvious from (i) that the larger T is the greater is the net present value of the sire, as was noted by N APIER and J ONES ( 197 6) in their example, and as is clear from the fact that a sire contributes more descendants the longer he is used. However, the problem with trying to define an optimum sire buying policy on the basis of this expression is that the comparison of breeding value is made with contemporary base bred males. If the nucleus is making genetic gains there will be young sires available for purchase which are genetically superior to the sires already in use. It is then likely that at some value of T the genetic superiority of young nucleus males ovei the oldest sires in use will be great enough to warrant the buying of these young sires as replacements for the old ones. In essence what is missing from the above treatment is a recognition of the cost of not replacing old sires by better young ones. The stimulus to the nucleus to produce genetic improvement is precisely the fact that if it does not do so it is in the interest of buyers to restrict their purchases of sires as far as possible.
Consideration of lag costs
One of the difficulties with the method outlined in the previous section is that the consequences of a single decision (sire purchase) over a long time period ate taken into account under the assumption that subsequent actions are fixed.
If we allow for possible variation in subsequent actions, then the value of present actions will change. For example, the effect of selection made at a given time depends on the generation length after selection has been made. To evaluate a policy we should consider an average over many periods. The simplest approach is to consider an equilibrium situation. If a producer continually buys sires from a nucleus making constant genetic gains, his population will eventually be improving at the same rate as the nucleus, but will lag behind by an amount depending on generation length and selection differentials (B ICHARD , 1971 ). The mean productivity of one population relative to another will depend on the diffe-rence between their lags, so that the lag of a population behind the nucleus should be a good measure of its profitability.
If G is the rate of genetic gain per time unit in the nucleus, C B is the genetic selection differential applied to breeding animals in the base, where the generation length is I B , the genetic lag A can be written, as shown by JAMES (1977), as : If D MB and d FB are genetic selection differentials applied to nucleus born males and base born females for use in the base; and if I MB and 1 F , are the average ages of male and female parents used in the base : Therefore : . If there were no lag at all, the returns from the base in each period would be increased by an amount NPBA, which we may therefore call the « lag cost ». The lag cost may then be written as : The value of 1 MB depends on the sire replacement rate. If there is no wastage and sires have equal numbers of progeny at ages a + 1, a + 2 , ... a !--T, I MB is a -f-'/ 2 (T -!-i). If the survival rate is W per time unit, then : The cost of sires per time unit is nC where n = N /T if W = Z and n = N(r -W) / ( I -W T ) if W ! i. The total cost per period, considering both lag cost and sire purchase cost, is : The second of the three terms in this expression is independent of sire buying policy, so the variable cost is : or when We shall suppose that sire cost, C, and sire selection differential, D m]3 , are fixed and find the value of T which minimises the variable cost. That is, we seek an optimum replacement rate for sires of a given type. We later take up the question of sire buying policies when cost and breeding value of sires are variable.
It is convenient to express the cost in units of NPBG, the increase in returns per time unit, and to express the sire selection differential as S, measured in units of rate of genetic gain, where S = D MB /G. Also, the cost of a sire relative to the increase in returns per sire per period will be denoted Q = C /PBG. With this notation : or, when We then find : For any given values of W and Q, there is an optimum number of periods of use of a sire which minimises total cost. This number is the solution of The second expression is the limiting form of the first as Wi, and shows that as Q increases, that is, sires become more expensive relative to the genetic benefits they confer, fewer sires should be bought each period. The result when there is wastage is not so obvious from the equation, but numerical results are given in table i, showing the optimum T values for a range of values of Q and W.
In general, solutions are not integers, but this is not important, since T = 4 .5 can be interpreted as using half the sires 4 times and half 5 times, though wastage makes this a little more complicated. When Q is small, so that sires are cheap relative to the rate of genetic improvement, sires should be turned over quickly, and the optimum is not much affected by the wastage rate, essentially because the rapid turnover does not allow time enough for wastage to produce effects. But if sires are expensive and should be used often, a high wastage rate means that there are many fewer older sires than when wastage rate is low, and thus the generation length and lag do not rise so rapidly with T. Therefore sires should be used more often if possible when wastage rates are high than when they are low. For example, when Q = 32 , a sire is used 8 times if W = i, but up to m. 3 times if W = 0 . 75 . However, the average age of sires used is lower (a -!-3 .55) when W = 0 . 75 than when W = z(a + 4.5).
Variation in price and breeding value of sires In the previous section we considered costs for sires of given price and breeding value. Except when sires were to be used very many times, wastage rate had little effect on optimum policy. In this section, to avoid mathematical complications, it will be assumed there is no wastage of sires. However, it will be assumed that price and breeding value are variable. The price of a sire is related to his breeding value by the function : where k(o) = i, so that Q a is the value of Q for a sire of average breeding value. The variable cost equation may then be written as : We can now consider variation in both S and T. First, which is the result of the previous section with Q replaced by Qok(S). Assuming k(S) is an increasing function of S, this implies better sires should be used longer than worse oneshardly a surprising result.
Second,
The effect of variation in S depends on the nature of the function k(S). The simplest assumption is that k(S) is a linear function : from which Therefore if T > Q o b, U decreases as S increases, while if T < Q o b, U increases as S increases. Thus for any given value of T it is best to have S either as large or as small as possible. There is no overall minimum, in the mathematical sense, with this form of k(S). The equations : define a saddle point, not a minimum. Another mathematical difficulty is that with a linear function the price of very poor sires will be negative, the nucleus paying the base to use very poor sires. This seems unrealistic, but if the slope of the line is not too great negative prices will occur only for values of S outside the range of the population and thus are irrelevant.
A numerical illustration of the relationship of U to S and T when k(S) is linear is shown in table 2 , where a = i, Q o = 20 and b = o.i. It is assumed that S may vary over the range ±10, corresponding to the top and bottom 5 per cent of nucleus males if the coefficient of variation of sire breeding values is 10 per cent and genetic gains are being made at the rate of 2 per cent per period.
The value of b then corresponds to the top 5 per cent of sires having twice the average price, while the bottom 5 per cent have zero price. In this case the solution of is given by The table shows clearly that T = 2 marks the point at which U does not depend on S, and also that T = 2 , S = -9 is a saddle point. It is also clear that the optimum value of T increases with S, the value of U at this optimum being lower as S increases. Under these conditions the best policy is to buy the best available sires and use them many times, the total number of times of use being given by the result of the previous section.
However, given this information, one would expect buyers to prefer sires with high breeding values, and the resulting demand would be expected to change the nature of the function k(S). In fact, one might expect that if there is a best policy, in terms of S and T, and this is known to buyers, there would be a greater demand for sires with the optimum value of S, with the result that the price curve k(S) would be pushed into a form in which there was no optimum value of S. Returning to the partial derivatives, we see that for any S, buyers should use such sires T times where T 2 = 2Qo k(S). We also want !! to be zero so that it is possible to compensate for buying a poorer sire by getting it cheaply enough.
The condition that au as = o is : Ob and making use of the relation between T and S : The solution of this differential equation under the condition h(O) = I is given by : Then we find : from which : Both of these derivatives are zero when T = S + V 2Qo , and with this relationship between S and T : -It is to be noted that k(S) has a minimum at S = -1 2Qo , being then zero, implying that below this point the price of sires increases as their breeding values fall. However, in the present context this is irrelevant since such sireswill never be bought. In fact, since the minimum use of sires is one period, the corresponding value of S is i -!/2Qo, for which C = !PBG. The zero price corresponds to T = o and thus is irrelevant. The relationship between U, S and T when k (S) has the equilibrium quadratic form is shown table 3 for a = i, Q o = 2 . It is noticeable that with this price function, good sires are used very many times because of their high cost. However, the minimum overall cost is the same, 3 . 5 NPBG, for all classes of sire provided the sires are used the correct number of times, S -!-2 . With this quadratic price function, the total cost can be divided into a lag cost of NPBG / + T ! 1 -S) B /2 / and a sire cost of NC /T which equals : When the optimum policy is used, T = S -! V 2Q o and the sire purchase cost is 1 / z NPBG (S -f-!2Qo). Thus the purchase costs are still higher for purchasers of genetically superior sires although fewer are bought. On the other hand lag costs are NPBG (a -!-VQ./2 — ! S), and are smaller when genetically superior sires are purchased. The increase in generation length caused by longer use of expensive sires is not enough to negate the benefits of their genetic superiority, so that the lag would be smaller in those populations which used very good sires but kept them for longer periods.
In this discussion it has been assumed that buyers compete, each knowing the optimum strategy and each having the same breeding objective. There is then no economic advantage in buying sires of any particular quality, provided they meet the minimum requirement S > i -!2Qo, and that sires are used an appropriate number of times. On the other hand, if the price function differs from this ideal form, there will be some class or classes of sires which will be more profitable to buy, and such classes can be identified using the type of analysis used here, as was done for a linear k(S).
Prices of boars in France
As seen above, the best policy for sire buyers depends on the form of the price function. As an example of a price function, prices of boars sold at testing stations in France in the year r9 77 1 7 8 were investigated. For these sales, the data available were the breed of boar (Large White or French I,andrace), the price paid, and the relative index values. Relative index value is calculated as an estimate of breeding value scaled to have an average of 100 and a standard deviation of 20 points. In addition, for each boar there was recorded the type of buyer (breeder, A.I. centre or producer). Prices paid by breeders and A.I. centres are not relevant to the present discussion and have been excluded from this study.
Excluded boars tended to have high index values, and though there was little comparative information, may have been somewhat more expensive than boars of similar index values bought by producers. Altogether, data were available for 6 1 sales held at 12 testing stations, giving prices for 6 97 Large White and 552 French Landrace boars. Since all boars with an index value below 100 are slaughtered, the available data are for boars with index values of ioo or more. There are few boars with index values above 140 , and most of these are bought by breeders or A.I. centres so our attention is concentrated on index values from ioo to 140.
For any one index value, prices showed a considerable range, and there were occasional very high prices. There also appeared on inspection to be some differences between sales in prices paid for boars of the same index value, but since our present purpose is not to make a detailed analysis of boar prices the data were pooled over all 6 1 sales. Then the median price paid for boars of a given index value ( 100 , ioi, 102 , ... 140 ) was determined and plotted for each breed. The median was preferred to the mean because it is less affected by occasional very high prices. The plot is shown in figure i, and the relation between median price and index value can be seen to be very similar in the two breeds. The curves are erratic near index values of 140 . This is mainly because the medians are based on very small numbers. However, since a high proportion of boars in this region are bought by A.I. centres and breeders, this may affect the prices paid by producers. In both breeds, price changes little as index values rise from 100 to about no, but then rises more rapidly. It has not been thought worthwhile to make a detailed statistical analysis of these prices. Rather, a simple quadratic equation which can describe both relationships without doing excessive violence to the data is plotted in figure i. The equation of the curve is : Clearly this simple equation describes the price-index relation reasonably well.
There is evidence (P. 5!!,!,I!x and L. Oi<T,iviE R , personal communication) that the rate of genetic improvement in French pigs is about 5 index points per year, and that an increase of one index point is worth about r. 5 o francs per pig.
Thus if we take our time unit as being 6 months, G is 2 . 5 index points per period, and B = 1 . 5 francs per point. Each boar bought could produce about 20 o piglets reared per period {P = 200 ) though this may be somewhat high on average. However, if we accept these values we have : PBG = 75o francs per period Then since the price of an average boar is 1 500 francs, Q o = 2 . Since S = (I -100 ) / 2 .5, the approximate price function (6) can be rewritten as : so that On the other hand, the theory developed in the previous section predicts that competition should tend to produce a price function in which :
This would give
The result would be extremely high prices for high-index boars. For example, the price of a boar with an index of 140 would be 121 5 0 o francs under this system rather than the observed value of 2 500 francs. It is thus clear that if the above estimates are correct, French pig producers are able to buy genetically superior boars very cheaply.
Assuming that boar prices can be adequately described by equation (6) : and therefore : Since S has an upper limit of about 20 , 1 is negative unless T is less than one sixth and therefore it pays to buy the best possible sires. With the existing price structure producers could spend more to buy the best available sires in competition with A.I. centres and breeders. Also, Supposing a producer buys boars with an index value of Z4 o, so that S = 1 6, giving a stationary value when T 2 = 20 / 3 or T = 2 . 5 8. Thus, using the above estimates, a producer would be advised to buy the best available sires and to use them for about 2 . 5 periods or i 5 months before replacing them with the best sires then available. Boar purchase costs would then be about I ooo francs per boar used per period. The above discussion suggests that genetically superior boars are cheap relative to average boars. It is also of interest to see whether average boars are cheap or expensive relative to their genetic value using the discounted gene now procedure for evaluation of a purchased sire. An average sire costs 1 500 francs and it is easily checked that the optimum usage period is two time units or 12 months. We shall then assume that such a boar has progeny born on average when he is 2 . 5 time units old. If it is assumed that the effects of sow selection in the producer's population on lag can be neglected the lag will be 2 1 B G when average boars are bought so that R = 2 1 B G.
Then the net present value of an average boar is : Letting r = 0 . 95 corresponding to a discount rate of 10 per cent per annum or 5 per cent per time unit, and letting Y = i we have : Sows are normally one year old at first litter with subsequent litters at 6 month intervals. We may then use the sow parity distribution reported by I,!GAUr,T, DAGO RN and T AS T U ( 1975 ) to find the average age of sows. The approximate age distribution of farrowing sows would then be : Age (6 month units) : 2 3 4 5 6 7 8 9 Percentage 27 21 17 13 9 6 4 3 .
The average age of dams would then be 4 . 05 time units. Since the average of sires is 2 . 5 time units this gives li3 = 3 . 275 time units so that : Using a discount rate of 5 per cent for a 6 month time unit, r = 1 ¡ LOS and then : from which J = 1 . 7010 . Therefore : N.P.V. = 2 2 88 francs That is, an average boar gives a sum of discounted returns in his descendants of about 3 7 88 francs while costing 1 goo francs, leaving a net value of about 2 2 88 francs to the buyer. Since average sires are profitable and better sires have been shown to be cheap relative to average sires, superior sires are clearly very profitable, provided that the assumptions on which this analysis has been based are correct.
Discussion
The analysis presented in this paper clearly has a number of limitations.
For the most part, attention has been concentrated on a steady state situation, whereas in practice sire buyers will often, perhaps nearly always, have to make purchase decisions in a non-equilibrium context. In principle this problem might be approached by use of dynamic programming methods, but it is not obvious how to set up an appropriately general model. In addition, the present investigation has considered sire purchase from a particular nucleus, whereas buyers usually have a choice between several, sometimes very many, sources of breeding males. If these are all improving at the same rate, differences between nucleus populations play the same role in the theory as differences within populations, and in principle this should lead to the same relation between price and breeding value between and within nucleus populations. If this is not so, a sire of given breeding value will be cheaper in some studs than in others, and the cheaper sires should be favoured. If the rate of genetic gain is not the same in every nucleus, then in the long run, that nucleus making most rapid progress should become dominant and supply the great majority of sires. However, if three are other nucleus populations which are initially superior, it may be best to buy sires from these in the early stages, buying from the nucleus making fastest progress only when it has surpassed its competitors, though this will clearly depend on prices.
The problem will be more complicated if the nucleus is producing breeding stock for commercial populations which do not have the same economic weights in all cases. Each sire would then have several breeding values, different ones for different potential buyers, and this would probably greatly complicate the effect of price on breeding policies. In fact, although it is hoped that the results of this study will prove useful, there is obviously much more to be done to clarify the sire purchasing problem.
It is interesting to note that when the theory is applied to purchase of French boars the actual decisions made buy buyers seem far from optimal. This would not be surprising, since the optimal policy is not easy to see, especially since it involves varying time of use of a boar in relation to his breeding value and price. However, there are other possiblilities for the discrepancy. One would be that genetic gains are not being obtained at the rate assumed, or at least they are not perceived as being obtained at that rate by sire buyers. This might be because of disagreement about the genetic changes occurring or because of disagreement about the economic value of the genetic changes which are taking place.
In addition, other economic factors may modify the price relationship. For example, in a time when the market for produce is poor, the incomes of producers will be low and they may lack the finance necessary to pay very high prices for superior sires. Again, if there is an excess supply of sires, there may be insufficient competition for the best ones to force those wishing to buy them to pay high prices.
A factor which has not been accounted for in the above analysis is the possible resale value of sires. It is unlikely that average sires would bring good prices when resold by producers after use for a few time units, but sires of very high breeding value used only for a short time by a producer could conceivably have a resale value of the same order of magnitude as less valuable sires bought directly from the nucleus, so that the net purchase price of highly selected sires would be less than the actual purchase price. The complicating factor is that the price on resale would decline with length of time the sire was used, and so if this factor were important, the optimum number of times to use a superior sire would be reduced. This aspect may deserve further study.
It has also been assumed that inbreeding may be neglected. In our context, this implies a producing population which is large enough so that if a superior sire is to be used many times it will be possible to avoid mating him to his daughters. When producing populations are too small for this the maximum use of superior sires will have to be less, and we would expect the price differential for high breeding value to be reduced.
However, although the present analysis has several limitations, it does suggest a method of planning sire purchase and use, and indicates the possibility that good sires may be rather cheap for the benefits they confer in some cases at least. | v3-fos |
2019-04-15T13:07:51.413Z | {
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} | s2 | Simulation model for the structure of Finnish agriculture
The simulation model for the structure of agriculture makes up one part of the Finnish food model. The structure of agriculture is described in the model by the agricultural population as well as by the number, the average size and distribution of all the farms, dairy farms, pig farms, poultry farms and non-animal farms. The agricultural population is calculated by forecasting its share from the total population as a function of GDP. The number of farms is derived from the agricultural population by assuming that the size ofa farm family is constant. The average size of the farm is derived by dividing the total acreage by the number of farms. The distribution of farms is forecasted by applying a logarithmic normal distribution function. The starting points for the structure of animal production are the consumption forecasts and self-sufficiency targets. The development of the average yield per animal is forecasted by applying a trend line. Also the average size of animal farms is forecasted by a trend. The number of animal farms is obtained simply by dividing the number of animals by the average farm size.
Introduction
The structure of Finnish agriculture has changed rapidly. The acreage of farms has decreased about 10 per cent in the 70's and the cultivated area has decreased even more due to soil bank, fallowing and afforestation policy actions. At the same time, the farm population has fallen considerably and with it the number of farms. Agricultural production has, however, not decreased in the same ratio; on the contrary it can be said that production has remained stable or has risen slightly. Animal production represents the major part of agricultural production with the most important still being milk production, the share of which is nearly half of agriculture's annual gross return. Considerable changes have occurred in milk production, falling in the 70's, but now it is remaining at the present level and may raise slightly in the future. An essential feature is, however, the rapid decrease of the number of dairy cows which is being compensated by the growth af average milk yield, so that production is about constant. Horses have fallen in number to little above zero but the numbers of pigs and hens have grown strongly.
The distribution of farms has changed considerably. The acreage per farm has not increased very much but small farms are dropping out of production and thus the large farms' share is increasing. Attempts have been made to control this distribution to some extent by making the establishment of large production units possible only if permission is granted.
The ongoing structural development of agriculture is not wholly accepted, since it means a decrease in farm population with rural areas becoming de-populated. There are, however, strong internal factors in the development which are difficult to overcome if this structural change is to be affected. Therefore it is interesting to study how development will continue without strong policy measures. The purpose of this study is to construct a mathematical model which can be used to follow the change of the structure of agriculture. The model is a part of a simulation model for the whole agriculture which is briefly described below.
Food and agriculture model for Finland
The purpose of the Finnish food model is to describe the interrelationships of the various parts of agriculture and serve as a policy tool for decision-makers in long term policy assessment and planning. It is not intended primarily for prognostication but rather for simulation of different development paths when different policy targets are set or different policy actions are taken.
The model is a recursive planning model, beginning from population and continuing to a structural model. Consumption is the major determinant in the model. It is a function of income and prices and together with self-sufficiency rates it determines agricultural production. This, in turn, effects the structure of agriculture as is explained later in this paper.
The yield level of crops is determined by the optimum use of fertilizers as the prices of fertilizers and products are given. The later ones are scenario variables whose evolution can be freely regulated. When production targets are set by the model the land needed can be calculated as the ratio of production and yield level.
Different versions of the model have been built, for example, the growth of Gross Domestic Product (GDP) has been considered in the present version as a scenario variable whose growth rate can be varied easily. Therefore, the GDP has been allowed to increase by a fixed percentage from year to year. In another version the non-agricultural production is a function of capital and labor force (see KETTUNEN 1 980). Since the growth of the economy is considered by the authors to be very uncertain the model of GDP is of minor importance to the functioning of the structural model.
Structural model for agriculture
3.1. Social structure of agriculture The social structure of agriculture is described in the model by the agricultural population as well as the number of farms, the average farm size and distribution. So far the agricultural population is calculated by forecasting its share from the total population as a function of GDP. The total population is assumed to grow by a fixed percentage (in this case 0.2 per cent) per year. The share of the agricultural population (AW (%)) is estimated as follows: (3.1) AW (%), = 0.03 + 0.87 c -1 -332GDP t It is presumed here that the minimum share would be 3 per cent but this minimum limit can be, however, easily adjusted within the model. The agricultural population (AW) is obtained by multiplying the total population by the percentage share: The number of farms is derived from the agricultural population by assuming that the size of farm family is constant (2.5 persons per family) and the number of farms (FARMS) is assumed to be the same as the number of farm families (households) The average size of the farm (ASF) (TAREA) by the number of farms: is derived by dividing the total acreage The net annual decrease of the total acreage is estimated to be 0.3 per cent.
The distribution of farms is forecasted by applying a logarithmic normal distribution function (see WALLENBECK 1979, p. 1 5 5-190). The farms are classified according to size using the following factors: total farms and farms without animals; hectares per farm, animal farms; animals per farm. Usually the distribution of farms is skew, i.e. there arc more of smaller farms than large ones. If the size distribution is drawn on a logistic scale, a frequency curve is obtained which is close to a normal distribution. When the average size increases the frequency curve moves to the right on the logistic scale but often it keeps its normal form. The fit of the logarithmic normal distribution to the distribution of the farm size was studied by HASSINEN (1980), and it was found that usually this assumption is valid in the Finnish case. In order to apply the log-normal distribution for the forecasts of the size distribution of farms, the distribution in the basic year and the function of the development of the average farm size must be known. Therefore the lognormal distribution can, in principle, easily be applied to the forecast of the size distribution.
The cumulative share of each size class of farm is obtained from the following: (3.5) F(xj) = Q(S '(lnxj (Inx S 2 / 2 ))), where F(x-) = forecast of percentage share of farms smaller than xj of all farms Xj = size class limit for i Q = standardized normal distribution S 2 = variance x = the average size of farms The estimate of variance is estimated from Q 1 = inverse of normal distribution (normal distribution tables are read backwards) Pnj = the cumulative percentage share of farms at the size class limit i Size class limits (Pnj and Pnj + n ) are selected so that their cumulative percentage shares arc approximately symmetrical to the 50 per cent level, i.e. closer to the lower and upper quartile.
The growth of GDP, is assumed to be 2.5 per cent in the basic scenario. This growth rate produces a forecast according to which the share of agricultural population in I 990 would be 7.5 per cent, the number of farms 147 900 and the average farm size 17.1 ha ( Table 1). The changes in the distribution of farms is presented in Table 2 for the years 1975 1990.
}.2. The structure of agricultural production Forecasting the structure of agricultural production is hampered by the lack of sufficient data regarding the level and development of specialization in different production lines. Therefore, methods applied for the forecasting of changes of production structure are usually simplified by assuming that specialization of the sector in question is fully realized. For example, every farm which has a single pig is counted into the pig farms irrespective of other possible production. This method obviosly results in a sum of all farm groups larger than the total number of farms. In this study farms are classified as dairy farms, pig farms, poultry farms and farms without animals. Since the supply of individual agricultural products is more flexible than the total supply of all agricultural products, it is obvious that changes in the production structure are also more flexible than the changes in the social structure of agriculture. Because of this flexibility, it is also possible to affect the structure by means of some specific agricultural policy. Therefore, making a long-term forecast of the structure of agricultural production without taking into account possible changes in consumption is unwise.
Experience has shown that the production of different agricultural products can be adjusted to some extent to the domestic consumption. Therefore, the starting point for the structure of animal production is the forecast of consumption of these products (ROUHIAINEN 1979). Production quantities are then derived by applying self-sufficiency targets and after production is determined, the number of animals can be derived. For that purpose, the forecast of the average yield per animal is made after which the number of animals is obtained by dividing production by the estimated average yield. In order to calculate the number of animal farms, the activities carried out on the average-sized farm should be specified and then the number of farms, which have animals, is simply obtained by dividing the number of animals by the average size of farms.
The development of the average size of animal farms is forecast simply by applying a trend line. Here, however, are the most serious weaknesses of the structural production model. It seems obvious that there is some kind of correlation between the volume of animal production and the average size of animal farms. For example, if milk production is limited it is necessary to reduce the number of dairy cows. It is likely that then propensity to stop milk production is the highest on the farms with a small number of dairy cows. Therefore in the production model it would be logical to expect a negative correlation between the number of cows and the average size of herds. On the other hand, if there is an oversupply of milk and the number of animals is reduced, there may be attempts to restrict the establishment of large production units or their expansion, slowing down the growth of average farm size. There appears to be some problems implicit in the calculation of this variable thus it is considered enough to use the linear trend method.
In the case of farms without animals, the model cannot be built from the production targets since a considerable part of crop production comes from farms which have also animal production. The calculation of the number of farms without animals is forecast therefore by applying a hyberbole function which is linked to the total number of farms.
Structure of the dairy sector
The starting point for the calculation of the volume of milk production is the consumption forecast of milk products. Production is estimated by setting a selfsufficiency target for milk after which production is obtained to the structure model from production model. To calculate the number of dairy cows corresponding to the production level required a model has been built which gives the milk yield per cow. The annual growth (AAY) of average yield is a scenario variable; The number of dairy cows (NC) is obtained by dividing production (Q m ) by the average yield: The calculation of the average herd size is estimated by applying a linear trend and having the annual growth of average herd size (A ACN) as a scenario variable; (3.9) ACN t = ACN, +(t -I)AACN It is difficult, however, to state without doubt that the forecast for the average herd size is correct. In the 70's the growth of herds was quite stable and represented a 0.32 increase per year. By projecting this growth to 1990 the average herd size would be 10.9 cows per farm. However, in 1977 the new milk production units have been of about 24 animal unit per farm.
The number of dairy farms (MF) is obtained by dividing the number of dairy cows by the average herd size: The average yield from dairy cows is estimated to increase by 78.6 kg per year, according to the linear trend from the years 1960-1978. Thus, at the end of forecast period, the average yield would be 5 328 kg per year. At the present time the average yield from inspection herds is even higher (5 359 kg in 1978), so that it is possible to achieve the estimated yield level. The yield increase is achieved by improvements in both the quality of the breed and husbandry techniques whilst the number of dairy cows is decreasing.
According to the food model the total consumption of milk products will decrease by 9 per cent in the period [1975][1976][1977][1978][1979][1980][1981][1982][1983][1984][1985][1986][1987][1988][1989][1990] (Table 3). If the self-sufficiency targets presented above are considered then there would be 488 000-577 000 dairy cows in 1990. The number of dairy farms would then drop to 45 000-53 000. The distribution of dairy farms depends on the development of average herd size and, according to the model, the share of herds of I-4 cows would drop to 18 per cent the share of s-lo cow herds would remain at 38 per cent and the share of larger units would grow a little; 31 per cent would be herds of 10-19 cows, 8 per cent would be herds of 20-29 cows and approximately 5 per cent of larger units.
The total consumption of beef is forecast to be 131 million kg in 1990. If the average slaughter weight is 200 kg, beef production based on dairy cows could be roughly estimated at 90-104 million kg. This would mean an under supply of 30-40 million kg, which could be filled by the expansion of the beef breed sector, since otherwise, imports would necessarily be rather high.
Pork production
The structural model for pork production is built without distinction between different lines of production. Pork production levels are obtained from the production model. The number of pigs (NP) required to maintain a specified production level (Qp) is derived by applying a coefficient k. The annual change of this coefficient (Ak) is a scenario parameter which depicts the change effected by breeding patterns; NP. The structure of pork production can be forecast with three alternative production targets, and adjustment to the production level occurs linearly within the periods, thus: 1) 100 per cent self-sufficiency in 1990 2) 110 " " " " 3) 120 The first difficulty in applying the model is to estimate the coefficient k which, in the past has varied significantly. It should fall a little because of the breeding and artificial insemination. In the 70's the coefficient k has fallen very rapidly, however, it is assumed that this decrease will be smaller in the future.
There are no annual statistics available for the calculation of the average size of piggeries, so a trend forecast is built, based on the statistics from years 1969, 1974 and 1977. If the development is the same in the future then the average size will be 147 pigs per piggery in 1990.
Pork consumption is forecast to increase by about 50 per cent by 1990 (Table 4) representing 1.4 million pigs in production. If the average size of piggeries is increasing, according to the forecasted trend the number of piggeries would fall to 9400. With a production target of 20 per cent above the domestic consumption 1.7 million pigs will be required and 11 300 piggeries in 1990.
Egg production
The structural model for egg production is analogous to the dairy model. Again, production is obtained from the production model and to calculate the number of hens a trend forecast is built for production per hen. The annual growth of egg production per hen (AAH) is a scenario parameter which can be changed: The average development of farm size (ASH) is forecast by a trend variable where the annual growth (AASH) is a scenario variable: The average yield per hen is forecast to grow by 0.147 kg per year according to previous trends (Table 5). At the end of the forecast period the production per hen would then be 15.1 kg. The growth of the average size of farms is assumed to continue as in the past, meaning that the average size would be 400 hens in 1990 and there would be 9500-11 400 farms in 1990. The size distribution shows that egg production is still practiced on small farms: 40 per cent of farms having less than 50 hens and about 7 per cent of farms with 1000 hens.
3.2,4. Non-animal farms
In case of non-animal farms the structure of the model cannot be built beginning from the production targets since a part of plant production comes from animal farms. Also, statistics from non-animal farms have been collected only from the The rapid growth of non-animal farms which occured in the period 1974-77 cannot continue if we consider the existing trend towards a reduction in the number of farms. Therefore, in order to avoid an unrealistic value for non-animal farms in the model a hyperbole function has been applied ( Fig. 1) There is a maximum share of 80 per cent for non-animal farms which is rather high. However, this limit has been selected since the forecast for 1990 is 5 5 per cent, which seems quite possible. If this factor is larger than one hundred per cent then the forecast is not logical and parameters have to be adjusted accordingly. The parameters for the model are logical, however, since the value of r for 1990 is approximately 95 per cent. | v3-fos |
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} | s2 | Genetic correlation between length of wattles and female body weight at sexual maturity in the foul
Two breeds (White Plymouth Rock and Light Sussex) and their F l crosses were used in this experiment in order to estimate phenotypic and genetic correlations between female body weight and their length of wattles at sexual maturity. Heritability estimates showed that both characters are highly influenced by heredity. The mean estimate of genetic correlation between the two characters was o.ig.
Introduction
Secondary sexual characters were proved to be correlated with egg productions and age at sexual maturity: PASVOG!1,et al. ( I95I ), PASVOG!r,( 195 2) and Cs A I, PERN and BONGAI!V ( 19 66). A YOUB and M ERA T ( 1975 ) proposed length of wattles of cockerels at 10 weeks of age as a criterion of selection for age at sexual maturity and egg production of their sisters. They obtained heritability estimates for the same trait.
The aim of this study is to estimate heritability of length of wattles for females at sexual maturity in other strains and genetic correlation between female body weight and this trait.
Material and methods
This experiment was carried out at Barrage, a Research Station belonging to the Agriculture Research Centre -Egyptian Ministry of Agriculture. Two seasons were available, namely 1974 -75 and 75 -7 6, using two pure breeds; white Plymouth Rock (PP), Light Sussex (SS) and their recriprocal Crosses. Sixteen sires were used, 8 sires from each strain. Each sire was mated to equal numbers of dams from each strain in order to produce the two pure breeds and their reciprocal crosses.
Body weight and length of wattles were recorded for females at sexual maturity ( 2 <^ weeks on the average). The data used in the analysis were as follows (Table i).
Genetic correlations were obtained from sire and dam: components of covariance estimated on fulland half-sisters.
Results and discussion 1 . -Means and variances Means, standard deviations and coefficients of variation are presented in table 2 , for each genetic group, and each character at sexual maturity. a) Body weight of females ; Body weight of the crosses were intermediate between parental breeds but closer to the heavier (PP). These findings suggest that mature body weight is depending largely on additive genes, with some degree of heterosis. Moreover, the two crosses showed less variability than the two pure breeds involved in this study, which is in agreement with many available references. b) Length of wattles: .' The length of wattles followed the same trend as found for mean body weight. The two crosses were approximately intermediate between the two parental breeds suggesting the important role of additive genes in the inheritance of wattle size.
. -Phenotypic co y relations
Correlations between female body weight and length of wattles at sexual maturity are presented in table 3 .
The estimates ranged from 0 . 12 to 0 .6 2 , suggesting a positive and significant correlation between the two characters.
3. -Heritabilities a) Body weight at sexuat maturity: .. Heritability estimates were obtained for the same two traits and are presented in table 4 . With respect to body weight they are within the range of many available references (K INNEY , ig6g). In general, heritabilities based on dams components of variance were greater than those obtained by sires components of variance. This may be due the presence of non additive genetic variance or maternal effect involved in the inheritance of body weight at sexual maturity.
b) Length of wattles:
Heritability estimates of length of wattles are comparable with the estimates found by Avoua and M ERA T ( 1975 ) for the length of wattles at 10 weeks of age.
Estimates from dam components af variance were generally greater than sire components of variance. These results confirm that length of wattles is appreciably influenced by heredity, although on the whole the heritability of this traits seems to be somewhat lower than that of body weight at the same age.
-Genetic correlations
Genetic correlations are presented in table 5 . Regarding the mean estimates being 0. 1 7 in 1974-75, 0.24 in 1975-76 and 0 . 19 from the pooled estimates, it may be stated that there was a positive correlation between mature body weight of females and their length of wattles in agreement with the phenotypic correlation. The negative values of genetic correlations may be due to sample error or scaling effect (FALCONER, 19 6 0 ). The higher value generally found for the sire component may correspond to the action of sex-linked genes. | v3-fos |
2019-03-19T13:05:44.707Z | {
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} | s2 | The effects of trace element supplements on blood levels of horses.
This study concerns the effects of Se, Cu, Co, Mn and Zn supplements on blood levels of Finnish warm-blooded trotters. The results revealed that the blood Se content corresponding to the feed Se content of 0.1 mg/kg is 0.17 + 0.03 mg/kg in the horse. Blood Se level was directly related to the feed Se content and the other feed trace elements had a similar effect on blood levels.
Introduction
The maximal performances demanded of race horses require that the various blood sub-factors fall within optimal, often very narrow, ranges (WILLIAMSON 1974). These sub-factors, macro-and microminerals, are influenced by the horses feed (KOSSILA et al. 1974, KÄÄNTEE and. In addition to the poor performances, deficiency conditions are reflected in diseases such as muscle dystrophy arising from lack of Se (LANNEK and LINDBERG 1975). Because the Se content of Finnish fodders is poor (KÄÄNTEE et ai. 1978), particular attention has been paid to the Se supplement of horse feed. Selenium, together with vitamin E and biotin, acts as a cell membrane protective factor (WROGEMANN and PENA 1976).
The effect of different Se compounds on the body Se level varies (KÄÄNTEE and KURKELA 1980). Both the body and feed levels of Se affect the behaviour of Cd, Hg, Cu, and Zn in the organism because of the interaction between various trace elements (DAVIES 1974).
In the studies performed the effect of feed factors on macro-and micro-minerals in the horse was investigated, with main attention focused on selenium.
Material and methods
Seven warm-blooded 4-B-year-old trotters in racing condition were used for the trials. The horses' weights varied between 340 and 410 kg and all were raced during the studies. The fodders and feeding periods are shown in Table 2. The feed Se level was altered by replacing part of the oats with barley rich in Se (0.68 mg/kg) during trial period 2. This increased the feed proportion of grain Se. During trial periods 4 and 5 the horses received 0.9 mg and 1.5 mg daily, respectively, of selenium as sodium selenite in a trace element mixture. The daily dose of trace elements and vitamins was: iron 600 mg, copper 2 5 mg, zinc 150 mg, iodine 0.15 mg, cobalt 3.0 mg, manganese 70 mg, vitamin E 750 mg, vitamin B, 24 mg, vitamin B 2 20 mg, vitamin B 6 1 2 mg, vitamin 812B 12 1 mg, folic acid 12 mg and vitamin C 500 mg.
The feed Ca, P and Se were determined at the Viljavuuspalvelu Oy company and the Oulu Research Laboratory of the Kemira Company (Table 1).
Hb was determined as cyanmethaemoglobin, scrum alkaline phosphatase by the HUGGINS and TALALAY (1945) method, SCOT and SGPT by the REITMAN and FRANKEL (1957) method, serum total protein by the WECHSELBAUM (1946) biuret method, serum vitamin 812B l2 by isotope dilution analysis and folic acid by the DUNN and FOSTER (1973) method. Scrum Ca, P, Mg, Na, K, Fe, creatinine and urate analyses were carried out with a SMA/60 Autoanalyzer in the Oulu Deaconess Institute Laboratory, and blood Se, Mn, Cu, Co and Zn determined by Perkin-Elmer atomic absorption spectrophotometer at Viljavuuspalvelu.
The blood samples were taken from the jugular vein during morning feeding on completion of each feeding period. On days prior to blood sample taking the horses did not take part in races or heats.
Results
The results of the blood Se analysis are shown in Table 2. They show that as the feed Se content rose the blood Se concentration also increased. A feed Se content of 0.1 mg/kg corresponded to a blood Se concentration of 0.17 ± 0.03 mg/kg. Table 3 shows analysis results of samples taken on completion of feeding periods. The use of the trace element mixture clearly increased blood Cu, Mn and Zn concentrations. The freely available refined salt raised serum Na values.
It was established clinically that during the use of the trace element mixture the general condition of the horses improved and the quality of the hair became better. Perspiration due to strain decreased and recovery was quicker. Racing performances also improved during the trial. Table 3 Analysed scrum and blood contents on completion of feeding periods (7 horses). The feeds given are in
Selenium
The blood Sc content of the horse depends on the feed Se content. The blood Se concentration of Finnish warm-blooded trotters has been measured as 0.18 ± 0.08 mg/kg (KÄÄNTEE ct al. 1978). Concepts of normal blood Se level differ greatly because of both variations in feed Se contents and inaccuracies in analytical methods employed. The Se requirement of animals is reported to be 0.1 mg/kg feed (NRC 1973).
The fodder Se concentration 0.1 mg/kg fodder was consisted of the grain Se, 0.03 mg/kg, and of trace clement mixture, 0.09 mg/kg. The blood Se poncentration was established to be 0.17 ± 0.03 mg/kg, which is of the same order as the normal value established in the previous studies (KÄÄNTEE et al. 1978).
The feed analyses carried out revealed the poor Se content of Finnish feeds. The Se content of basic feed was 0.03 mg/kg, which is clearly less than the critical Se level of 0.05 mg/kg below which not even large supplements of vitamin E can replace the lack of Se (JONES et ai. 1977).
On basic feeding the blood Se content was 0.08 ± 0.04 mg/kg. Muscular dystrophy has been established in horses with blood Se levels of 0.05 mg/kg or under (LINDHOLM et ai. 1978). On basic feeds, however, the blood Se content was above this critial level. In horses fed on Swedish domestic feeds the blood Se level varied between 0.01 mg/kg and 0.08 mg/kg (values calculated on the basis of the GSH-Px activity values of the HENNICHS and PEHRSON 1979 trial). The values established in the present study were slightly higher because brans rich in Se and Promeca were used. In Sweden, a Se supplement of 4.4 mg has been added to domestic feeds 2-4 times monthly to satisfy the Se requirement (HENNICHS and PEHRSON 1979). In the present trial daily Se supplements of 0.09 mg and 1.5 mg were added to supplement the normal feed of Finnish trotters. The feed Se content then rose to the NRC (1973) animal requirement level and blood Se content to values of 0.17 ± 0.03 mg/kg and 0.19 ± 0.04 mg/kg, respectively. It has been established in hens that grain Se raises body and blood Se contents considerably better than sodium selenite added to feed mixtures (KÄANTEE and KURKELA 1980). This was not found in the present material. The barley used was rich in selenium, containing 0.68 mg/kg grain. The selenium content of test feed 2 was 0.1 3 mg/kg with grain Se, but the blood Se content was 0.14 ± 0.04 mg/kg. The selenium content of test feed 5 was almost the same at 0.14 mg/kg. Most of the supplied selenium was sodium selenite from the trace element mixture; 0.03 mg/kg was grain Se and 0.11 mg/kg came from the trace element mixture. The blood Se content was 0.19 ± 0.04 mg/kg. The differences in blood Se contents were probably due to factors caused by gastrointestinal bacteria influencing selenium absorption (the bacteria transform selenium compounds) as well as to the effects of vitamin C contained in the trace element mixture. (COMBS and PETO 1976) Copper The Cu requirement of the horse is s-B mg/kg feed, according to OLSSON (1 969). DREPPER (1972) recommends 10 mg Cu/100 kg bodyweight for racing horses. Low Cu contents have been found in the timothy grass of Northern Finland (LAKANEN 1969). The Viljavuuspalvelu Company reports the mean Cu content of the hay of 1977-1978 as 4.4-4.6 mg/kg, of barley 5.5-5.1 mg/kg and of oats 3.9-5.3 mg/kg. The hay and oat Cu contents established by KOSSILA et al. (1972) were 6.7-7.4 mg/kg and 9.8-6.8 mg/kg, respectively, i.e. considerably higher than the values reported by Viljavuuspalvelu. The serum Cu content of horses in the material of KOSSILA et al. (1972) varied between 470 and 1140 ug/kg. According to KOLB (1967), the normal value for horses is 1300 ,«g/kg. MULLER-REH (1972) established the plasma Cu content of the horse to be 820-1210 g/kg while the feed Cu content varied between 1 1 and 3 1 mg/kg dry feed. According to EKMAN et al. (1975) the normal plasma Cu content is 1 530 ± 380 ng/kg. In the material studied the whole blood Cu content on feed 1 was 779 ± 395 fs-g/kg and on feed 2 749 ± 369 /ig/kg. The content trebled following the giving of the trace mineral mixture to 2044 ±1613 /<g/kg. The daily Cu supplement was 2 5 mg, and thus the daily copper requirement of 60 mg was fully satisfied when feed Cu (5 mg/kg) was also taken into account.
Zinc
The zinc requirement of horses is according DREPPER (1972) 40 mg/100 kg and that of Mn 20 mg/100 kg. KOSSILA et al. (1974) found Zn contents of 0.34 1.18 mg/kg in serum of horses that had received feed exceeding DREPPER's norms. According to SCHEUNEURT and TRAUTMANN (1965), there is less zinc in serum (2.0 mg/kg) than in erythrocytes (12.0 mg/kg). MULLER-REH (1972) established the plasma Zn concentration of the horse as 0.5 5-0.67 mg/kg while the feed Zn content was 0.22-0.44 mg/kg. In the material studied, whole blood was used for determining the 2n content in order to avoid errors due to haemolysis. The blood Zn concentrations obtained were 2.67 ± 0.78 and 3.00 ± 0.63 mg/kg and after a daily supplement of 150 mg Zn 3.16 + 0.61 mg/kg. The Zn supplement used had no significant effect on the blood Zn value.
Manganese
The blood Mn concentration doubled with a daily 70 mg supplement. According to the figures reported by Viljavuuspalvelu, the Mn contents of hay (71-84.7 mg/kg), oats (56.7-60.7 mg/kg) and barley (27.5-24.0 mg/kg) are sufficient to supply the 20 mg/100 kg requirement for a horse suggested by DREPPER (1972). MULLER-REH (1972) proposes 30-50 mg/kg as the recommended Mn content of horse feed.
In the material studied the horses' blood Mn concentration varied markedly because of feed factors. On feed 1 (hay + oats) the blood Mn content was 38.0 ± 13.8 Mg/kg. On feed 2 where part of the oats was replaced by barley poorer in Mn, the blood Mn content fell clearly to 28.8 ± 12.3 ,u g/kg. The Mn content of barley, according to Viljavuuspalvelu, is about half that of oats. With the daily trace element supplement of 70 mg the blood Mn level rose to 79.8 ±41.8 /tg/kg. Iron Both positive and negative interactions occurs between the various trace elements in the body. Certain interactions may generate diseases like anaemia, which may be due to iron, copper or cobalt deficiency, or excessive zinc or selenium intake (VUORI 1979). There is negative interaction between Cu and Zn as well as between Mn and Fe. Cu has positive interaction with Fe. A feed rich in Zn can give rise to conditional Cu deficiency. Which in turn affects Fe metabolism (DAVIES 1974).
In the present study, relatively large trace element supplements were used in association with feed 4. Clinically, the supplements were not established to be harmful. On the basis of analysis results, the greatest increases were found in blood Cu and Mn levels, but scrum Fc fell slightly even though the trace element mixture contained Fe. This may have been due to the effects of Mn.
The Hb values and serum alkaline phosphatase, GPT, GOT, STP, Ca, P, Mg, K and Fe of the material studied were at normal levels for Finnish warm-blooded horses (KÄÄNTEE 1977). As can be seen in Table 3, these blood parameters remain within fairly narrow ranges in a healthy race horse.
Sodium
Serum Na values at the lowest limits of the 'normal' range were established. The normal values for serum Na given in the literature material compiled by BEST (1979) range between 125 and 152 mmol/1, with most authors mentioning 139 141 mmol/1 as the ideal concentration. The Na requirement of the body is strongly affected by perspiration as well as by secretion of gastric and intestinal juices. A horse engaged in heavy racing and training requires 8 5 g refined salt according to NRC (1973) Such a high daily requirement demands a salt supplement in the feed or free access to salt, as was the case with feed 5. Serum Na content rose to the level of 141 mmol/1 demanded by maximal performance recommended by WILLIAMSON (1974) Folic acid In his studies ALLEN (1978) established that the requirement and use of folic acid by training and racing trotters was greater than that of inactive horses. Folic acid is particularly excreted from the body in the sweat. The serum folic acid content of training horses was 3.3 fx g/1 (1.5-6.1 ug/1) and in inactive animals 10.6 ug/1 (6.4-15.8 Afg/l).
In the present material, the serum folic acid content, or that of the basic form of tetrahydrofolic acid which acts as coenzyme in the transfer of hydroxymethyl groups, in training horses was 5.93 ± 1.2 ,«g/l at the beginning of the trial. This level approximates those measured by ALLEN. With the trace element mixture the horses received a 12 mg folic acid supplement daily. This raised the serum level to 7.33 + 2.3. /<g/l, which was clearly lower than those established by ALLEN in inactive horses. The folic acid supplement used in the material studied has to be regarded as recommcndablc because of increased consumption due to the heavy training of the horses.
Vitamin 6, 2 In the material studied the serum vitamin 812B 12 content was 1.84 ± 0.19 ng/ml and after a daily vitamin 812B 12 supplement 2.01 ±0.19 ng/ml. Thus the vitamin supplement had no significant effect on the serum vitamin 8, 2 level of the horses. The values determined approximated those established by SALMINEN (1975) in Finnish horses, i.e. 1.54 ± 0.16 ng/ml. SECKINGTON et al. (1967) found 1.11 ±O.l ng/ml to be the mean serum vitamin 812B l2 content among 2 5 horses.
Summary
In this study variations in blood parameters of 7 Finnish warmblooded trotters were investigated in association with changes in the feed trace element content. The latter were achieved by replacing part of the oats of the basic feed by barley rich in Se, and by giving a trace clement mixture in association with the basic feed.
When the Se content of horse feed was 0.03 mg/kg the blood Se concentration was 0.08 ± 0.04 mg/kg. With a feed value of 0.1 mg/kg the blood concentration rose to 0.17 ± 0.03 mg/kg and with a feed content of 0.14 mg/kg to 0.19 ± 0.04 mg/kg. The blood Cu value (779 ± 395 /<g/kg) tripled when a daily 25 mg copper supplement was used. The Mn content 38 ± 13.8 ,Mg/kg rose to 79.8 ±41.8 ,Mg/kg with a daily 70 mg Mn supplement. The blood Zn value of 2.67 ± 0.78 mg/kg rose to 3.19 ±0.61 mg/kg when the horses received a daily 150 mg zinc supplement. | v3-fos |
2019-03-19T13:09:02.363Z | {
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} | s2 | Physiological effects of Pekilo single cell protein on pigs
The physiological effects of Pekilo microfungus biomass (Paecilomyces varioti) grown in sulphite spent liquor solution was studied in pigs. Sixteen Finnish landrace gilts weighing about 22 kg were divided into two groups of eight animals each. The animals were fed a barleyand oat-based diet supplemented with vitamins and minerals. The soybean and fish meal mixture used as a protein supplement for the control group was replaced with Pekilo at a level of 13 % of the feed weight in the experimental diet used up till farrowing. The experimental diet used during lactation contained 15 % of Pekilo. The feed consumption and weight gain of the animals were recorded. The experiment lasted almost one year and included the growth, gestation and lactation periods of the sows. The weight gain of the piglets was observed. After weaning of the litter the sows were analysed for blood haemoglobin, haematocrit and white blood cell count, plasma glucose, serum bilirubin, ASAT, ALAT, urea, uric acid, allantoin, total protein and albumin/globulin ratio. The allantoin, uric acid and creatinine contents of a single urine sample were determined quantitatively, and the pH, glucose and protein contents semiquantitatively. The urine density was measured. There were no significant differences between the two groups with regard to weight gain, feed consumption, feed efficiency and reproductive performance. Serum urea and plasma glucose levels were significantly lower in the Pekilo-fed group than in the control group but were within the normal limits for swine. The serum allantoin level and the urine allantoin/creatinine ratio of the Pekilo group were significantly above the control group values. No differences were found between the two groups in the other blood and urine analyses. The use of Pekilo biomass as protein supplement in the diet of breeding sows was found to have no detrimental effects as assessed on the basis of weight gain, feed consumption, feed efficiency, reproductive performance and certain blood and urine parameters.
Introduction
To help to alleviate the world-wide shortage of proteins new single cell products have been developed. Detailed recommendations for the study of the harmlessness of new protein products were drawn up by the Protein-Calorie Advisory Group (PAG) composed of representatives of United Nations organi-zations (Pag 1972). This Group was dissolved at the end of 1977. In the United States all single cell protein products must fulfill the Food and Drug Administration (FDA) requirements on food additives.
Pekilo biomass was developed in Finland and is produced at the Jämsänkoski Mill of the United Paper Mills Ltd. by growing the microfungus Paecilomyces varioti in sulphite spent liquor solution (Romantschuk 1975). Protein content of the Pekilo mass varies somewhat according to the growth rate of the organism. In industrial production the crude protein content of Pekilo seems to stay near to 50 % of dry matter (Salo 1979). Pekilo mass additionally contains, on dry matter basis, 32-36 % carbohydrates, mostly hemicellulose (Salo 1977), and 2-4 % fat (ether extract) as well as minerals and B vitamins (Romantschuk 1975, Salo 1979. The nucleic acid content of Pekilo is 10 to 11 % of dry matter (Salo 1979).
In order to determine the nutritional properties of Pekilo numerous shortterm experiments have been carried out on rats, pigs, calves and chickens (Ahlström et al. 1968, 1969, Lampila et ai. 1971, Poutiainen 1973, Laksesvela and Slagsvold 1974, Alaviuhkola et ai. 1975, Farstad et ai. 1975, Barber et ai. 1977. Experiments on the suitability of Pekilo as animal feed performed in nine different countries were reviewed in the International Pekilo Symposium held in 1978 (Kiiskinen 1979). The addition of Pekilo to livestock feed was approved in Finland in 1971(cf. Forss 1974. Its acceptability for human concumption still requires much research work, particularly as regards its long term effects.
The long-term experiment into the effects of Pekilo on the reproductivity of sows being carried out at the Swine Research Station of the Agricultural Research Centre, Hyvinkää, provided an opportunity for obtaining additional information about the effects of relatively long-term feeding of Pekilo biomass on the general condition, liver, kidneys and protein metabolism of swine.
Material and methods
Sixteen Finnish landrace gilts, 9 to 12 weeks old, were divided into two groups of eight animals each. The pigs were born at the Swine Research Station between December 9, 1976 and January 6, 1977. At the beginning of the experiment the average weight of the control group animals was 22.4 kg and of the experimental group 22.2 kg. They were fed at first in groups of four, and later of two animals. During gestation and lactation the sows were fed individually. Weight gain was followed by weekly weighings. Breeding was begun when the sows weighed 100 kg, at an age of about 7 months, using Finnish landrace boars. The piglets were weighed as newborn and at 3 and 5 weeks. From the age of 1 week onwards they had free access to the sow's feed mixture. The litter was weaned when 5 weeks old.
The barley-and oat-based control diet was supplemented with fish meal and soybean meal. A small amount of skim milk powder was added to the control diet during lactation. In the experimental diet Pekilo biomass was the source of supplementary protein. Table 1 shows the basic composition of Pekilo biomass and Table 2 the composition of the feed mixtures. The first batch of Pekilo lasted for about half of the test period, after which Pekilo mass B was used. The control diet and Pekilo diet were similar in crude protein, crude fat and crude fibre contents as well as energy sources -The animals were fed twice daily. Daily rations were gradually increased from 0.7 kg per animal at the beginning to 2.7 kg per animal after fifteen weeks. The animals had free access to water. Blood and urine samples were taken from the sows on the fifth day after weaning of the litter. The blood sample was drawn from the ear vein with an injection needle 34 hours after morning feeding. The single urine sample was collected when the animals urinated.
The blood haemoglobin was determined as cyanmethaemoglobin (Van Kampen and Zijlsta 1961) and the haematocrit by centrifuging ((Microhematocrit centrifuge, Clay-Adams, Inc.) the blood in a capillary tube. White blood cells were counted mechanically (Coulter Counter, Model ZF, Coulter Electronic Ltd.) in 1 % acetic acid and the differential count was made from blood smears stained by the May-Griinwald-Giemsa method (Hyvärinen et ai. 1972). The glucose was determined from plasma (Schmidt 1961). Bilirubin (Jendrassik and Grof 1938), ASAT, ALAT (Anon .1974), urea (Fawcett and Scott 1960), uric acid (Kageyama 1971), total protein (Weichselbaum 1946) using commercial test combinations (Boehringer Mannheim GmbH, Mannheim) and allantoin (Sumi et al. 1976) were determined from the serum. The albumin/globulin ratio was calculated following electrophoretic fractionation of the serum protein in SDS polyacrylamide gel (Fehrnström and Moberg 1977) (LKB, Bromma 2117 Multiphor). The intensity of the stained protein fractions was measured with a densitometer (Digiscreen Scanner R, Gelman, Model 39381).
The presence of glucose and protein in the urine and the urine pH were determined with test strips (Combur 8 Test, Boehringer Mannheim GmbH, Mannheim). Urine density was measured with an areometer. The uric acid and allantoin contents of the urine were determined by the same methods as for serum. The creatinine content of the urine (Clark and Thompson 1949) was determined for calculations of the uric acid/creatinine and allantoin/ creatinine ratios. A spectrophotometer (Perkin Elmer Double Beam, Model 124) and a fluorescence spectrophotometer (Perkin Elmer, Model 203) were used for the spectrophotometric and fluorometric measurements, respectively.
The mean and the standard error of the mean (SEM) were calculated from the results. The significance of differences between groups was tested with the Student's t-test. Table 3 shows the mean weight gain and feed efficiency (feed consumption in kg/weight gain in kg) in the Pekilo and control groups during the first 19 weeks of the experiment. There were no statistically significant differences between the groups as regard to the weight gain of the animals or their feed utilization.
Results
The breeding data of the sows are presented in Table 4. Some matings had to be repeated in both groups, and one sow in the Pekilo group did not become pregnant in spite of several matings. Histopathological examination of the ovaries, uterus, liver, heart and kidneys (Department of Pathology, State Veterinary Medical Institute, Helsinki) did not reveal any changes that could account for the failure to conceive. The mean age of the animals at the time of conception was 266 days in the control group and 261 days in the Pekilo group. There were no significant differences between the two groups with regard to the number of newborn piglets, birth weight, perinatal mortality, or weight gain of piglets during suckling. No external malformations were evident. Mortality before the age of 5 weeks was 11 % in the control group and 17 %in the Pekilo group. Most deaths occurred during the first few days after birth. Farrowings were not observed, which is normal practice at the station. One of the Pekilo group sows acquired mammitis and three piglets in Table 3. Initial weights of the pigs and their Weights after 19 weeks on the experimental diets as well as daily weight gains and feed efficiencies of the animals. her farrow died, evidently from starvation. The farrow was partly on artificial feed (Plasma Nasu, Hankkija) and the pigs were weaned at the age of 4 weeks. The blood analysis results of the sows in the two groups are given in Table 5. The normal blood values of swine are included for comparison purposes. These data, however, should be accepted with some reservation in view of differences in the methods of analysis and in the pysiological condition of the animals tested. Significant differences between the control and Pekilo groups were not found for the blood haemoglobin and haematocrit values, the white blood cell count, serum ASAT and ALAT activities, bilirubin, uric acid and total protein contents or in the albumin/globulin ratio. The glucose content of the plasma of the sows given Pekilo was significantly (P < 0.01) lower than that of the control group animals. The serum urea content was also significantly (P < 0.05) lower and the allantoin content higher (P < 0.01) than than in the control group.
The urine pH of all the animals was about 5. Protein was not found in the urine samples. Glucose was present in moderate amounts in the urine of two control animals and abundantly in the urine of one Pekilo animal. There was considerable individual variation in the urine density, but the group values did not differ significantly. The mean of the urine density was 1.009 g/cm 3 in the control group and 1.008 g/cm 3 in the Pekilo group. The results of the quantitative urine analyses are listed in Table 6. The allantoin and uric acid contents of samples from a single urination did not differ significantly in the two groups. The allantoin/creatinine ratio, on the other hand, was significantly higher (P < 0.01) in the Pekilo than in the control group.
Discussion
In the present experiment the kind of protein supplement used in the diet Pekilo or fish meal and soybean meal mixture did not have a significant effect on the growth of pigs. In earlier feeding experiments the Pekilo used as protein supplement also had no significant growth-limiting effect in pigs (Alaviuhkola et al. 1975, Barber et al. 1977. In the light of the results obtained in this experiment, the use of Pekilo biomass had no detrimental effect on the reproductive performance of the sows. The test material, however, was too small to allow any definite conclusion The slightly faster growth rate of the Pekilo pigs during the suckling period was presumably due to the smaller average size of the litters in comparison to the control group. Significant differences were not found between the Pekilo and control group sows with respect to blood haemoglobin, haematocrit, white cell count, serum ASAT and ALAT activities, bilirubin and protein contents and albumin/ globulin ratio. In a short-term feeding experiment with pigs Farstad et al. (1975) observed some differences in the blood values of animals receiving various amounts of Pekilo in their diet either with soybean protein or as the sole source of protein, as compared with animals given soybean protein alone; the values lie, however, within normal range for pigs. The serum urea level in the Pekilo group was lower than that in the control group. The result is similar in trend to that obtained by Farstad et al. (1975). Although a difference was seen between the plasma glucose contents of the Pekilo and control groups the values lie within normal limits.
The rather low serum uric acid contents of the swine were very close to the lowest limits of detection of the analysis method used. Uric acid, which is a product of nucleic acid metabolism, is metabolized further to allantoin in swine. Roth and Kirchgessner (1977) have demonstrated that serum allantoin content as well as excretion of allantoin and uric acid in urine increase in proportion to the amount of nucleic acid in the diet of pigs. However, the effect of nucleic acid content in the diet on the uric acid excretion of swine was rather small. In the present experiment the difference between allantoin contents in urine of the two groups was evident only after the concentration differences between the urines were eliminated by relating the allantoin values to the urine creatinine values. The daily urine volume of swine may vary normally from two to six liters (Cornelius and Kaneko 1963). In the absence of metabolic cages it was not possible to collect the 24hour urine from the animals. No difference was found between the urinary uric acid levels of the Pekilo and control groups; the values were dispersed over a wide range in both groups. The pH and the mean urine density of the samples from both the Pekilo and the control animals corresponded to those normally encountered in swine (Cornelius and Kaneko 1963). The glucose present in the urine of a few animals may have been due to postprandial hyperglycaemia. The taking of urine samples could not be timed exactly.
This experiment gave information about the effects ot long-term feeding with comparative!}' small amounts of Pekilo biomass on some blood and urine parameters that give information about protein metabolism and the state of the liver and kidneys. The results obtained coiroborate the results of short-term feeding experiments carried out earlier and indicate no detrimental effects. Further studies are needed to obtain evidence on the absence of such effects from the long-term use of Pekilo, especially with regard to possible carcinogenic, mutagenic and teratogenic effects. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Pyrethrum flowers and pyrethroid insecticides.
The natural pyrethrins from the daisy-like flower, Tanacetum or Chrysanthemum cinerariifolium, are nonpersistent insecticides of low toxicity to mammals. Synthetic analogs or pyrethroids, evolved from the natural compounds by successive isosteric modifications, are more potent and stable and are the newest important class of crop protection chemicals. They retain many of the favorable properties of the pyrethrins.
Pyrethrum Flowers and Pyrethroid Insecticides
by John E. Casida* The natural pyrethrins from the daisy-like flower, Tanacetum or Chrysanthemum cineraruifolium, are nonpersistent insecticides of low toxicity to mammals. Synthetic analogs or pyrethroids, evolved from the untural compounds by successive isosteric modifications, are more potent and stable and are the newest inportant class of crop protection chemicals. They retain many of the favorable properties of the pyre-Uwns.
Most insect pests of crops, livestock, and man have been easily controlled for the past 35 years by relatively inexpensive organochlorine and organophosphorus compounds and methylcarbamates. Control of pest insects is progressively more difficult and costly because of increasing restrictions on some of the major insecticides of these types. They are considered to have unfavorable persistence. environmental impact and/or toxic effects on higher animals including man. There is an urgent need for alternative chemical or biological control methods for pest control. Pyrethroids developed within the past seven years are helping to meet this need. These insecticides are structural modifications of one of the oldest insect control agents, the remarkably effective but unstable pyrethrins from pyrethrum flowers. The newer pyrethroids have greatly improved potency and stability. It is appropriate as the use of pyrethroids expands to examine their origin, properties and safety aspects as compared with the pyrethrins and their prospects for filling current and projected gaps in insect control capabilities.
Pest insect control until the 1940's was moderately successful with the use of primarily compounds from natural sources either directly or after simple extractions or other treatments. These "first generation" insecticides were inorganic arsenicor fluorine-containing toxicants or botanicals such as nicotine, rotenone, and pyrethrins. Except for the *Pesticide Chemisiry and Toxicology Laboratory, Department of Entomological Sciences, University of California, Berkeley, California 94720. pyrethrins, they were displaced in the 1940's and 1950's by synthetic organic or "second generation" insecticides which provided nearly complete control at reduced cost due to high potency or persistence or both these properties. There are four principal classes of second generation insecticides, i.e., organophosphorus compounds, methylcarbamates, chlorinated hydrocarbons, and pyrethroids; all act as nerve poisons. The first two classes inhibit acetylcholinesterase and thereby disrupt synaptic transmission. The others probably act at nerve membranes principally by altering sodium conductance mechanisms. Several of the chlorinated hydrocarbons (e.g., DDT, aldrin, and dieldrin) have been restricted or banned because of unacceptable persistence, effects on wildlife, and evidence of possible carcinogenic activity. Some of the important organophosphorus and methylcarbamate insecticides are very hazardous to manufacture, formulate and apply due to their high acute toxicity when ingested, inhaled or absorbed through the skin. Most of the pyrethroids appear at present to have generally favorable persistence and toxicological characteristics.
The first and second generation insecticides act on systems important for survival in both pest and other organisms including mammals. They therefore lack the selectivity which is theoretically possible with hormones or antihormones, agents that disrupt cuticle or chitin formation, or other types of insect growth regulators. These "third generation" insecticides have not yet been perfected for extensive use, and there are definite limitations in the types of pest February 1980 infestations where such slow-acting compounds are likely to prove acceptable. Chemical control of insects depends almost completely at present on first and second generation insecticides including pyrethrins and pyrethroids. The common names of important compounds are given in Table 1.
Pyrethrum Flowers
In the early 1800's pyrethrum flowers were used by Caucasian tribes and in Persia to control body lice. The flowers were first produced commercially in Armenia in 1828. Production started in Dalmatia (Yugoslavia) about 1840 and was centered there until the first World War, in Japan until shortly before the second World War, and in East Africa since then. More than half of the world's current production comes from Kenya, with important amounts from Tanzania, Rwanda, and Ecuador. Insect powder was first imported into the United States in about 1860, and several unsuccessful attempts were made over the next 90 years to produce the flowers commercially in this country. Since about 60 years ago the flowers were extracted with kerosene or similar solvents to give liquid sprays more effective than the powders.
The pyrethrum plant of commerce is the daisy, Tanacetum cinerariifolium (Trev.) Schultz Bip.
[Pyrethrum cinerariifolium Trev. and Chrysanthemum cinerariifolium (Trev.) Vis.], a herbaceous perennial of the family Compositae. The producing areas are near the equator, from 1800 to 4000 m in altitude, and with rainfall of 50 to 200 cm spread throughout at least seven months of the year. Under these growing conditions, flowering continues for seven to 11 months each year. In Kenya alone, more than 85,000 families are engaged in growing pyrethrum. Worldwide, perhaps 200,000 farmers are involved in pvrethrum culture. Pyrethrum production is currently estimated at about 15,000 tons of dried flowers per year, about half the amount currently needed for the world market. The dried flowers contain 1-2% pyrethrins by weight, averaging about 1.3%; so, the production of pyrethrins is about 150 to 200 tons per year. Present annual production averages about 560 kg dried flowers per hectare, although plant selection and improved propagation and cultivation techniques make it possible to produce 900 kg of 1.8% or higher dried flowers annually in some growing areas.
The pyrethrins are localized in the secretory ducts of the achenes, where they are protected from photodecomposition and isolated so they are not toxic to insects feeding on or visiting pyrethrum flowers. The flowers are hand picked when four or five rows of disc florets are open, and each flower contains about 3-4 mg pyrethrins. After drying in the sun or mechanically, the flowers are ground and extracted with hexane. Evaporating the hexane yields a dark, viscous oleoresin concentrate containing about 30%o pyrethrins. The concentrate is either diluted with plant or mineral oil to 25% pyrethrins (oleoresin extract) or purified by methanol extraction and charcoal treatment to produce a dewaxed and decolorized refined extract. This purification removes components which earlier gave allergic responses evidenced as dermatitis in humans sensitive to ragweed pollen.
Pyrethrum extract is important to control of pest insects in the household, in barns, and in stored products, and for direct application to man and livestock. Before the second World War, powdered pyrethrum flowers or pyrethrum extract were employed for control of agricultural and horticultural insect pests, ause largely superseded in the 1940's by the more effective and simpler chlorinated hydrocarbon and organophosphorus-insecticides. Compared with these synthetic organic insecticides, the persistence of the pyrethrins, even with various additives to retard photooxidation, is not adequate for crop protection or silviculture. The present uses for pyrethrum are well established and dependable methods of insect control, but for very specific purposes not likely to change in the foreseeable future.
Environmental Health Perspectives
The selection of resistant strains, a problem with most other insecticides, has had little impact on the use pattern of the pyrethrins.
The pyrethrins knock down houseflies, mosquitoes, and other flying insects rapidly and, at appropriate doses, the insects die a few minutes or hours later. Instructions for space sprays or aerosol applications through the 1940's cautioned users to be neat and tidy, and to sweep the flies outside the house after knockdown; under these circumstances the user was oblivious to the recovery of the flies. The hyperexcited state preceding or associated with knockdown is also useful in other ways. It repels and disorients biting flies and mosquitoes which therefore bite less frequently. It flushes cockroaches from their hiding places to contact lethal doses of the insecticide. Three developments helped establish and maintain these uses for pyrethrum. The first was an alternative method for delivering pyrethrins to control mosquitoes by incorporating ground pyrethrum flowers with other ingredients into mosquito coils, which, burned throughout the night, generated a smoke that repelled, expelled, knocked down, or killed mosquitoes in human habitats. The second in 1941 was the aerosol can or "bomb" which, although now used to disperse many types of household and industrial agents, was originally perfected to deliver pyrethrum extract. It produces droplets below 30 ,um in diameter, essential for maximum effectiveness and economical use of the pyrethrins. The final development was an additive or synergist, piperonyl butoxide, which was discovered in 1949. Although essentially noninsecticidal, it increases the potency of pyrethrum by more than fourfold when added at two to ten parts of synergist per part of pyrethrins. The pyrethrins-synergist combination is much more economical than the insecticide alone, since the synergist costs less than 5% per unit weight of the pyrethrins. The synergist also increases the likelihood that insects knocked down will subsequently die rather than recover. In addition to piperonyl butoxide, another type of synergist, MGK 264, has also been important for many years.
Pyrethrum is generally considered to be the safest insecticide and was once labeled as "nontoxic to humans and pets." This labeling is no longer allowed, so it is difficult for the user to differentiate the relative safety of various household insecticide products. After use for more than a century, there are very few cases of human illness associated with exposure to pyrethrum, and most of these are early reports of dermatitis or allergic reactions due to impurities no longer present in the purified extract. Pyrethrins were once used at three consecutive daily doses of 10-20 mg per adult or 5 to 10 mg per child to control intestinal worms without reported ill effects.
February 1980
However, it is known that accidental oral or dermal exposure to high doses of pyrethrins can cause a temporary numbness of the tongue and lips. Pyrethrum extract has low acute toxicity to mammals and birds despite a very high toxicity to insects, other invertebrates and fish (lethal concentrations of a few parts per billion in water). Tests with laboratory mammals indicate that pyrethrum, even at high doses or combined with piperonyl butoxide, does not produce carcinogenic, mutagenic or teratogenic effects. Any pathological changes observed at high doses are in the liver.
The pyrethrins are very unstable in light and air, limiting the areas where they are effective but also providing a safety factor against the accumulation of hazardous residues. Uses of pyrethrum and its synergists are regulated by restrictions under the Environmental Protection Agency and by tolerances for levels in food and feed under the Food and Drug Administration. The tolerance values for post harvest applications to various plant products are commonly 1-3 ppm for pyrethrins and 8-20 ppm for piperonyl butoxide. In several cases, these compounds are exempted from the requirement for tolerances because their safety relative to the levels likely to be present under normal conditions is acknowledged.
Chemistry of the Pyrethrins
Pyrethrum extract contains six closely related insecticidal esters, collectively referred to as the pyrethrins, which differ only in the terminal substituents in the side chains of the acid and alcohol components. The acid is a substituted cyclopropanecarboxylic acid and the alcohol a substituted cyclopentenolone. Three alcohols are involved, pyrethrolone, cinerolone and jasmolone for the pyrethrins, cinerins, and jasmolins, respectively ( Table 2). The two acids are chrysanthemic acid for the I series and pyrethric acid for the II series. The main structural features of these compounds were elucidated between 1910 and 1916 but not reported until 1924 by Hermann Staudinger and Leopold Ruzicka, two Swiss chemists each later awarded a Nobel Prize for pioneering discoveries in several fields of chemistry. Important They could probably be obtained, if needed, by total synthesis, by reconstitution from the acids and alcohols derived from pyrethrum flowers, or by isolating the natural materials using various combinations of column adsorption and partition chromatography, gas-liquid chromatography and high pressure liquid chromatography. The pathways used by pyrethrum flowers in biosynthesis of the acid moieties of the pyrethrins from mevalonic acid are well established and of the alcohol moieties are partially understood. The pyrethrates originate biosynthetically from chrysanthemic acid or chrysanthemates by oxidation of the trans-methyl group of the isobutenyl substituent, followed by biomethylation [Eq. (1)]. Oxidation of an isobutenyl methyl substituent is involved in biosynthesis of pyrethrates in pyrethrum flowers and metabolism of chrysanthemates in mammals and insects. The R' substituent may be hydrogen as in 192 chrysanthemic acid or an alcohol moiety as in the pyrethrins. The oxidases and dehydrogenases require pyridine nucleotide cofactors as indicated. Biomethylation utilizes S-adenosylmethionine as the methyl donor. Pyrethrate-hydrolyzing esterases require no cofactors.
The biological activities of the pyrethrum constituents depend on the structures and stereochemical characteristics of both the acid and alcohol components. Pyrethrins I and II are considerably more potent than the cinerins and jasmolins. The chrysanthemates (I) are generally more potent for kill and the pyrethrates (II) for knockdown. Thus, pyrethrum contains a combination of an excellent knockdown agent (pyrethrin II) and a potent insecticidal component (pyrethrin I). The pyrethrins have three chiral centers and therefore eight different optically active forms are possible (Fig. 1). There is also geometrical isomerism (E or Z) in the side chain of Environmental Health Perspectives pyrethrolone Pyrethroid stereochemistry is illustrated with pyrethrin II. Asterisks designate chiral or asymmetric centers. TI indicates the remainder of the molecule beyond the bracketed portion. Partial structures give optical isomers in the bottom row and geometrical isomers at the sides of the upper row. Arrows indicate names for the transformations and the chiral center or double bond where inversion takes place. Two isomers are possible, the natural one in the pyrethrin II structure and the alternative one in the partial structure, in each of the five cases. This makes 25 or 32 possible isomers. Pyrethrin I lacks the methoxycarbonyl group at the upper left so it has only 24 or 16 possible isomers. the alcohol (chrysanthemates) or of the acid and alcohol (pyrethrates) increasing the number of possible stereoisomers to 16 for the chrysanthemates and 32 for the pyrethrates. Although these isomers have not all been prepared and tested, the available evidence strongly suggests that the naturally occurring configuration is likely to be the most potent one.
History of Pyrethroids
Synthesis of naturally occurring compounds and their bioactive analogs is a normal part of natural products chemistry research. These investigations are important in structural elucidation, often using simplified compounds as models. If model chemicals for the pyrethrum constituents are insecticidal, they are pyrethroid insecticides. The principles by which second generation insecticides were later discovered were already recognized over 60 years ago, since Staudinger and Ruzicka prepared about 100 candidate pyrethroids between 1910 and 1916, although none was particularly insecticidal. After correcting some details of the pyrethrins structures assigned by Staudinger and Ruzicka, LaForge, Barthel, and Schechter derived a simpler synthetic analog, in which an allyl group replaced the pentadienyl side chain of the alcohol moiety. This compound, allethrin, the first commercial pyrethroid, culminated February 1980 (I) wartime research in both the United States and England seeking pyrethrum substitutes to minimize dependence on the natural material produced in areas where shipping channels and therefore available supplies might be disrupted.
Few new pyrethroids were discovered following commercialization of allethrin until developments about 15 years ago in the laboratories of the Sumitomo Chemical Company (Osaka and Takarazuka, Japan) and of Michael Elliott, Norman Janes and their co-workers at Rothamsted Experimental Station (Harpenden, England). The acid moiety was standardized as the now commercially-ROe >,t pyrethrins IS a available chrysanthemic acid and new alcohol moieties were examined. Pyrethroids developed at this time served essentially as pyrethrum substitutes without expanding the general scope of use. Variations of the acid moiety were then made, using the best alcohol moieties available. At present, dozens of laboratories in many countries are examining thousands of potential pyrethroids each year. There are four primary optimization goals at present: high effectiveness as knockdown agents for flies and mosquitoes (i.e., pyrethrum substitutes); maximum potency as broad spectrum insecticides; adequate stability for plant protection; reasonable cost rela- tive to the use levels for adequate pest control.
It is sometimes difficult to decide whether or not a synthetic insecticide is a pyrethroid. Generally, new compounds may be considered pyrethroids if their biological properties depend on stereochemical features similar to those of the pyrethrins. The most active compounds are carboxylic acid esters with the carbons adjacent to the carboxyl group bearing appropriate substituents positioned, ifthey are at chiral centers, in a configuration corresponding to pyrethrin I. Structural optimization is normally achieved by sequential isosteric replacements of critical substituent groups, as illustrated in Eq. (2) for the alcohol moiety and Eq. (3) for the acid moiety. Selecting appropriate replacement groups is not as easy or obvious as it might appear. Each novel moiety was discovered in tests of many hundreds or thousands of esters, most of which proved to be essentially noninsecticidal. Some of the acid and alcohol moieties are closely related to those of the natural esters, while others seem far removed, e.g., chlorophenylcontaining acid moieties and cyano-containing alcohol moieties. One test for a pyrethroid acid moiety is to determine if it is most potent with the normal pyrethroid alcohol moieties; the same argument applies for pairing candidate alcohol with acid moieties. The acid moieties of some pyrethroids bear a close structural resemblance to a portion of molecules of the DDT type, raising the question of whether pyrethroids and DDT might act in part at similar or the same neuroreceptors. Although there are many similarities of action between pyrethroids and DDT, the relevant neuroreceptors are not adequately defined so specific neurophysiological tests cannot be used to differentiate unequivocally pyrethroids from nonpyrethroids.
Pyrethroids as Pyrethrum Substitutes
Five pyrethroids are used in much the same manner as pyrethrum extract (Fig. 2). They are highly insecticidal but not persistent enough for agricultural use. Three are primarily knockdown agents and the other two are very potent for kill. Household sprays are usually a mixture to mimic the action of pyrethrum. They contain a knockdown agent, a more insecticidal component, and a synergist, normally piperonyl butoxide. With the exception of kadethrin, these compounds are chrysanthemates.
The knockdown property requires rapid penetration conferred by the polarity of either the acid or alcohol component. Instability results from susceptibility to photooxidation at allyl, isobutenyl, furan and thiolactone substituents. For example, the S-Bioallethrin, the most potent isomer of allethrin, has the same optical configuration as pyrethrinl. It is generally less active than the pyrethrins but more stable due to the less easily-oxidized alcohol moiety side chain. The other two knockdown pyrethroids, in contrast to the pyrethrins and allethrin, contain elements in addition to carbon, hydrogen and oxygen, i.e., nitrogen in tetramethrin of Sumitomo Chemical Company and sulfur in Kadethrin of Roussel-Uclaf (Paris). Kadethrin is the most potent knockdown agent, even more active than pyrethrin II, but is very labile due to the photoinstability of both the furan ring and the thiolactone moiety.
1{esmethrin has insecticidal potency equal or superior to the pyrethrins on a wide variety of pests. The cyclopentenolone nucleus of the pyrethrins is replaced by the sterically equivalent furylmethyl unit and the pentadienyl side chain by a benzyl group. CI / IO Phenothrin is derived from resmethrin by replacing the furan ring by a phenyl group and the methylene bridge by oxygen, resulting in a more stable but usually less active compound. Both insecticides lack significant knockdown properties and the IR, trans isomers (i.e., bioresmethrin and biophenothrin) are more potent with some species and the IR, cis isomers with others. Synergists are of little or no value with resmethrin and phenothrin at normal ratios of synergist to insecticide. Resmethrin was discovered in Rothamsted and offered for commercialization by the National Research Development Corporation (NRDC) based in London. Phenothrin was discovered independently in England and Japan.
Pyrethroids for Crop Protection
Four pyrethroids are currently used for crop protection: permethrin, cypermethrin, decamethrin, and fenvalerate, compounds obtained by replacing photolabile centers in earlier esters with alternative and more stable structural units (Fig. 3). These pyrethroids are derived from phenoxybenzyl alcohol first synthesized for other purposes in 1935 or from a-cyanophenoxybenzyl alcohol known since 1973. The acid moiety of permethrin was first investigated by Jihri Farka's in Prague in 1958. He prepared the allethrin analog with enhanced insecticidal activity compared to the chrysanthemate. It took 15 years for this dichlorovinyl acid to appear once again in the literature, when Elliott showed its importance as a permethrin (1973) cypermethrin (1975) decamethrin (1974) fenvalerate (1976) Environmental Health Perspectives pyrethroid acid having derived it by isosteric modifications. Permethrin, containing this photostable component, was the first pyrethroid with properties appropriate for crop protection. The a-cyano derivatives, cypermethrin and decamethrin, also first prepared at Rothamsted, are extremely active insecticides. The cyano substituent in the S-configuration increases the potency by about 3-to 6-fold. The R-configuration is essentially inactive. Decamethrin and the equivalent IR, cis, aS isomer of cypermethrin are the most potent pyrethroids at present. The research of Sumitomo Chemical Company culminating in fenvalerate involved a series of discoveries, i.e. the useful properties of phenoxybenzyl alcohol, of its a-cyano analog, and of the indicated non-cyclopropanecarboxylic acid as an excellent substitute for chrysanthemic acid analogs. Fenvalerate is generally in the range of 0.3 to 2 times the potency of permethrin, depending on the pest species.
Pyrethroid Metabolism and Environmental Degradation
Two ways have been used to enhance the stability and therefore the potency of pyrethroids such as pyrethrin I. The first involves adding a synergist or antioxidant to retard metabolic or photochemical oxidative reactions. The second and much more effective procedure replaces substituents susceptible to photochemical or metabolic degradation with altemative groupings that confer greater overall stability to the molecule. Much of the safety of the pyrethrins is attributed to their instability. Thus, the stabilizing process could potentially generate compounds persisting in mammals and acting as environmental contaminants. The pyrethroid should be protected from abiotic (mainly photochemical) degradation and insect metabolism but susceptible to metabolism in mammals and environmental sys- piperonyl butoxide tems. The author and his colleagues at Berkeley have emphasized research on metabolism and environmental degradation, as have Miyamoto, Ohkawa, and co-workers of Sumitomo.
Metabolic detoxification is a major factor limiting the insecticidal activity of the pyrethrins and other chrysanthemates. Houseflies metabolize pyrethrin I, S-bioallethrin, and biotetramethrin by oxidation of a methyl group in the isobutenyl substituent to the corresponding carboxylic acid, a pathway paralleling the first steps in the biosynthetic conversion of chrysanthemic acid or its esters to pyrethric acid or pyrethrates in pyrethrum flowers [Eq. (1)]. Houseflies also oxidize allethrin in the alcohol component, probably at the double bond and the methylene group of the allyl moiety. These reactions are effected by the housefly microsomal mixed-function oxidase system when fortified with reduced nicotinamide-adenine dinucleotide phosphate (NADPH), the critical cofactor.
Synergist action involves inhibition of pyrethroid detoxification resulting in greater persistence in insects and higher potency. The microsomal mixedfunction oxidase system metabolizes both the pyrethroid and the synergist, e.g., allethrin and piperonyl butoxide. Sites of oxidation are indicated by arrows in Eq. (4). Piperonyl butoxide, both in vivo and in vitro, inhibits housefly metabolism of allethrin and other chrysanthemates, the synergist in the process undergoing metabolism at methylene substituents adjacent to oxygen atoms. The synergist is usually a better metabolic inhibitor in insects than in mammals.
Metabolic considerations played an important role in designing pyrethroid acid and alcohol moieties for enhanced insecticidal activity. Oxidation at an isobutenyl methyl group was circumvented by the dihalovinyl replacement or by shifting to the fenvalerate acid moiety. Pyrethroids are also detoxified by hydrolytic processes. Insect esterases generally microsomal mixed f unction oxidase detoxification products oxidase inhibition (4) hydrolyze trans-chrysanthemates faster than cischrysanthemates, providing a partial explanation for the greater toxicity of the cis-isomer in some cases. Suitable esterase inhibitors synergize the insecticidal activity of several pyrethroids but such combinations are not commonly used. The a-cyano substituent also retards esterase hydrolysis and possibly oxidative detoxification as well. Thus, the structural features used in optimizing the insecticidal activity of decamethrin are those designed to resist metabolism, i.e. a dihalovinyl group, the cis-isomer about the cyclopropane ring, and the cyano substituent. Even these modifications have not completely overcome the limiting effect of decamethrin detoxification in houseflies, since it can be synergized at least tenfold by a very high level of piperonyl butoxide or related synergist.
Mammals also metabolize pyrethroids by oxidation and ester cleavage. The same detoxification reactions account for the low toxicity of pyrethrin I and allethrin to mammals and the need to use a synergist for increasing their toxicity to insects. Fortunately, piperonyl butoxide as normally used gives little if any increase in toxicity of these rethrins to mammals. However, high synergist levels to block mammalian detoxification by oxidases (piperonyl butoxide) or esterases (organophosphorus compounds) generally increase pyrethroid toxicity. Caution must be exercised in using synergists because of such potential hazards. Structural modifications to stabilize the pyrethrins to insect metabolism could produce very hazardous compounds if they stabilized them in a parallel fashion to mammalian metabolism. Fortunately this is not the case for pyrethroids studied so far, e.g. decamethrin). Although the dibromovinyl group is not oxidized in mammals, there are still five sites of oxidation at methyl and aryl groups, the 4'-position being major, and ester hydrolysis is also important [Eq. (5)].
Decamethrin metabolism in rats and mice involves hydroxylation at either methyl group or any one of three aromatic positions. Ester cleavage by esterase action or possibly oxidative processes yields the acid and alcohol fragments. The cis-hydroxymethyl derivative is detected only after ester cleavage. The cyanohydrin breaks down to hydrogen cyanide and the aldehyde which is then oxidized to the acid. The two carboxylic acids are excreted with or without conjugation with glucuronic acid or amino acids such as glycine and taurine. The hydroxymethyl and phenolic derivatives are conjugated in part as sulfate esters. The liberated cyanide is quickly detoxified by conversion to thiocyanate which is excreted or temporarily bound in the stomach and hair prior to excretion. In soil a portion of the cyano moiety of related compounds is hydrated to the amide. Metabolism studies of this type made on pyrethrins I and II and all the synthetic analogs discussed above clearly show that structural modifications can be made for enhanced insecticidal activity and photostability while maintaining rapid biodegradation in mammals. There are structure-dependent differences in the persistence of pyrethroid residues in mammals and birds; for example, although the residues are low, the more metabolically-stable cispermethrin persists longer than trans-permethrin in fat, milk and eggs.
Environmental movement and fate were of little concern with the unstable pyrethrins and early chrysanthemates but are of considerable importance with the more stable pyrethroids used for crop protection. As with DDT, the newer halogen-containing pyrethroids are;highly liposoluble, almost insoluble in water and persist on surfaces due to low vapor pressure (the pyrethrins and permethrin are viscous liquids and decamethrin a crystalline solid). Air movements are not likely to disperse these pyrethroids except during application or shortly thereafter. They are quite persistent on plants due to a combination of retention in leaf cuticular waxes (so that they are not washed off by rain), low volatility, and resistance to photochemical degradation. Studies at Berkeley show that photodecomposition of the pyrethroids shown in Figure 3 involves isomerization at the cyclopropane ring [Eq. (6)], ester cleavage, decarboxylation, diphenyl ether cleavage, oxidation to benzoic acid derivatives, and dehalogenation. These occur slowly enough that if only abiotic factors were involved these pyrethroids would be some of the most persistent organic insecticides. Only the IR isomers at the left are insecticidal. X refers to methyl, halogen or other substituents. This is one of many processes involved in pyrethroid photodecomposition, usually yielding less active products.
Environmental Health Perspectives
It is fortunate that environmental cleansing involves biotic as well as abiotic processes since the degradation rate of agricultural pyrethroids is greatly increased once they enter biological systems. Plants metabolize these pyrethroids, on partitioning out of cuticular waxes, by ester cleavage (trans isomer more rapid than cis), methyl and aryl oxidation and conjugation reactions as in mammals (except that glucoside rather than glucuronide conjugates are formed in plants). Although there is no evidence for plant metabolites that are hazardous, the residue analyses often consider several metabolites and photoproducts in addition to the parent compounds. Pyrethroids are not expected to undergo a high level of biological magnification on passing through food chains. They are nearly immobile in soils due to their low water solubility, rapid adsorption and minimal vapor diffusion. Although contamination of aquatic systems is a serious potential problem from direct application or erosion of treated soil, it is not likely to occur by diffusion or leaching. Thus, under field conditions the pyrethroids are rapidly absorbed into stream banks, pond sediments and organic matter to decrease their concentration in water. Soils high in microbial activity extensively metabolize pyrethroids within a few weeks by ester and diphenyl ether cleavage, hydration of the cyano moiety and other reactions ultimately leading to carbon dioxide. Pyrethroids do not seem to affect soil microorganisms adversely.
Pyrethroid Toxicology
Pyrethroids are generally broad-spectrum insecticides. They control a large variety of insects, although the effective dose may vary greatly between the most and least sensitive species. In stored products protection a synergist is commonly added. In food and fiber production the pyrethroid is often used in the same fields as one or more other insecticides, miticides or ovicides. To protect susceptible honeybees, pyrethroids must be applied at times and in amounts to minimize pollinator and hive damage. Predator and parasite kills may lead to resurgences of pests no longer controlled by their natural enemies. Pyrethroids are not effective in controlling soil insects possibly due to soil binding and metabolism of the compounds. Crustaceans and beneficial aquatic insects are potential non-target victims of pyrethroid uses to control mosquito larvae and other dipterous larvae of medical importance.
Resistance has previously curtailed the use of almost every type of insecticide and poses a serious threat to the future of pyrethroids. Cross resistance does not appear to be a problem between pyrethroids and organophosphates or methylcarbamates. However, previous selection of houseflies with DDT for a recessive factor conferring knockdown resistance (kdr) carries with it a cross-resistance to pyrethroids. Houseflies on Danish farms developed pyrethroid resistance when pyrethrins and pyrethroids replaced chlorinated hydrocarbon and organophosphorus insecticides. One pyrethroid-resistant field strain was subsequently selected in the laboratory with bioresmethrin to a resistance factor of 1400-fold. Despite no previous exposure, this strain was more than 60,000-fold resistant to decamethrin. Thus, the most potent of all insecticides on a normal strain has almost no effect on this resistant strain. This is the most dramatic example available of pyrethroid resistance. Some of the housefly resistance mecha-February 1980 I I ar , hy X') FIGURE 4. Housefly resistance mechanisms of three distinct types are recognized from studies involving crosses of resistant unmarked fly strains with susceptible flies carrying markers on appropriate chromosomes. Bioassays and the visible markers are used to associate resistance factors with individual chromosomes. Recessive factors on chromosome 3 confer reduced penetration to many insecticides and knockdown resistance (kdr) to pyrethroids and DDT. The detoxifying mixed-function oxidases or incompletely dominant mfo of chromosome 2 are also associated with resistance to many other types of insecticides. Pyrethroid resistance is greatly enhanced by recessive modifier characters conferred by chromosome 2 and to a lesser degree by 1 and 5. nisms are considered in Figure 4. Low levels of field resistance are also known in local areas with mosquitoes, Australian cattle ticks, some lepidopterous pests on cotton, and a few other pest species. It may be possible to forestall pyrethroid resistance by restricting their use to minimal doses and numbers of applications and by more fully utilizing integrated pest management systems. Routine application of pyrethroids at high doses, as done in the past with other insecticides, practically guarantees that resistance will reach levels where economic use of pyrethroids is no longer possible. Fish are sensitive to pyrethroids at part per billion levels, so great care must be taken in treating agricultural crops and forests to avoid contaminating lakes and streams and commercial fish-producing areas. The high sensitivity of fish is possibly related to the ability of their gills to concentrate pyrethroids which then disrupt nerve-controlled respiratory mechanisms. Birds seem to be unusually tolerant and have survived high oral doses of several synthetic pyrethroids. Although the mechanism of avian insusceptibility is not known, chickens rapidly metabolize and excrete orally-administered permethrin.
Mammals appear to be relatively tolerant to pyrethroids, so in this respect these synthetic insecticides are welcome alternatives to some of the other second generation insect control agents. Selectivity is often expressed as a ratio for the amount of insecticide per gram of body weight to kill 50% of a group of orally treated rats divided by the comparable value for various topically-treated insects. This selectivity ratio averages about 4000 for many pyrethroids but is less than 100 for various chlorinated hydrocarbon, organophosphorus and methylcarbamate insecticides. A few compounds (pyrethrin II and kadethrin) administered intravenously are toxic to rats at levels equivalent to their potency on susceptible insects. The relatively low toxicity to mammals following oral, dermal or inhalation exposure therefore results largely from factors preventing entry into the nervous system, such as metabolic detoxification. Excessive exposure to certain pyrethroids may result in skin irritation in sensitive individuals.
Lifetime feeding studies with mammals are at least as important as acute toxicity observations in evaluating the safety of pyrethroids. Some of these studies have been completed and others are still in progress on each pyrethroid proposed or in use for crop protection. Tolerance values or the maximum allowable residues in food and feed will be based on the dietary levels found to have no effect, the amount of residues normally present when the compounds are used in accordance with good agricultural practice, and a safety factor to correct for possible differences in sensitivity of humans and the laboratory mammals.
Pyrethroids are nerve poisons, but their mode of action at the molecular level remains obscure. They cause repetitive discharges in arthropod nerve due to interference with axonal sodium and potassium channels. The repetitive firing is attributed primarily to prolongation of the turning off of the increase in sodium conductance and secondarily to the suppression of the increase in potassium current. It is not clear which symptoms in insects or other animals-are due to effects on the central or the peripheral nervous system or both. Pyrethroids are more toxic to insects at low than high temperatures, as is also the case with DDT. Many types of isolated nerve preparations from insects and other arthropods are highly sensitive to pyrethroids, but none of the investigated systems so far is an adequate model of the effects on organisms. In pyrethroid poisoning of various insect species, fish and mammals, there is probably no need to invoke a fundamentally different primary mode of action. Poisoning of rats and mice is related to but not necessarily dependent on the levels of some pyrethroids in the brain.
Structure-activity studies, particularly with sy-nergist-treated insects, help to define the configuration of the physiologically-important nerve receptor. The flexible pyrethroid molecule with its high stereospecificity must adopt a conformation in which all structural features essential for potency are appropriately oriented with respect to each other and to a complementary chiral receptor. The most sensitive and relevant receptor must be isolated and defined pharmacologically as a prelude to understanding pyrethroid mode of action at the molecular level.
Summary
Pyrethroids are the most potent lipophilic insecticides. They are also the most expensive per unit weight. The cost is increased by producing the single, most potent optical isomer using advanced techniques of synthesis and resolution. Less active isomers and byproducts can often be converted to a useful isomer or intermediate in a recycling process. The pyrethroids may potentially provide an excellent cost/benefit ratio in agricultural pest insect control, in part because they persist sufficiently to require relatively few applications. This economic situation would change drastically if resistance phenomena required increases in pyrethroid doses of twoto ten-times. The potency of pyrethroids also means a smaller environmental burden of the parent compound and its photoproducts and metabolites, with their possible undesired effects. Thus with decamethrin, a single compound of high chemical and isomeric purity, application at 10 g/hectare for pest control gives an initial deposit of 1 mg/M2. Most other types of pesticides, because of lower potency, require deposits of 50 to 200 times as high. Insecticides as active as some pyrethroids are known among the chlorinated hydrocarbons, organophosphates, and methylcarbamates, but with the latter compounds this remarkably high insecticidal activity is usually accompanied by unacceptable mammalian toxicity.
Pyrethroids do not provide new or unique approaches to insect control. They are strictly alternatives to or replacements for current compounds. Synthetic analogs have been used for 30 years as pyrethrum substitutes without diminishing the demand for the natural product. However, the pyrethrum industry in various countries must become better organized and more efficient in production and distribution continually to compete with the ever-increasing number of synthetic alternatives, although these take time to develop such a proven safety record. The more stable pyrethroids are being increasingly used to replace DDT and other chlorinated hydrocarbons. Both classes include long residual contact insecticides effective on many of the same pest complexes. The current pyrethroids are not phytotoxic, so their use results in higher yields than are obtained with other equally-effective but phytotoxic insecticides. Pyrethroids are not suitable replacements for organophosphates and methylcarbamates as plant systemics because of low water solubility or as soil insecticides due to soil binding, metabolism, and low vapor pressure. The future of pyrethroids as contact insecticides and stomach poisons will depend on what further restrictions are placed on the present insecticides, the comparative seasonal cost for pest control with pyrethroids and with other compounds, and the final risk assessments based on the toxicological findings.
Advances in the past seven years establish pyrethroid insecticides as one of the major classes of pesticide chemicals. They also indicate that theoretically additional structural modifications can increase their potency more than 10-fold further and/or reduce the seasonal pest control cost by a similar factor. Alternative pyrethroids are available for introduction if there are toxicological problems with the current compounds. Structure optimization is now focusing on new properties in addition to potency, low mammalian toxicity, competitive price and suitable persistence. These goals are: diminished toxicity to fish or to honeybees, predators and parasites; broader spectrum of activity including mites and aphids to reduce the need for pesticide mixtures; effective on strains resistant or cross resistant to current pyrethroids; potent as ovicides for insect and mite eggs; effective as nematocides and anthelmintics. How many new pyrethroids can be justified and might it be practical to develop? At current or anticipated costs probably no more than four to eight additional pyrethroids could be developed for agricultural use over the next ten years on a worldwide basis. It is therefore important to use the current pyrethroids at doses and in a manner to minimize the selection of resistant strains and thereby conserve this valuable resource for control of pest insects in the years and hopefully decades ahead.
The author is indebted to the Rockefeller Foundation for appointment as a Scholar-in-Residence at the Bellagio Study and Conference Center at Lake Como, Italy where this article was prepared. It is similar to a lecture presented on receiving the 1978 Kenneth A. Spencer Award of the American Chemical Society. | v3-fos |
2019-03-19T13:05:05.635Z | {
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} | s2 | Nutritive value of full-fat rapeseeds for growing pigs.
The digestibility and nutritive value of full-fat, ground rapeseeds of two low-erucic acid, low-glucosinolate varieties, Candle (Brassica campestris) and Regent (B. napus), were determined for three pigs, weighing about 40 kg each. The ME values were 18.72 and 20.26 MJ ME/kg DM, the NE values 1,58 and 1.70 FU/kg DM (FU = 0.7 kg starch) and the DCP values 155 and 201 g DCP/kg DM, respectively, for the two varieties. Nitrogen balances in the same trials were 21.0 and 22.0 g N/d. Twenty-eight freshly weaned piglets averaging 11 kg in weight were fed during four weeks 1) a control diet containing barley and skim milk powder, or 2) the control diet with 10 % finely ground Candle seeds incorporated. The calculated energy and DCP values of the diets were alike. The growth rates of both groups were equal, as were the palatabilities of the diets. The feed conversion figures were 2.0 and 2.1 FU/kg liveweight gain, respectively. Accordingly, the energy value of Candle seeds was a little poorer for piglets than for the older growing pigs.
Introduction
Rapeseeds are usually cultivated for the food oil industry and only the defatted meal is used for animals. The whole seeds can also be fed to animals, of course. Low-erucic acid, low-glucosinolate rapeseeds have been successfully included at 10 % level in the diets of laying hens, from 10 to 20 % in the diets of broiler chickens ) and up to 21.5 % in the diets of turkeys (Moody et al. 1978). High-glucosinolate seeds, however, have decreased the production of poultry at the 10-20 % inclusion level (Woodly et al. 1972, Olomy et al. 1975a, 1975. Low-glucosinolate seeds have proved to be a useful protein and energy source also in lamb milk replacers (Seoane et al. 1976), and have been found to improve the milk production of dairy cows (Anon. 1978).
In the present study the full-fat ground rapeseeds of to low-erucic acid, low-glucosinolate varieties. Candle (Brassica campestris) and Regent (B. napus), were investigated as feeds for pigs. In the first trial the digestibility and nutritive value of the seeds was determined for growing pigs. In the second trial freshly weaned piglets were fed diet containing 10 % Candle rapeseeds.
Materials and methods
The diets contained 20 % of milled rapeseeds, barley as a basic component, and skim milk powder sufficient to bring the crude protein content to 120 g DCP/FU. Minerals and vitamins were incorporated in the diets at standard levels. The composition of the rapeseeds is set out in Table 1. The digestibility trials were performed with three castrated pigs weighing about 40 kg. The pigs were kept in digestibility cages in a room maintained at 13-18°C. A vermicide was given to the animals at the beginning of the trial.
The daily ration of 1.6 FU was given as two meals. Barley, rapeseeds skim milk powder, and mineral and vitamin mixtures were weighed separetely, and given mixed with two volumes of water. The pigs ate the ration immediately and were then offered water.
The preliminary period was 15 days, and the collection period 5 days. The faeces and urine were collected in the morning and samples of fixed amount were taken for analysis. Sulphuric acid was added to the urine collection pail to keep the acidity under pH 3. At the end of period spatterings of feed were collected from a plastic sheet placed in front of the trough, and subtracted from the total amount of feed given.
The dry matter determinations were made at 103°C, and the samples for analysis dried in vacuum at 50°C. Feeds and faeces were milled with a sieve of 0.5 mm.
The feed analyses were performed according to standard procedures. The determinations of glucosinolates and tannins were made at the State Institute of Agricultural Chemistry.
The digestibility of the barley meal was determined in a separate trial and the digestibility coefficients for skim milk powder were taken from Feed Tables (Eriksson et al. 1972). The digestibility of rapeseeds was then calculated by subtraction.
The metabolizable energy and net energy values were calculated using factors and models of NJF's Feed Tables (Anon. 1969).
Results and discussion
The digestibility coefficients and calculated energy and DCP values are presented in Tables 2 and 3. Information about nutritive value of full-fat rapeseeds for pigs is scant. The only figure found was the ME value of 21.1 MJ/kg DM in the Feed Tables of the DDR (Nehring et al. 1970), which is slightly higher than the value here for the Regent seeds. The difference may be due to the fact that the present seeds were ground fairly coarsely, some whole seeds being visible in the meal. (A separate trial revealed that the pig is unable to digest unmilled rapeseeds.) However, high variation in rape varieties or seed batches seems to occur. For instance, the Swedish Feed Tables give a ME value of 5.11 Mcal/kg DM for ruminants (Eriksson et al. 1972), and the Norwegian Tables 4.41 Mcal/kg DM (Breirem & Home 1970).
In the present study the Regent seeds (R. napus) were found to be a little better in nutritive value than the Candle seeds (B. campestris) (Tables 2 and 3). Sibbald and Price (1977) have reported an even larger average difference between B. napus and B. campestris seeds (5.08/4.52 Meal ME/kg DM for poultry).
The Candle variety, however, has a thinner seed coat than the older varieties and thus a better energy value. Mutztar et al. (1978) have found its AME value to be similar to that of Tower for poultry (4.4/4.5 Meal, respectively).
The nitrogen balances for Candle and Regent seeds were alike, 21.0 and 22.0 g N/d, respectively. The protein of the seeds constituted only about 25 % of the protein content of the rations, however.
The FU value of full-fat rapeseeds for growing pigs proved to be about one and a half times, and the DCP value/kg DM almost two times the value of barley. An additional advantage of the rape seeds compared with barley is the better amino acid composition and higher linolic acid content (see Nehring et. al 1970.) From the cost point of view, however, barley is a better feedstuff than the rapeseeds.
Materials and methods
To obtain further information on the value of rapeseeds, a four-week growth trial was conducted with 28 freshly weaned pigs. The piglets were chosen because with older pigs the high oil content of the diet is reflected in lowered quality of the carcass.
The age of the pigs at the beginning of the trial was from 4.5 to 6 weeks and the average weight 11.1 kg. The pigs were given a vermicide and divided according to weight, age and sex into two equal groups, each of which was further divided and placed into two pens according to weight. The weight gain of pigs was followed individually and their feed consumption per pen.
The ration of the control group consisted of barley, skim milk powder, minerals and vitamins. The experimental diet differed from the control ration only in a 10 % rapeseed content; proportions of barley and skim milk powder were reduced so that the crude protein contents of the diets equal (Table 4).
The rapeseeds were ground together with barley using a 2 mm sieve. The milling was successful and no whole seeds were detected in the meal.
The feeds were given as two meals daily, mixed with warm water. Between meals the dry meal was put in the troughs. b Calculated using rapeseeds feed values as presented in Table 3.
Results and discussion
The pigs were weighed twice at the beginning and end of the trial and twice during the trial. The growth rates of the two groups were very alike, the average daily gain being 485 g for experimental group and 487 g for control group (Fig. 1). The palatability of the experimental feed was better than the control feed in the dry form, but when mixed with water no difference was found. No sickness was recorded during the trial.
The consumption of feed was also almost equal. The average daily intakes were 921 g and 902 g for the experimental and control groups, respectively. The utilization of feed was a little better in the control group: an average of 2.0 FU/kg liveweight gain against 2.1 FU/kg in the experimental group. The energy value of the experimental diet thus proved to be a little lower than the value calculated for the 40-kg pigs ( Table 4). The 10-25 kg piglets thus utilized the rapeseeds somewhat less efficiently, despite the fact that the rapeseeds fed to piglets were ground much more finely than those used in the digestibility trial. | v3-fos |
2016-05-15T15:08:51.291Z | {
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} | s2 | Udder conformation and its heritability in the Assaf (Awassi × East Friesian) cross of dairy sheep in Israel
A survey of udder conformation in 544 ewes, 2-5 years old, and 269 hoggets after first lambing, all belonging to the Assaf Cross (Awassi x East Friesian) was performed. Twentyseven per cent of the ewes possessed udders unsuited for milk fractionation. Analyses made in a half-sister group of udder shape, teat location, teat length and teat thickness showed ha of 1.1 j: 0. 45 , 0.¢2 ! 0,23 0.38 ! 0.26, o.z3 ! 0.25 , respectively. No significant correlation was found between udder shape and milk production.
Introduction
In machine milking ewes, good fractionation of milk yield is a desirable trait. Machine or hand stripping delays the process of milking and thus sometimes not all the milk is taken from the ewe. SAG I and M ORA G ( 1974 ) showed the pattern of milk fractionation could be related to udder conformation. Ewes with udders with low teats showed better performance than ewes with udders having high teats. Although the use of a mechanical udder support improved milk fractionation, there is still an advantage to ewes having udders with low teats (S A G I , 197 8).
The great variation that was found in udder conformation in the Arr!assi breed in Israel by S HARAV et al. ( 19 6 2 ) strengthens the idea that there is room for a breeding program that will improve the udder conformation in the dairy ewe.
During recent years, the Assaf cross (Awassi x East Friesian) was introduced into Israel owing to its high prolificacy compared to the Awassi breed (GooT, rg66). There are now over 5 ooo heads of this cross in the country, which constitutes about 10 p. 100 of the total dairy sheep. This work presents a study of the frequency of udder types in the cross and estimates the genetic part of the phenotypic variability. The possible connection between udder shape and milk production is investigated.
Material and Methods
A survey of udder conformation was performed in the Assaf dairy flock belonging to Moshav Moledet, near Afula, Israel. This flock is maintained under intensive management and kept indoors during the whole year. Nearly half of the ewes lamb twice a year. The flock consisted of nearly i o0o dairy ewes; in this work 2 6 9 hoggets after first lambing and 5 44 2 -to-5 -year old ewes were included.
Udder examination took place between 20 to 6 0 days after lambing during the first milk recording in the lactation. In order to classify the udder conformation its shape was characterised by 4 sub-traits, with 4 grades in each category, according to table i. Estimation of milk production during the first 100 days of the lactation was based on three successive monthly milk recordings. During the days of the recording the lambs continued the usual regime of suckling the residual milk, remaining after the milking. Heritability of udder shape calculations were performed on 451 half-sisters between 2 to 5 years old that were in the flock at the time of udder typing. The ewes were daughters of 19 rams and the size of the half-daughter groups ranged between 9 and 47 ewes.
The calculations of heritability and of mother-daughter correlation were performed after excluding the undefined cases (grade 4 in table I ) and after reclassification of the sub-traits as follows : Heritability calculation of the udder conformation was performed according to HILL and SMITH (1977).
Results
The frequency of the sub-traits of the udder conformation as defined in table i is presented in table 2 . Table 2 shows that 27 per 100 ewes have udders which are classified as undesireable, according to S AGI ( 197 8), in relation to milk fractionation. Comparing the data from hoggets and ewes it can be seen that, while there is no difference in teat length and thickness, there is a significant (P < 0 . 05 ) difference in udder shape and location of the teats.
Values of heritability of the sub-traits are presented in table 3 . Heritability values that were calculated were found to be high and medium despite the fact that typing of the udders was done by sight only.
Among the ewes that were examined there were 68 mother-daughter pairs. The frequency of udder sub-traits in the two groups was examined by X 2 -test and in the case of teat location there was a significant connection (P < 0 . 05 ).
The lack of significant correlation in the other udder sub-traits among the mother-daughter pairs seems to be due to the relatively low frequency of some of the ratings in this sample.
The mean:!: S.D. milk production of the hoggets and ewes during the first 100 days of the lactation was ig2 ! 5 1 liters and 223 = 6 2 liters, respectively. No significant differences were found either between ewes from different ages ( 2 to 5 years) or between ewes raising singles or twins. Testing the differences between the mean milk production of the ig halfsister group showed that the differences are not significant, F,,/ 3 ., = 0.75. No significant correlation was found between the udder shape of the ewe and her milk production.
Discussion
Poor milk fractionation of the dairy ewe can be explained in part by udder conformation. Adaptation of the udder to the demands of machine milking can be achieved both by genetic and mechanical means (Fr,nMnrrT, rg 74 ). By putting together the factors of good udder type and mechanical support, the machine milk fraction can be brought up to 71 p. ioo compared to the case of bad udder conformation without support which releases only 44 p. 100 during the machine milking period, according to S A GI ( 197 8).
In the present study, we found that in the Assa/ cross in Israel 27 pet 100 of the ewes have unsuitable udders as regards milk fractionation. The high values of heritability obtained for udder shape and teat location indicate that improvement could be achieved by selection. Moreover, the typing of the udder can be done by simple methods suitable to farm conditions. It is necessary that the typing takes place as early as possible in the life of the ewe; however, it is known that the udder conformation changes either between or within lactations (I ATSCH , 1977 ). Thus, typing procedures ought to first take place at the beginning of the second lactation and must be performed during successive lactations.
In this study no connection was found between the udder shape and milk production of the ewe. It seems that selection for udder shape is relevant mainly to the milk fractionation. Résumé La conformation de la mamelle et son héritabilité chez les brebis laitières Assaf (Aw-assi y Frisonne de l'Est) en Israël Un examen de la conformation de la mamelle chez 554 brebis de type Assaf (Awassi X Frisonne de l'Est), âgées de 2 -5 ans, et 2 6 9 antenaises après leur premier agnelage, a montré que 27 p. 100 des animaux possédaient un type de mamelle défectueux entraînant de mauvaises | v3-fos |
2017-07-29T04:25:16.736Z | {
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} | s2 | Identification of the X chromosome of the domestic pig (Sus scrofa domestica)
As the banding patterns of the X chromosome and chromosome No. 8 of the domestic pig are very similar, this identification problem has been studied. Depending on the metaphase stage, the banding pattern of the two chromosomes differ very much. Revised landmarks for the X chromosome and for chromosome No. 8 are presented, but it is not possible to use the landmarks on long chromosomes from the early metaphase stage.
Identification of the X chromosome of the domestic pig has been difficult from the early investigations of the karyotype. Different authors have described the X chromosome as meta-, submeta-, or telocentric, HuI,oT (ig6g). After introduction of the banding methods, GusTAvssoN et al. ( 1972 ) and H ANS E N ( I g 72 ) described different banding patterns of the X chromosome by the Q-band technique; B!RG!R ( 1972 ), EC HAR D (Ig73), HANS!N-MELAND!R et al. (1974), PACE et al. (Ig75), ,!YSA (Ig75), MICH!I, MANN et al. (1977), and !'!IYAK! et al. ( 197 8) have described different banding patterns by the G-band technique. Some authors found one distinct band on the middle of the short arm (p), other found two and three bands on p. From one to four bands have been described on the long arm (q) with one or two of them, but not in all cases the same, having a higher intensity. A diagrammatic representation of the Q-, G-, and R-band patterns of the pig chromosomes was published by H ANS E N ( I g 77 ), but still some identification problems have persisted.
The only chromosome in the pig karyotype, which is difficult to distinguish from the X chromosome by banding methods, is chromosome No 8. In attempting to solve this problem, the G-band pattern of the X chromosome and chromosome No 8 were studied in different metaphase stages of leucocytes.
The G-band technique was chosen because this technique gives more distinct bands than the Q-band technique, HA N SE N (1975).
Materials and methods
Blood cultures from pigs of different strains of Danish I,andrace were prepared according to the method described previously, H ANS E N ( 1972H ANS E N ( , 1977. It must be stated that the culture time was 4 8 hours, because it seems that the number of prometaphase stages at q.8 hours is a little bit higher than for 72 hours. The G-band staining was carried out according to the method described by W ANG and FE D O R OFF (1972), slightly modified.
The chromosomes were subdivided in groups according to the system by L E V AN et al. ( 19 6 4 ), and arranged in karyotype according to the Reading Conference ( 197 6), which does not follow the Levan system precisely. Landmarks are indicated by L, short arms by fi and long arms by q. The numbering of bands is according to the Paris Conference ( 1971 ), and Su!ptement (ig 75 ). The terms distal and proximal refer to the position of bands or parts of arms in respect to the centromere.
Results
Depending on the length or the degree of contraction of the chromosomes, the banding pattern becomes more or less distinct. For that reason the early metaphase stages are very useful for banding purpose. Figure z shows pair No. i,No. 8 and the sex chromosomes from four male pig leucocytes in different metaphase stages. Chromosome pair No. i demonstrates the metaphase stage by means of the length. Figure 2 shows the diagrammatic representation of the bands and the landmarks on chromosome No. 8 and the X chromosome. Figure 3 shows a karyotype based on cell No. 175 -7 , representing an early metaphase stage with long chromosomes.
The description of the banding patterns of the X chromosome and chromosome No. 8 is given for a) contracted, b) medium contracted, and c) long chromo- somes. Pair No. 19 . The X chromosome. I, : on contracted and on medium contracted chromosomes two bands on each side of the middle of q. On long chromosomes each of these bands are subdivided into two bands. (p) : on contracted chromosomes two bands are visible; a proximal pale band, and on the middle or just distal to it a band of medium intensity. On medium contracted chromosomes, three bands : a pale proximal and a pale distal band, and on the middle, a medium band. On long chromosomes the proximal and the distal band are subdivided into two very pale bands, (q) : on contracted and medium contracted chromosomes four bands are visible. A distal and a proximal pale band, and two medium to intense bands on each side of the middle, Xq 2 i and Xq 3 i. Band Xq 21 is the most intensely stained band. On long chromosomes each of these intense bands are subdivided into two bands. The distal pale, Xq 33 , is very often visible as a small distinct dot on each of the chromatids.
Pair No. 8. I, : on contracted and medium contracted chromosomes, a proximal band on q. On long chromosomes this band is subdivided into two bands. (p) : on contracted chromosomes two bands; a proximal pale band, and a « distal medium to intensely stained band. On medium contracted and on long chromosomes the cc distal )) band is in fact a median band, which is subdivided into two bands, 8fiI4 and 8pi6. Band 8yq. is the most heavily stained one. The very long and pale telomere region on 8! usually disappear on contracted chromosomes, Fig. I cell 15 8-1 . On medium contracted and long chromosomes the telomere region is sometimes difficult to identify, too, because the region entangles or doubles up with the distal band. Band 8y q is placed on the middle of P, and 8 P 1 6 just distal to the middle, when the telomere region is identifiable, As shown in Fig. z, one of the chromosomes of pair No. 8 are sometimes shortened and more heavily stained compared to the homologue. This heavy staining of one of the chromosomes usually causes indistinct banding patterns, and for that reason the identification of the chromosome is sometimes difficult.
Because the frequency of cells with long chromosomes is not very high in cultures, the diagrammatic representation of the banding patterns in Fig. 2 are based on medium contracted chromosomes as a previous paper, H ANS E N (i 977 ). Table I indicates the bands which on medium contracted chromosomes serve as landmarks on the X chromosome and on chromosome No. 8.
Discussion and conclusion
In conventional Giemsa stained metaphase plates it was impossible to identify with certainty the X chromosome of the pig, because it belongs to the metacentric group, H ULOT (ig6g). Because of the great similarity of the banding pattern of the X chromosome and pair No. 8, these can be difficult to destinguish by the banding methods, too. The reasons for that are several : a) similar morphology; b) similar banding pattern of !; c) next to the centromere a heavily stained band on q; cI) overfluorescence /overstaining.
In the first description of the Q-band pattern of the pig karyotype by , the X chromosome was described with a bright band on the middle of the short arm, and two bright bands on the long arm. At the same time H ANSEN ( I g 72 ) described the X chromosome as follows : (p) distal half distinct, proximal half pale. (q) three bands. In a later paper H ANSEN ( I g 77 ) found on q two main bands on the middle, and a very narrow pale band next to the centromere. BURGER ( I g 72 ) and S YSA ( I g 73 ) described a slightly brighter fluorescence on the middle of p, and H ANSEN -M ELANDER et al. ( 1974 ) found one band on the middle of !, and two bands on q. In a later paper S YSA ( 1975 ) described one distinct narrow band on the middle of the short arm. In certain plates he found this band accompanied by narrow bands on both sites. On q he found four bands, the second one most intensely stained. The present results confirm the median band on the short arm as described by G ( 19 8 0 ). However, very distinct, but pale bands are present proximally as well as distally to the median band on !, and both of the pale bands are subdivided into two bands on long chromosomes. Especially, the distal very small band, Xp I 6, is lost on contracted chromosomes, and for that reason only two bands are visible, see Fig 8 0 ). On long chrom.osomes band Xp I 6 can be twisted, and give a heavy staining at the end of P. Possibility for these reasons E CHARD ( I g 73 ) and PACE et al. ( I g 75 ) described two bands on !. The long arm shows two very distinct median bands as described by GusTAVSSON et al. ( 1972 ), EC HARD (1973), HAO!I,-TORN et al. ( 1973 ), HANS!N-1VIELANDZ;R et al. ( I g74) and HANSEN (1977). But the present results show a distinct narrow band proximally as well as distally to the two median bands, i.e. on medium contracted chromosomes a total of four bands on q. This is in accordance with S YSA ( 1975 ), with Fig. 3 in the paper by H ANS E N -ME LAND E R et al. ( 1974 ), and in part with HA N SE N ( I g72) and LIN et al. ( 19 8 0 ), because the last two authors only describe three bands. As demonstrated in Fig. I cell 175 -7 , each of the two broad bands on q, Xq 2I and Xq 31 , are subdivided into two bands on long chromosomes. On chromosom.e No. 8 the very long telomere region of p is often twisted or doubled up with band 8p 1 6, see e.g. Fig. I cell 1 6 2 -7 . Possibly for that reason band 8fiI6 is usually described on the distal part of !, GusTAVSSO N et al. ( I g72). When the telomere is visible, band 8!14, which has the highest intensity of staining, is placed exactly on the middle, and 8P 1 6 just distally to the middle of !, see Fig. I cell 175 -7 . The banding pattern of the long arm is very characteristic, because of the decreasing intensity of the bands from 8q 2 i to 8q 27 , which is in contrast to Xq. The banding pattern of 8q has also been described in different ways in the literature.
It seems that bands which serve as landmarks only can be used as landmarks on contracted and medium contracted chromosomes. On long chromosomes these landmarks are very often subdivided, and the intensity of the subdivided bands are usually equal. Furthermore, bands which are negative on contracted or medium contracted chromosomes often show one or two very pale, but distinct bands, on long chromosomes.
The karyotype in Fig. 3 is in accordance with the agreement of the Reading Conference ( I g 7 6), because it was decided to put chromosome No. 5 inthetemporarily used pig karyotype into the right position, i.e. into the group of metacentric chromosomes, if further measurements confirm the results by H ANS E N ( 197 6, I g77). Recently I,IN et al. ( 19 8 0 ) have measured the relative length of the pig chromosomes, and the results agree very well with the previously and the revised results by H ANS E N ( 19 8 0 ). From the present results it is obvious that the criteria for identification of chromosome No. 8 and the X chromosome are very different in cells from the early to the late metaphase. If these are observed it is easy in well spread metaphase plates to identify with certainty the X chromosome of the domestic pig. The landmark system, however, is only usable on contracted and medium contracted chromosomes. | v3-fos |
2018-12-22T00:46:56.417Z | {
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} | s2 | Use of Agricultural News by Metropolitan Newspapers
News has been defined in many different ways. It is a relative concept, changing with time, place, and social conditions. Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License. This research brief is available in Journal of Applied Communications: http://newprairiepress.org/jac/vol63/iss4/8 County staff members also filled out questionnaires relative to staff time spent on the teaching centers. A total of 786 questionnaires was collected from center users. However. only 253 of these were used in the final analysis because the others indicated group usage. The study was only interested in testing the merit of self-teaching centers used by one or two people at a time. Responses were highly favorable. with 81 percent of the users rating the self-teaching centers as a "good" or " above average " way to present information . About the same number rated the information they received as "above average " in usefulness or "very useful." When given a choice of projection systems, 90 percent of the users chose the video cassette system. A third of the viewers said the main reason they used the center was to learn a process. Participants spent an average of 10 to 15 minutes using the centers. The study indicated that three variables were significant in determining the degree to which clients will use self-teaching centers in county Extension offices. Twenty-six percent of the variance could be attributed to county populations and traffic flow in the Extension office, while 28 percent was attributed to publicity alone. The two counties with the most regular publicity and promotion effort had the largest number of center users. Radio , newspaper, and newsletters were the most common vehicles for promoting the programs. Mark Ec/ov and John Pates South Dakota State University Use of Agriculture News by Metropolitan Newspapers News has been defined in many different ways. It is a relative concept, changing with time, place, and social conditions. The importance of farm news was obvious when a majority of the people of the United States made their living from agriculture. Today, with only a small percentage of people living on the farm . agricul32 1 Hays: Use of Agricultural News by Metropolitan Newspapers Published by New Prairie Press, 2017 lural news probably is seen 10 be much less important by most urban editors. Melvin Mencher, in his book, News Reporting and Writ ing , defined news as "information that people need to make rational decisions about their fives, " Given the fact that the production of food and fiber af· fects the lives of everyone , would that not make agri· cul tural news important to urban readers as well as those directly associated with agriculture? How do large urban newspapers treat agricultural news? In a pilot study , students in an agricultural communications independent study course at the University of Illinois conducted content analyses of randomly selected issues of three big city dailies in an effort to find some answers to questions like these. Newspapers selected were the Chicago Tribune , the Los Angeles Times , and the Washington Post. The study covered February , March , and April , 1980. Five of the 11 selected issues of the Tim es were found to contain nothing that could be identified as agriculture or farm news. Another five issues had one ag riculture story each , and one issue had two. None merited front page; placement ranged from page three to page 20. In the Post , eight of the dozen issues studied had no agriculture news. The four others had one story each-one of these reporting the death of a U.S. Department of Agriculture official. One story was on page one, The Tribune proved most fruitful. Two of 12 selected issues had one agriculture story eaCh , three others had two stories each , and one issue had three stories. One story received front page placement. What types of agriculture or farm news did the metropoli tan dailies use? The type found most often had economic implications . Examples from the Times included " Heavy Rains affect Strawberry Farmers" (Feb. 21) and "Japanese to buy Grain from U.S ." (Mar. 21). Other Post stories concerned environmental issues and " impact" stories such as an Iowa grain elevator scandal. In the Post , all agriculture stories except the death not ice related to economic issues. Most important (as indicated by placement) was a front page article on the economic cr isis faced by Maryland tobacco farmers. 33 2 Journal of Applied Communications, Vol. 63 [1980], Iss. 4, Art. 8 http://newprairiepress.org/jac/vol63/iss4/8 DOI: 10.4148/1051-0834.1834 Another was a story on the Commodities Credit Corporation appearing in the business section , while one story reported the effect of higher food prices on consumers. Three of the 11 Tribune stories were found in the " Agriculture / Commodities" columns of the business section. Each of these dealt directly with the national farm economy and covered such things as the Soviet grain embargo, land prices, and inflation. Several other stories reported on food prices and clearly were aimed at the consumer rather than a farm audience. An example of the latter was " Poultry, Pork: Consolation for Beef, Produce Prices " appearing in the food section (Mar. 6). ft is expected that this modest study will be repeated and that, with the accumu lation of sufficient preliminary data, hypotheses will be developed for testing through more extensive research activity. 34 Robert Hays University of Illinois 3 Hays: Use of Agricultural News by Metropolitan Newspapers Published by New Prairie Press, 2017
County staff members also filled out questionnaires relative to staff time spent on the teaching centers.
A total of 786 questionnaires was collected from center users. However. only 253 of these were used in the final analysis because the others indicated group usage. The study was only interested in testing the merit of self-teaching centers used by one or two people at a time.
Responses were highly favorable. with 81 percent of the users rating the self-teaching centers as a "good" or " above average " way to present information . About the same number rated the information they received as "above average " in usefulness or "very useful." When given a choice of projection systems, 90 percent of the users chose the video cassette system. A third of the viewers said the main reason they used the center was to learn a process. Participants spent an average of 10 to 15 minutes using the centers.
The study indicated that three variables were significant in determining the degree to which clients will use self-teaching centers in county Extension offices. Twenty-six percent of the variance could be attributed to county populations and traffic flow in the Extension office, while 28 percent was attributed to publicity alone. The two counties with the most regular publicity and promotion effort had the largest number of center users. Radio , newspaper, and newsletters were the most common vehicles for promoting the programs.
Use of Agriculture News by Metropolitan Newspapers
News has been defined in many different ways. It is a relative concept, changing with time , place, and social conditions. The importance of farm news was obvious when a majority of the people of the United States made their living from agriculture. Today, with only a small percentage of people living on the farm . agricul-32 lural news probably is seen 10 be much less important by most urban editors.
Melvin Mencher, in his book, News Reporting and Writ ing , defined news as "information that people need to make rational decisions about their fives, " Given the fact that the production of food and fiber af· fects the lives of everyone , would that not make agri· cul tural news important to urban readers as well as those directly associated with agriculture? How do large urban newspapers treat agricultural news?
In a pilot study , students in an agricultural communications independent study course at the University of Illinois conducted content analyses of randomly selected issues of three big city dailies in an effort to find some answers to questions like these.
Newspapers selected were the Chi ca go Tri bune , the Los Angeles Times , and the Was hington Post. The study covered February , March , and April , 1980. Five of the 11 selected issues of the Tim es were found to contain nothing that could be identified as agriculture or farm news. Another five issues had one ag riculture story each , and one issue had two. None merited front page; placement ranged from page three to page 20.
In the Post , eight of the dozen issues studied had no agriculture news. The four others had one story each-one of these reporting the death of a U.S. Department of Agriculture official. One story was on page one, The Tribune proved most fruitful. Two of 12 selected issues had one agriculture story eaCh , three others had two sto ries each , and one issue had three stories. One story received front page placement.
What types of agriculture or farm news did the metropo li tan dailies use? The type found most often had economic implica tions . Examples from the Times included " Heavy Rains affect Strawberry Farmers" (Feb. 21) and "Japanese to buy Grain from U.S ." (Mar. 21). Other Post stories concerned environmental issues and " impact" stories such as an Iowa grain elevator scandal.
In the Post , all agriculture stories except the death notice related to economic issues. Most important (as indicated by placement) was a front page article on the economic cr isis faced by Maryland tobacco farmers. | v3-fos |
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} | s2 | Irrigating Corn in Extreme West-Central Kansas
Declines in groundwater levels in this area have sharpened interest in irrigation efficiency. During the last three years we evaluated irrigation amounts and timing influence on corn yields Res_ults that timing Cit arngat1on wtll maantaan corn ytelds and use less water. Our 3-year test was with furrow irrigation on Ulysses silt lo·am. All plots received a preplant irrigation. Each in-season irrigation was approximately 7.5 inches. Application efficiency may range from 50 to 75% for furrow irrigation. Phosphorus was applied uniformly each year. We compared irrigation schedules. usi ng preplant only ·and at three soil-moisture tensions with two nitrogen fertilizer levels, two plant population lev:els, and three commercial corn hybrids. Whe n to irrigate was determined by monitoring soi_l mo.isture tension. Tension is the re lative .difficulty of extracting moisture from the soil. Watering when the soil water tension was 0.8
Declines in groundwater levels in this area have sharpened interest in irrigation efficiency. During the last three years we evaluated irrigation amounts and timing influence on corn yields Res_ults that timing Cit arngat1on wtll maantaan co rn ytelds and use less water.
Our 3-year test was with furrow irrigation on Ulysses silt lo· am. All plots received a preplant irrigation. Each in-season irrigation was approximatel y 7.5 inches. Application efficiency may range from 50 to 75% for furrow irrigation. Phosphorus was applied uniformly each year. We compared irrigation schedules. usi ng preplant only · and at three soil-moisture tensions with two nitrogen fertilizer levels, two plant population lev:els, and three commercial corn hybrids.
Whe n to irrigate was determined by monitoring soi_ l mo. isture t ension. Tension is the re lative .difficulty of extracting moisture from the soil. Watering when the soil water tensio n was 0.8 bar a! 2 feet deep ":'atering an 2.7 t1mes a se·ason: tw1ce m 1973 and f\..... _.'e times in 1972 and 197 4 (Table 1). Watering when soil water tension was 0.8 bar produced the highest yield, 134 bushe[s an acre (Figure l ). When pre-plant irrigation filled the soil profile to six feet, then either two or three in-season irrigations (depending on the year) produced maximum yields. Irrigating up to eight times a yea r produced no higher yields during three years, 1972-197 4. lrrig'atin'g preplant only, produced 85% as much grain as the top yield with 43% as much applied water.
The 0.8 bar treatment required that the first in-season irrigation be applied July 7; later irrigations w ere 22 days apart (3-year average). Irrigating when soil moisture tension was lower required earlier ·and more freque nt irrigations. The 0.6 bar tension required irrigation by June 30 with later irrigations ave raging 16 days apart; the 0.4 bar tension, by June 19 a nd 10 days apart. The intervals b etween irrigations varied with p lant-use and rainfall. An entire fi e ld cannot be watered so precise ly as our plots were; however, our results should be used as a manageme nt guide to timing or scheduling irrigation.
Regardless of other treatments , 160 lbs/ A nitrogen was required a nd resulted in 139 bushels an acre versus 113-bushel average with 80 lbs/ A of nitrogen ( Figure 1 ). So, it wou ld be a mistake to sharply reduce nitrogen applied, anticipating a water shortage. Nitrogen rates might be reduced slightly but our 50% nitrogen reduction cut yields too much.
The effect of plant population d epended on other treatments (Figure 2). The higher population was fa vored where nitrogen w as adequate . However, when nitrogen was short (80 lbs/ A), the high population reduced y ield. That was more apparent when both water and nitrogen were in short supply. Most surprising was that plant population did not affect yields for the pre-plant only treatment. We expected the lower population to be favored under pre-plant irrig·ation only.
The three commercia l hybrids averaged about the same over the three years. They responded differently but their responses va ried from year to year giving no clear-cut conclusions . It is im- portant to recognize that hybrids react individually to various environmental conditions.
Results of our tests are summarized in more detail in Table 1. Highest yields were from 2 or 3 in-season irrigations with 24,000 plants ar acre and 160 pounds per acre of applied nitrogen. Limited in-season irrigation is most practical when a moderate to large amount of available water is stored before planting.
Soil moisture tension can be monitored by tensiometers or electrica I resistance blocks. Tensiometers, limited to tensions below 0.85 bar, cost about $20 each. Tensiometers used in this test were obtained from Soil Moisture Equipment Corporation, Santa Barbara, California.
Information in this report is for farmers, producers, colleagues, industry cooperators, and other interested persons. It is intended to help in irrigation management. It is not a recommendation but represents three years' research at one location.
Publications and public meetings by the Kansas Agricultural Experiment Stati·on are available and open to the public regardless of race, color, national origin, This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. | v3-fos |
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} | s2 | Analysis of veterinary-recorded diseases in first lactation cows
Blood groups and other genetic markers offer an efficient aid in the studies on breed struc- tures, migration of genes and on phylogenic relations between populations. Highlights of results obtained during twenty years of research are listed and discussed. numerous breeds of many infectious the to tropical diseases and this of genetic Records on veterinary treatments of r i,!97 first-lactation cows sired by 50 Swedish Red and White (SRB) and 34 Swedish Fviesian (SLB) AI bulls were analysed. 22.9 and 28.7 p. 100 of the SRB and SI,B cows respectively were treated some time during their first lactation. Udder problems were the most frequent disease for both breeds, though more frequent in the SLB. Five growth models fitted to the weight-age data of F. bull calves from to different Fyiesiall. strains, were compared for their suitability to discribe the shape of the growth curve. The most accurate model was Richards function followed by the von Bertalanffy, Gompertz, logistic and Brody functions. All models underestimated the 17-18 month weights. The only significant differences between strains in growth parameters were found with the von Bertalanffy model. The importance of the individual and maternal components of calving performance in a population of German Simmental cattle were to be studied and genetic parameters were to be estimated using the model of WILL HAM. Calving data of calves of testing bulls for analysis of the individual component were collected in the first phase by special recording. Frequencies for classes of calving performance were: 18 p. 100 without assistance, 35 p. 100 with little assis- tance, 44 p. 100 with considerable assistance and 3 p. 100 surgical assistance. Heritability for the individual component was h2 - .14 with a considerable standard error because of the rela- tively small data set (s2h1 -= .14). In the second phase, daughters of the same testing bulls were recorded at their first calving. From these r,2oo he:fer calvings parameters for the maternal component will be estimated using also the data of the first phase. For the third phase, the interior pelvic measurements of the heifers are currently also recorded in order to study this component of the maternal effect in more detail.
Although the l3os indicms breeds of South Africa (like the A frikaner and other Sanga types) are classified as such on account of their anatomy, physiology and adaptability, their Y chromosome is not acrocentric like that of the Brahman and related indicacs breeds.
They have the typical submetacentric Y chromosome found in 80 S taurus breeds. A unique haemoglobin type, Hb I, has also been observed in the South African litdicus breeds, previously confounded with the Hb C occurring in the Brahman, Santa Gertrudis and their Asian ancestors. Blood groups and other genetic markers offer an efficient aid in the studies on breed structures, migration of genes and on phylogenic relations between populations. Highlights of results obtained during twenty years of research are listed and discussed.
RESULTS
South Africa with its numerous breeds of livestock and many infectious diseases could prove to be an ideal outdoor laboratory to investigate the resistance to tropical diseases and to correlate this resistance to the ever increasing number of genetic markers. Records on veterinary treatments of r i,!97 first-lactation cows sired by 50 Swedish Red and White (SRB) and 34 Swedish Fviesian (SLB) AI bulls were analysed. 22. 9 and 2 8. 7 p. 100 of the SRB and SI,B cows respectively were treated some time during their first lactation. Udder problems were the most frequent disease for both breeds, though more frequent in the SLB.
In g!neral, differences among herds appeared to be the most important source of variation.
Significant effects of year, month, and age at calving were also found for several diseases.
Differences between daughter groups were significant for teat injuries, mastitis, any kind of teat or ud:ler disease, foot, leg, or locomotory diseases, veterinary treatments whatever the origin, and culling rate in the SRB breed, while only teat injuries, foot, leg, or locomotory diseases, and the veterinary treatments whatever the origin, were significant in the SI,B breed. Heritability estimates varied between zero and q.(i p. !oo. The highest value was obtained for udder or teat diseases in SRB.
A difference of 10 p. too in transmitting ability for veterinary treated daughters in first lactation was shown to exist between the extreme bulls within each breed, despite the fairly small number of bulls exceeding ioo daugliers, which was chosen as a minimum.
CLASSIC VS. DESIRED GAIN INDEX
F. PIRCHNER Lehrstuhl Jiiv Tierzucht der Technischen U n i vcrsität A7iiaaehen, Freising-WeihenstePhan Selection indices are among the most sophisticated tools of modern breeding theory. The optimal utilization of information leads to maximum gains in a given situation. However, extent and kind of information can be changed. The genetic change in milk and beef performance depends to a large extent on the resources allocated to estimate breeding values of the two respective trait complexes.
Desired gain index, suggested by Pezek and Baker, can take this problem into account and bring alxmt changes in desired proportions. In view of the fact that agreement between supply and demand is of paramount importance to overall efficiency of the economy, it appears that application of such techniques to achieve gains of various traits in correct proportions, deserves attention.
NONLINEAR MODELS DESCRIBING THE DIFFERENCE IN GROWTH CURVES OF CATTLE STRAINS
Ewa PTAK Department of Genetics and Animal Breeding A gricultural Academy, Krakow jPoland Five growth models fitted to the weight-age data of F. bull calves from to different F y iesial l . strains, were compared for their suitability to discribe the shape of the growth curve. The most accurate model was Richards function followed by the von Bertalanffy, Gompertz, logistic and Brody functions. All models underestimated the 17 -1 8 month weights. The only significant differences between strains in growth parameters were found with the von Bertalanffy model. The importance of the individual and maternal components of calving performance in a population of German Simmental cattle were to be studied and genetic parameters were to be estimated using the model of W ILL HAM . Calving data of calves of testing bulls for analysis of the individual component were collected in the first phase by special recording. Frequencies for classes of calving performance were: 1 8 p. 100 without assistance, 35 p. 100 with little assistance, 44 p. 100 with considerable assistance and 3 p. 100 surgical assistance. Heritability for the individual component was h 2 -.1 4 with a considerable standard error because of the relatively small data set (s 2 h 1 -= . 14 ). In the second phase, daughters of the same testing bulls were recorded at their first calving. From these r, 2 oo he:fer calvings parameters for the maternal component will be estimated using also the data of the first phase. For the third phase, the interior pelvic measurements of the heifers are currently also recorded in order to study this component of the maternal effect in more detail. | v3-fos |
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} | s2 | Quantitative analysis of cholesterol in foods by gas-liquid chromatography.
In order to develop a simple and exact analytical method for cholesterol determination in foods by gas-liquid chromatography, several experiments were carried out in collaboration with several universities and institutes. For the extraction of lipids from foods, it was decided that chloroform-methanol (2:1) extraction was the most suitable procedure. Since pretreatment procedures using methods such as thin-layer chromatography and florisil column chromatography to purify the unsaponifiable matters reduced the recovery of cholesterol, and good results were obtained without applying the pretreatment, these procedures were concluded to be unnecessary. Gas chromatograms obtained with free sterol showed results similar to those of the trimethylsilyl ether derivative and acetate. 5-alpha-Cholestane is used as a good internal standard.
The Japanese Resources Bureau, Science and Technology Agency, is planning to reform the standard tables of food composition in Japan, and in this program it was decided to add the cholesterol contents of several foods to the tables. We were requested to develop a simple and exact analytical method for cholesterol determination in foods.
The determination of cholesterol is usually carried out by colorimetric methods (1,2), but these methods are unsuitable for cholesterol in foods because of the presence of a considerable amount of several interfering sterols in some foods such as shell-fish (3,4). Therefore, we decided to study the determination of cholesterol by gas-liquid chromatography(GLC) which had been known to be suitable for sterol separation (5). This study was carried out in collaboration with several universities and national institutes. solvents. Short-necked clam (Tapes philippinarum) and top-shell (Batillus cornutus) were chosen as samples, because such shell-fish are known to contain several kinds of sterols (3,4). Table 3 shows that a mixture of chloroform and methanol in the ratio of 2:1 (v/v) is the most suitable solvent system to extract the lipids more exhaustively from foods. The lipids extracted from foods by each solvent system were saponified in ethanolic alkali solution and the unsaponifiable matter was extractd with diethyl ether. Cholesterol was estimated by applying the unsaponifiable matter to GLC.
Since the results indicated that the assayed values of cholesterol were the highest when the chloroform-methanol extraction procedure was applied to both the samples of short-necked clam and top-shell (Table 3), the extraction procedure using the chloroform-methanol system was employed in subsequent experiments. Experiment 2. Investigation on the effect of pretreatment Several procedures such as thin-layer chromatography (TLC) (6), florisil column chromatography (7), and the precipitation with digitonin (8) have been reported to remove impurities from the unsaponifiable matter in the determination of cholesterol. Therefore, the effects of pretreatment procedures on the assayed values of cholesterol in foods were compared. The three methods mentioned above as pretreatment procedures were investigated. First, synthesized triolein containing 70.7mg of cholesterol per 100g oil was used as a sample for the collaborative test. When the recovery of cholesterol was examined by applying the method without any pretreatment, four laboratories gave a satisfactory result within 92 to 103 ( Table 4), while that obtained from one laboratory (E) was 84%, which might be an exception. On the other hand, when florisil column chromatography was used as a pretreatment procedure, the recoveries were very low except for that from one laboratory (B). Reduced recoveries were obtained also by Lab. (A) with TLC and precipitation with digitonin as pretreatment procedures.
In the next experiment, oyster sterols were used as another sample for collaborative testing, because the lipids are known to contain several sterols as impurities (3). A sample for the collaborative test was made by mixing 5.983 mg of the oyster sterols with soybean oil. When the cholesterol content was determined, deviation among the laboratories was smaller than that obtained from the above experiment (Table 4). 502 T. KANEDA et al. In order to see the pretreatments of the TLC and the digitonin precipitation, egg yolk lipids and oyster lipids were used which were similarly prepared by chloroform-methanol extraction. As shown in Table 5, the recoveries of cholesterol from yolk lipids tend to decrease with the application of TLC or the digitonin precipitation method. On the other hand, the results from the oyster lipids showed that reduced values by applying the TLC pretreatment were not clearly observed, whereas the digitonin precipitation method resulted in slightly decreased values.
In other experiments, sometimes it was noticed that these pretreatments yielded low recoveries of cholesterol. From these results, it was concluded that the pretreatment procedures were unnecessary for the GLC determination of cholesterol in foods.
Experiment 3. Effect of derivatization for the GLC determination of cholesterol
Since it is known that sterol acetates and the trimethylsilyl ether derivatives are useful for GLC analysis, effects of derivatization on the determination of cholesterol were investigated. Methyl esters of lard fatty acids were mixed with cholesterol to make a concentration of 0.1%, and this was used for the Table 5. Effect of pretreatment procedures on the assayed values of cholesterol. collaborative test. Table 6 shows the results from the four laboratories. When the recoveries were tested with the free form of cholesterol, good results showing 99% and 103% were obtained from two laboratories ( Table 6). On the other hand, acetylation and trimethylsilation were also confirmed to be useful for the GLC determination of cholesterol. The results from Experiments 2 and 3 showed that derivatization was unessential for the GLC determination of cholesterol in foods, because good separation of cholesterol from other impurities could be achieved without derivatization, and the recovery tests always gave good results. Therefore, it was concluded that the unsaponifiable matter of samples should be applied directly to GLC in order to save procedure time.
Experiment 4. Collaborative test for the determination of cholesterol in foods by the proposed method
Collaborative tests for the determination of cholesterol in several foods were performed by using the proposed method. A recovery test was first performed on a sample of lard with a known added amount of authentic cholesterol (40.9mg/100g oil). As shown in Table 7, five laboratories obtained good results showing recoveries between 93 and 105%, whereas one laboratory (B) received a high recovery value which should be excluded. The cholesterol contents of Kotama-gai (a type of clam) and Euphausia sp. were also determined by the proposed method. As shown in Table 7, deviation among the respective data obtained from the laboratories was small except for the datum on Kotama-gai from one laboratory (E). Table 8 shows the assayed values on the other samples carried out by two laboratories. The respective data were similar to each other. From these a Recovery (%) of added cholesterol is shown in parentheses . b Excluded from the calculation of the mean value. | v3-fos |
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} | s2 | Polyol mixture supplementation in the diet of breeding sows and piglets
In a factorial 2x2 experiment the use of a polyol mixture (sugar alcohols) in the diet of sows and piglets was investigated. The trial was performed with 26 sows divided into two groups. The polyol sows were fed 180 g polyol mixture daily, and for polyol group piglets the polyol supplementation in creep feed was 5 %. The average number of piglets per litter in the control group was 10.3 and in the polyol group 9.2. The addition of polyols to the diet of sows had only a small effect on the performance of piglets. The piglets receiving polyols in creep feed gained during 1— 21 days post partum 21.1 % more than controls and during the rest of the lactation period 7 % more (P > 0.05). The incidence of diarrhoea in piglets of polyol groups was slightly higher compared with controls. The consumption of creep feed supplemented with polyols tended to be higher than consumption of control feed. Colostrum and milk samples were taken for analysis within 12 h after feeding and 7 and 21 days thereafter. The protein and lactose contents of colostrum and milk were slightly higher and the fat content lower from sows receiving polyols, but the differences were not significant. The mineral and amino acid composition was also analysed.
Introduction
The mixture of polyols or sugar alcohols, is a by-product of xylitol production from birch tree hydrolysate. Recent reviews by Touster (1974) and Tuori and Poutiainen (1977) have dealt with various aspects of the metabolism of polyols.
Polyols apparently have important antimicrobial effects. Xylitol exercises an inhibitory effect on dental caries by decreasing the bacterial growth on teeth (Schein and Mäkinen 1975). The fermentation of polyols by rumen microbes is slow compared with monosaccharides (Poutiainen et ai. 1976). The findings of Korhonen et ai. (1977) and Mäkinen et ai. (1979 a) indicate an increase in lactoperoxidase activity in milk and saliva of cows on polyol feeding. Lactoperoxidase enzyme is antimicrobial against intestinal infections in the newborn (Reiter 1978). Difficulties in piglets during the suckling period and after weaning are most frequent!}" associated with gastrointestinal disorders due to bacterial overgrowth, usually of Escherichia coli. Recent findings on antimicrobial effects of polyols and lactoperoxidase enzyme were thus a motivation toward the present study.
The satisfactory growth of piglets in the period before and after weaning depends on a high voluntary intake of creep feed. The palatability of the feed is of great importance, and feed consumption increases when sweetening in gredients, e.g. sugar, are added. Study of the possible use of polyol mixture as a sweetener was the other purpose of this work.
The study is part of a research project directed at the utilisation of sugar alcohols in the nutrition of domestic animals. The effect of polyol supplementation on piglet performance and composition of the colostrum and milk of sows was investigated. The enzyme activity of sow milk during polyol feeding has been reported by Mäkinen et ai. (1979 b).
Materials and methods
The experiment was carried out with 26 Landrace sows of the Swine Research Station. All sows were fourth or more parity animals at the beginning of the experiment. Three weeks before farrowing the sows were allocated at random to the control or polyol group. The sows received before farrowing 3.2 kg gestation ration daily, plus 180 g of polyol mixture in the polyol group and 100 g of barley in the control group daily. During the lactation period the sows were fed a lactation ration in proportion to the number of piglets (2.8 kg + 0.3 kg/piglet feed daily). The sows in the polyol group continued to receive 180 g of polyol mixture and the control sows 100 g barley during the lactation period.
After farrowing, the control and polyol groups were divided into two subgroups with respect to the creep feed for piglets. The four groups were as follows: Group 1 Control sows, control piglets, 5 litters Group 2 Control sows, polyol piglets, 5 litters Group 3 Polyol sows, control piglets, 5 litters Group 4 Polyol sows, polyol piglets, 5 litters The creep feed for piglets was supplemented with either 5 % of polyol mixture or 2.5 % of a mixture of glucose and fructose. The creep feed was offered to piglets after one week of age and the feed consumption was measured beginning at three weeks of age. The piglets were weighed at birth and weekly thereafter. A check for diarrhoea was made daily. Weaning took place at 5 weeks.
Feed analyses were made by standard methods (Paloheimo 1969) and sugar alcohol composition of the polyol mixture was analysed with a gas chromatograph (Carlo Erba 180).
Milk samples from 26 sows, 13 of the control group and 13 of the polyol group, were obtained for chemical analysis by intramuscular injection of 10 IU of oxytocin, followed by manual expression of milk from several teats. The samples were collected at 12-16 hours, 7 days and 21 days after farrowing and strored at -2o°C until analysed. After thawing the milk samples were analysed for total solids, ash, fat by the Gerber method, nitrogen by the Kjeldahl method, protein by multiplying the value of nitrogen by 6.38, and lactose by the chloramine-T method. The amino acid composition was analysed with a gas chromatograph (Hewlett Packard 5710 A), the mineral content with an atomic absorption spectrophotometer (Varian Techtron AA 1000), and phosphorus by the method of Taussky and Shorr (1953).
The average weight gain of piglets for each group was tested by two-way variance analysis.
The data of milk composition was tested by one-wa} ? variance analysis. The differences between treatment means were tested by the Tukey-test (Steel and Torrie 1960).
Results and discussion
The composition of the diets is presented in Table 1. The ash content of creep feed in the control ration was 4 percentage points higher than in the polyol ration and also the DCP percentage and FU-value were lower in the control diet. This difference is probably due to a mistake in the preparation of the creep feed mixture. The average dry matter percentage of the polyol mixture was 56.8 and the sows were fed 102 g DM of polyol mixture daily, corresponding approximately to 0.5 g polyols per kg live weight. In creep feed the percentage of polyol supplementation was 5.0 or 28 g polyol DM/kg feed.
Ihe mean values of sow and litter performance are given in Table 2 and Figure 1. The litter size differed in the two groups: the average number of piglets per litter in the control group was 10.3 and in the polyol group 9.2. The percentage of stillborn piglets in the control group was 6.8 and in the polyol group 10.2. In one litter in group 4 the number of stillborn was six of fourteen born. If this litter is excluded the correspoinding percentage is 6.5. Litter mortality from birth to 21 days was one piglet in group 1 and one in group 2; afterwards no death of piglets occurred.
The different size of litters caused some anomalies in the results. The treatments had no statistically significant effect on growth of the piglets when the number of piglets per litter was treated as an independent variable. The supplementation of polyol mixture in the diet of sows had only a small effect on the performance of piglets. The growth rate of the litters of sows receiving polyol supplement was in the period I -2l days post partum 6.6 percent better, but the number of piglets should be take into account, which was on average 1.1 piglets less. During the whole lactation period the difference was quite small. The piglets receiving polyol mixture in the creep feed gained during I-2l days post partum 21.1 per cent more than piglets in the control group and during the rest of the lactation period 7-B % more.
In the whole experimental period the response of growth rate to polyol supplementation amounted to an average 10.1 % increase. Reduction in the number of piglets per litter should be taken account when evaluating the results. The health of sows and piglets was normal during the experiment. The incidence of diarrhoea was 7,6, 8, and 11 in 5 litters in groups 1,2, 3 and 4, respectively during the rearing period. Piglets in two litters of the polyol group had to be treated clinically. Other cases of diarrhoea were quite slight. The polyol feeding did not raise the lactoperoxidase content of sow milk (Mäkinen et ai. 1979 b). The general composition of colostrum and milk of sows receiving polyol supplement and sows on control feeding is presented in Table 3. The values are averages of 13 samples taken at each sampling. The colostrum samples were taken 12-16 hours after farrowing. The composition changed very soon post partum. The variation among samples of colostrum was quite wide: 15.7 22.5 % for total solids, 0.56-0.92 % for ash, 2.9-9.1 % for fat, 4.9-12.3 % for protein and 3.6-6.9 for lactose. The colostrum of sows given polyol supplementation tended to be richer in all constituents except fat in comparison with sows on the control ration.
The protein content fell rapidly after farrowing, 3-3.5 percentage points during the first week of lactation. During the same time the percentages of ash, fat and lactose rose. Polyol sows produced higher milk content of protein and lactose but lower fat content than the controls. However, the differences were not statistically significant owing to the wide variation among samples. The ash content at seven days post partum was lower in the polyol group (P < 0.05). The lactose content was on average 0.1 percentage point higher sows in the polyol group than controls, possibly because of the polyols give in the diet.
The values found in the present study for sows colostrum and milk composition are in agreement with values given in the literature. The values for total solids and protein in colostrum are much lower than those reported by Braude et al. (1947), Elliot et al. (1971 and Fahmy (1974) but this is mainly due to the different sampling time post partum. In the present study the samples were taken 12 -l6 hours post partum whereas values given in literature are usually for samples taken during farrowing. The values of samples at 7 or 21 days post partum agree closely with those reported by Rook and Witter (1968), Elsley (1971), Mahan et al. (1971, O'Grady et ah (1973) and Chen et al. 1978).
The composition of minerals in the colostrum and milk of sows is shown in Table 4. The values are averages of 13 samples taken at each sampling.
The phosphorus content was lower in colostrum than in milk, and the calcium in colostrum was only half of the content in milk. The calcium content in Table 4. Mineral composition of colostrum and milk of sows.
Control diet
Polyol diet colostrum of the polyol group was 0.10 g/kg lower compared with the control.
The value of calcium in milk was 1.5-1.6 g/kg, which was low compared with the values given by Elliot et al, (1971), Fahmy (1972) and Chen et ah (1978). The values of phosphorus were comparable with those reported in the literature. Magnesium content was the same in colostrum and milk, but at 21-days post partum was significantly lower (P < 0.05) in the potyol than the control group. The magnesium values were a little below the values given by Elliot et al. (1971), Fahmy (1972) and Chen et al. (1978). The potassium content in colostrum was 1.26-1.32 g/kg and it decreased as the lactation progressed. Magnesium and potassium contents were lower at every sampling time in sows on the diet supplemented with polyol mixture. The amino acid composition of colostrum and milk from sows is shown in Table 5. Values are reported as grams per 100 grams of milk protein and are averages of the five samples taken at each sampling time. The amounts of all amino acids of colostrum were greater in the polyol group than in the control group. A similar result was found for milk at seven days postpartum, except for the values of glutamic acid and tyrosine which were higher in the control group. At seven days postpartum the total amount of amino acids in colostrum was 12.2 % and in milk 7.6 % greater in the polyol group than the control group. At 21 days postpartum the values in milk were 1.9 % smaller for the polyol group. The protein content of milk in the polyol group was 0.53, 0.17 and 0.17 percentage points higher than in the control group at the different sampling times; 12 h, 7 d and 21 d postpartum, respectively. The values obtained in this study correspond quite closely with values reported in the literature (Elliot et al. 1971, Elsley 1971 Tutkimuksessa selvitettiin polyolinlisäyksen (sokerialkoholiseos) vaikutusta porsaiden kasvuun, rehunkulutukseen ja ripulin esiintymiseen sekä emakonmaidon koostumukseen. Koe käsitti 26 pahnuetta ja oli faktoriaalinen 2x2, jossa emakot saivat 180 g polyoliliuosta tai 100 g ohraa normaalin rehuannoksen lisäksi ja porsaat porsasrehua, joka sisälsi joko 5 % polyoliliuosta tai 2.5 % glukoosi-fruktoosiseosta. | v3-fos |
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} | s2 | Effects of the m gene for muscular hypertrophy on conformation at one year of age in beef cattle
University of California herd, were studied, including 54 , born during Spring 1970 , genotypically distributed as 7mm, 33 m-!and 14 + + (m being the symbol for the autosomal allele for doublemuscling). Conformation was evaluated at yearling age by scoring superficial muscles for bulginess and the lateral shape of the rump, and combining the scores into an index (h). Differences between mm and m + and between m-! and -!-!-, were significant (P ! o.ooi). Comparing m+ with !-+, the expression of the m gene was enhanced in bulls; the interaction between genotype and sex was significant (P < 0.05 ). The phenotypic variance of heterozygotes (m-!) exceeded that of normals (+ +) for 12 (P < 0.5 ) and 365-day liveweight (P < 0.05), height at withers (o.i ! P < 0.25 ) and cannon bone (metacarpus) circumference (P ! 0.25 ). For each of these four traits, mtM—m-), the absolute difference between means, was more than twice as great as I m + + + I. -
Introduction
This paper reports differences in body conformation for double-muscled (mm), heterozygous (m+) and normal ( ' + +) beef cattle at one year of age. Comparisons between the phenotypic means and variances associated with the three genotypes are presented.
Beef cattle manifesting the genetic condition of double-muscling (muscular hypertrophy) are characterized by extraordinarily bulging muscles of the shoulder and thigh, a very rounded rear end (lateral aspect), and a fine-boned skeleton; the enlarged muscles and reduced subcutaneous fat give the intermuscular grooves particular prominence. Double-muscled cattle have a greater proportion of muscle in the body, less bone and viscera and much less fat compared with normal animals. The condition appears to be transmitted according to a monohybrid autosomal mode of inheritance (OLIVER and C ARTWRI G HT , 19 68; R OLLINS et al.,I97z).
Reviews of the literature on double-muscling have been made by L AUVERGNE el l ll., ( 19 6 3 , I9 68); OLIVER and CA R T WR i& H T ( I g68); and BO YA jE AN et al. ( 1971 ).
Materials and methods
Fffects of the m gene for muscular hypertrophy on body conformation at one year of age were observed mainly in the background of the Aberdeen Angus breed, but also the Here f ord and Shorthorn. All three breeds are of a similar size. RoI,I,irrs et al. ( 1972 ) described the experimental herd of the University of California at Davis (U.C. Davis).
An animal is classified as double-muscled (mm) or non-double-muscled on the basis of its phenotype. A non-double-muscled animal is classified as m+ or + + on the basis of pedigree or progeny test.
Conformation, interpreted as the fullness or bulginess of muscles and lateral shape of the rump, was measured at yearling age by scoring superficial muscles and rump shape and combining the scores into an index (I 2 ) (R OLLINS et al., 1972 ). I,iveweight, height at withers and cannon bone (metacarpus) circumference at 3 65 days of age, were measured also. Yearling conformation scores (, 2 ) were available on 95 calves which were all born at U.C. Davis during 19 6 9 or 1970 . However, estimations of the differences in conformation between genotypes mm, m+ and !--! were restricted to the 1970 calves because experimental conditions in that year were more controlled. These calves were genotypically distributed as 7 mm, 33 m + and 14 + + .
Most of the 1970 calves were born within a 2 +-month period during the Spring of that year, and were weaned at approximately six months of age. After weaning until one year of age calves were fed ad libitum, the roughage : concentrate ratio of the rations being 75 :2 5 and 70 : 3 0, for bulls and heifers, respectively.
As sires of the I g 7 o mm and m!-calves, 6 44 and 8 5 6 were chosen from amongst the several bulls at U.C. Davis who were carrying the m gene, because they were the only double-muscled (mm) Angus bulls available, who were also both unre-lated to the cow herd and to each other. Sire 6 44 was a Red Angus bull, son of a Certified Meat Sire (CNIS), and half-brother to a CMS. Sire 8 5 6 was an Angus bull, purchased as a double-muscled calf, the offspring of a commercial dam and an unknown sire. As sire of the !---!-calves, Angus bull Co 5 was chosen primarily on the basis of his cc normal n appearance, i.e. because there was no suspicion that he was heterozygous. Co 5 also had a CMS classification.
No distinction will be made henceforth between Angus and Red Angus since the breeds differ only in colour.
To qualify for the tittle Certified Meat Sire, a minimum of 10 progeny of the bull must meet or exceed standards set by Performance Registry International, P.O. Box i 33 , Joplin, Missouri 6 q8oi, U.S.A. These standards implicitly include liveweight at 3 65 days of age.
It was necessary to combine straightbred (AA) and crossbred (XB) data for m+ and -f-+ calves. However, crossbreds are expected to show a greater 3 65-day liveweight relative to syraightbreds, and this effect on weight might well carry over to the other yearling traits studied, viz. I 2' height at withers and cannon (metacarpus) circumference. The effect of genotype (m--! or !-!-) on conformation is confunded with this effect of crossbreeding unless the data are adjusted. Crossbred data were therefore adiusted to a straightbred basis by using adjustment factors derived from the difference between means (XB — AA) within genotype, and the ratio of phenotypic standard deviations (a m+ /6 ++ ) for each trait.
Results
The distributions of the index of yearling conformation (1!) for mm, m!-and + + calves are shown in Table i.
The clear separation of mm and m+ distributions indicates distinct differences in conformation. The modal values for the m+ and -f--j-distributions are different, although the distributions overlap in both sexes, the overlap being greaterin heifers than in bulls.
For each genotype there is variation in expression of conformation (muscularity) around the modal value, the variation being greater in m + than in ++ or mm.
The trin:odal nature of the distributions of I 2 provides evidence for the monohybrid autosomal mode of inheritance of the m gene for double-muscling. This has been discussed by R OLLINS et al. lI9!2). The trait 1! clearly reflects genetic influence on conformation. These genotypic differences in conformation and expressivity of doublemuscling will now be examined in detail in order to explore their causes and validity.
1 . -Comparison of means of / 2 1 2 phenotypic means are given for the three genotypes in Table 2 and the analyses of variance for the comparisons between mm and m+ and between m-fand + + are given in Table 3 .
Homozygote (mm) calves had a significantly (P< o.ooi) lower 1! score than m+ (a lower 1! score indicates more muscling). Bulls had more bulging muscles than heifers (P < 0 . 025 ) although there was no evidence of an interaction between genotype ans sex. Heterozygote (m+) calves had a significantly (P < o.ooi) lower 1, score than + +, and bulls had more bulging muscles than heifers (P < o.oz).
The interaction between genotype and sex was significant (P < 0 . 005 ), evidence that the expression of the m gene was increased in bulls. Since normal (+ +) bulls tend to show greater muscle development than eifers, the genetic background of heterozygous (m+) bulls may enhance the effect of the m gene.
In the double-muscled (mm) animal, the m gene manifests pleiotropic effects, including increasing the fullness (bulginess) of the musculature and depressing 3 6 5 -day liveweight, height at withers and cannon (metacarpus) circumference ( Table 2 ). The effect of the mm phenotype compared with normal, (mm — + +) X 100 I( + +), is more marked for I 2 and cannon circumference than for the other traits.
The m+ phenotype, compared with ++, shows adistinctly (P < o.ooi) more bulging conformation of the musculature (Table 2 ). However, contrary to the effect of the mm genotype, the m + compared with -!--!-shows a slightly (P > o.q.) increased liveweight and slightly increased height at withers (P > 0 . 2 ). The effect on cannon circumference is in the same negative direction as the effect of mm, but much reduced in amount (P > o.i).
If the effect of the m gene is to change the mean for a trait, compared with the normal mean (+ +), then the extent of that change may be reflected in the change of variance, or, on a linear scale, in the change of standard deviation. In table the four yearling traits are compared with respect to the effect of the m gene on the mean and the standard deviation and the relationship between them.
Averaging over both sexes for (mm -++) X 1 0 0 1 ( + + ), the expression of the mm genotype shows most in 1, and cannon circumference, then liveweight and least effect on height at withers. In all traits mm was reduced nemerically, relative to !-1.
The expression of the m-! genotype compared with normal, (m+ — Q) X 100 I( + + ), averaged over both sexes, is greatest for 1,. The mean 3 65-day liveweight of heterozygotes was increased -f-3 p. cent with a corresponding, but smaller, increase in height at withers. The cannon circumference was least affected, but it was reduced -I p. cent.
Discussion
This study demonstrated that it was possible to treat bovine conformation at one year of age as a quantitative genetic trait. The trimodal distribution of the index of conformation (1!) provided evidence for the monohybrid autosomal mode of inheritance of the double-muscled condition. It was thereby possible to observe the effect of the mutant m gene on conformation at yearling age.
The expression of the homozygote (mm) genotype showed more in 1! and cannon circumference and less in 3 6 5 -day liveweight and height at withers ( Table 2 ). The mm calves were heavier (-!-7 p. cent) than -!+ calves at 1 8 0 days (weaning) but their post-weaning growth rate was markedly depressed (— 17 p. cent) due to a reduced (— m p. cent) feed intake, compared with non-mm controls (NoTT and R OLLINS , 1979 ). The genetic potential for post-weaning growth is greater for double-muscled than for non-double-muscled animals (R ATMONDT , Ig6 I ; NE UVY and V I SSAC, 19 62; HA N SET and DEMOUI,7N, 1965; ANONYMOUS, 1 9 66; F A I, I E Z , 19 66; T RILL AT ( 19 67) cited by BOYAJ!AN et al., 1971 ). Thus, in a favourable environment, where the diet contains a high enough proportion of concentrates, the expression of m in the homozygote is towards increasing liveweight at 3 6 5 days.
On the development of the long bones of the skeleton, the m gene in the homozygote (mm) has more effect on later-developing width than on earlier-developing length, and more effect on later-developing proximal bones (e.g.) humerus than on earlier-developing distal bones (e.g. metacarpus) (V ISSAC , I g6S; R OLLINS et al., I g6g; H AN SET and ArrsA y , 1972 ). The. opposite is true for the d gene for Snorter dwarfism in cattle. This pattern of effect of the m gene, together with the lower yearling liveweight of mm compared with --!-+, explains, in part, the observation of less effect of mm on height at withers and more effect on cannon circumference.
For each of the four traits measuring conformation, I mm -+ +1, the absolute difference between the homozygote (mm) and heterozygote (m+) means, was more than twice as great as 1 m+ -+ + the absolute difference between the heterozygote (m-!) and normal (++) means. However, the m gene in the heterozygotes (m+) had a greater effect on the mean for conformation of muscling (1 2 ) and 3 65-day liveweight than on skeletal characters ( 3 6 5 -day height at withers and cannon circumference), i.e. the partial dominance (incomplete recessiveness of the m gene was greater in traits reflecting muscle growth than in those measuring bone growth. The action of the m gene in the heterozygous condition may be different from its action in the homozygote, at least because of the incompletely dominant allele (+). However, the expression of the m gene, both in the heterozygote and in the homozygote, probably depends upon background modifying genes (genes of individually small effect) besides the environment. A consideration of the background modifying genes provides a possible explanation as to why the m gene in the heterozygote has a greater effect on 1! and liveweight, both of which reflect muscle growth, and less effect on height at withers an cannon circumference, both measures of skeletal growth.
The expression of a mutant gene in homozygotes and heterozygotes, and the dominance relations between two alleles, can be modified by selection (CASTLE and P H I L I L PS, I9I4 ; WRIGHT and CHASE, 193 6; FORD, 1940 ;FISHER and HOI,T, 1944 ). Thus it would be expected that the m gene would have a greater effect on the mean in the heterozygote (m-!) for traits in which selection criteria had been in the same direction as the effect of the m gene, and less effect where selection had been in the opposite direction. This expectation is realised when the magni-. tude of the different effects of the m gene on the four yearling traits are compared (Table 2 ).
In this experiment, the m gene in heterozygous condition was studied in the background of the British beef breeds which have been selected primarily on growth rate and carcass characteristics; reflecting weight gain and muscle development, with much less emphasis on the skeleton. Since selection for weight and muscle development in beef cattle and the expression of the m gene are both in the same direction, the background modifying genes may enhance the expression of the m gene in heterozygous condition for muscle conformation (1!) and 3 6 5 -day liveweight. Bulls generally have muscles which are more bulging than heifers, and thus for similar polygenic reasons the effect of the m gene on the mean for yearling conformation (I 2 ) of heterozygotes was greater in bulls than in heifers.
In recent years, beef cattle selection has emphasized larger mature size (size of frame at yearling age which is related to height at withers), longer legs and heavier bone (thicker cannon bones) (A MER . ANGUS Assoc., I g6 5 ; E LI nos et al., ig6g; LONG, 1971 ). The m gene, in the homozygote (mm), depresses height at withers and reduces cannon circumference. Since the selection for skeletal characters in beef cattle and the expression of the m gene are both in opposite directions, the background modifiers may attenuate the expression of the m gene in heterozygous condition for 3 6 5 -day height at withers and cannon circumference. Nevertheless, despite the slightly increased mean liveweight, the effect of m un heterozygotes was to reduce cannon circumference, an effect in the same negative direction as the effect of m in homozygotes (mm).
Evidence from the literature indicates that the more bulging conformation of the double-muscled (mm) compared with non-double-muscled animals can be explained, in part, by a generalized but not uniform muscular hypertrophy together with a shortening of the long bones (B UTTERFIELD , i 9 66; V ISSAC , rg68; R OLLINS et al., ig6g; VISSAC et al., 197 1 ; HA N SET and A N SA Y , 197 2). The heterozygote (m+) showed distinctly (P < o.ooi) more bulging conformation of the musculature (1!) compared with normal (++), which might be due to a similar hypertrophy of the musculature, with gradients of muscular hypertrophy in parts of the body, in the same direction as the expression in the double-muscled (mm) animal but much reduced in amount. The corresponding superiority of the m+ animals for carcass traits, compared with + +, was discussed by T HI E SSEN (I973) ! For the overall trait of double-muscling, it appears that in the Italian Piedmont breed the average phenotype of the heterozygote (m+) lies closer to the doublemuscled (mm) phenotype, whereas in the major British breeds, e.g. !4!g'MS, the heterozygote lies closer to the normal ( + + ) phenotype. One possible interpretation of the difference can be given in terms of the evolution of dominance; thus in the Piedmont breed, where double-muscling is selected for, the m gene would tend to become dominant, but in breeds where it was selected against, the m gene would tend to become recessive (I, AUVER G N E et al., 19 63).
That the phenotypic variance of a major gene influencing a quantitative trait can very depending upon the background modifying genes, was shown by WRIGHT and CHASE ( 193 6). Their results lead to two expectations : first, that in a genetic background favouring muscle development, the variation in expression of the m gene un heterozygous condition would be large for conformation of the musculature (1!) and for 3 65-day liveweight, and that it would be greater than the variation for normal (-!-!); second, that in an intermediate genetic background, i.e. some modifiers encouraging skeletal growth, other modifiers discouraging it, the variation in expression of the m gene in heterozygotes would be small for 3 6 5 -day height at withers and cannon circumference, but that it would also be greater than the variation for normals (+-!). The variance estimates given in Table 4 show that these two expectations are met.
It is expected, according to one view, that the heterozygote for a mutant gene would be more variable than the wild type (FISHER, zgq.8; p. 6z). Similary, the presence in the genome of a mutant gene can be expected to result in an increase in the phenotypic variance (WanDrrrGTON, rg 4 2; MIC HI E, 195 8). The explanation for this may lie in terms of the evolutionary adaptation of genes. By replacing a -r-allele with m in the heterozygote, the genome of that individual is being changed, which allows gene-gene and gene-environment interactions which have not been adapted to one another by selection; thus phenotypic variation is expected to increase.
It can be seen that for all four traits Fisher's rule am + > c;2 ++, applies (Table . The magnitude of the difference between the variance estimates, 2 +/ I depends upon the different genetic backgrounds for the different traits, and is probably related to the selection history of beef cattle and to the magnitude of the effect of the m gene in the homozygote (mm), already mentioned. The increase in standard deviation (% m+ -a ++ ) x ioo /a ++ , is related directly, but not in proportion to, the change in the mean, (w-t-— -!--!) X 100 1( + +) (Table 4 ), which suggests that the modifying genes affecting conformation and weight are different from those affecting the skeleton; this would be expected since muscle and bone are different tissues.
Since, in this study, the m gene behaved as expected, it should be possible to predict the results of deliberately putting the m gene into different genetic backgrounds, which had been selected on the basis of different criteria, for instance meat breeds and dairy breeds. It would be predicted that there would be enhancement of the expression of double-muscling in the meat breed, but attenuation in the dairy breed. The results of crossing double-muscled (mm) and normal (+-!-) Charolais bulls with meat-, rustic-and dairy-breed type females in France (V ALLS O RTIZ et al., 1972 ) suggest that the predicted pattern of results may be emerging. The penetrance of the double-muscled character varied in each breed type depending upon whether previous selection had been favourable or unfavourable to muscle development (VISSAC,197 2).
There are possible genetic and environmental factors affecting estimates of phenotypic means and variances for 1 2 and the other three traits : i. As the -!--!-sire was not mated to nay mm cows, within-sire comparisons of m+ and -!-!-animals cannot be made. To the extent that the sires of the m+ and !--i-animals were polygenically different, the distributions of I 2 ave biased by sire effects.
4 . The h scores were not adjusted for differences in liveweight. Although the heavier animal might be expected to show greater muscular development, the physiologically older animal would also be expected to be laying down more subcutaneous fat than the younger and lighter animal which would reduce the prominence of the superficial muscles, and the development of the lower round would make the lateral view of the animal look more rectangular or « normal ». The consequence of greater physiological age might be to increase the I, score (less apparent bulginess of muscles). So a higher weight does not necessarily mean a lower 1! score (greater apparent bulginess of muscles). 5. For both bulls and heifers the range of 3 6 5 -day weights for -!--f-animals lay within the range for m+ animals, i.e. the lightest and heaviest non-doublemuscled animals were m+. The wider range and larger variance for the m+ might be due to an interaction between the m gene and the environment, the result depending upon whether the background modifying genes were favourable or unfavourable to growth. 6. In the homozygote (mm), the effect of the m gene on conformation is marked, but double-muscled calves tend to be less viable suggesting that the m gene allows greater susceptibility to environmental stress. In the heterozygote (m+), with a + allele to mask the effect of the m gene, the susceptibility may usually, but not always, be less. When the environmental susceptibility is less, the resulting heterozygote (m-!) phenotype is a more bulging conformation of the musculature than the normal (-!--!-) range. Many of the m+ calves expressed such a phenotype. Although the relationship between 1, and liveweight is not direct, poorly growing animals would nevertheless be expected to show a rather flat musculature.
Conclusions
Conformation at one year of age was treated as a quantitative trait and the effect of the m gene on that trait was observed.
With conformation interpreted as the bulginess of superficial muscles and the lateral shape of the rump, combined into an index (I,), there was a marked (— 73 .8 p. cent) difference between homozygote (mm) and normal (-f-+) animals, and a smaller (— 20 . 5 p. cent) but significant (P < o.ooi) difference between heterozygotes (m+) and normals (!-+) (a lower 1 2 score indicates more muscling). The effect in m+ was greater in bulls than in heifers (P < 0 . 005 ).
Comparing m+ with + +, the effect was greater for 1 2 and liveweight (!-3 . 0 0 p. cent) than for height at withers (-f-1 . 2 p. cent). The effect on cannon circumference (— 1 . 0 p. cent) was in the same negative direction as mm but much reduced in amount. Apart from I 2 , none of the differences in conformation between M+ and !--!was statistically significant, which reflects the role of small sample size and rather large variation, but they may be biologically significant since the expression of the m gene in heterozygous condition was similarly small for other characters studied (NoTT, 1973 ;NoTT and RO LLIN S, 1979).
For each of the four traits measuring conformation, I mm -m+ I , the absolute difference between means, was more than twice as great as I m+ -+-! I.
The effect of the m gene in the heterozygote (m!-) on the mean and standard deviation i.e. the partial dominance (incomplete recessiveness), was greater for muscle conformation (1,) and liveweight than for skeletal traits. This difference appeared to be related both to the selection history of beef cattle, which has favoured muscle development and weight gain, longer legs and thicker cannon bones, and to the magnitude of the effect of the m gene in the homozygote (mm). Considered together, the results of this study support the monohybrid autosomal mode of inheritance for double-m.uscling.
The size of the difference between m+ and !-+ means for muscularity (1!) and liveweight at one year of age, and the overlap between m!-and -!-+ distributions for these traits, indicated that current commercial selection standards for beef cattle, which emphasize a bulging musculature in the live animal and heavier weights at younger ages, would unconsciously favour the heterozygote.
Received for publication in August ig8o.
F I S H E R R. A., 195 8. The genetical theovy of natural secletion. Dover Publ. Inc., New-York. | v3-fos |
2019-03-19T13:04:22.145Z | {
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} | s2 | The effect of selenium fertilizers on the selenium content of barley, spring wheat and potatoes
When Se-cnriched fertilizers were given to barley and spring wheat on fine sand soils in Southern Finland the Se content in the grain was raised as follows: 50 g Sc/ha increased the Se content by around 50 fig/kg; 250 g Sc/ha by appr. 400 U g/kg; and 500 g Sc/ha by a little more than 900 fA g/kg. The selenium content of potatoes rose to Se/kg dry matter when the soil was enriched with 500 g Se in the form of Na 2 per hectare. The selenium content ofpotatoes was found to be very low when no added selenium was given. The selenium content of potatoes did not fall when the potatoes were boiled.
Introduction
Selenium is not an indispensable nutrient for crops. Nevertheless, there have been experimental attempts at raising the selenium content of agricultural produce; in the USA (CARY and ALLAWAY 1973), in New Zealand (GRANT 1965, DAVIES andWATKINSON 1966, and in Denmark (GISSEL-NIELSEN 1971, 1975. TheSe quantities used in the experiments have varied greatly, ranging from a few grammes when crops were sprayed (GISSEL-NIELSEN 1971) to several kilogrammes per hectare when transmitted through the soil (CARY and ALLAWAY 1973). When the selenium has been transmitted through the soil, selenium fertilizer quantities in the region of 100 g Se/ha have proved sufficient to give the desired Se level for fodder crops and hay, 50-100 u g Se/kg (GRANT 1965, GISSEL-NIELSEN 1977. It has been found that much smaller Se quantities give the same results when crops are sprayed if the spraying is done at the right time (GISSEL-NIELSEN 1977). Pure selenium, selenate and selenite were used in the experiments to increase the selenium content of the fertilizers. GISSEL-NIELSEN (1977) has conducted many experiments and he holds that selenite is the best source, even though selenate may be more effective than selenite for raising the selenium content of crops. The same researcher has also demonstrated (GISSEL-NIELSEN 1977) that the sulphate content of the soil has a much greater influence on how selenate is transmitted than on how selenite is transmitted.
The present study made use of selenite only. The selenite was employed to raise the selenium content of the fertilizers and for crop spraying purposes. The study aims 6 495 JOURNAL OF THE SCIENTIFIC AGRICULTURAL SOCIETY OF FINLAND at reaching a preliminary assessment of the manner in which the selenium content of barley, spring wheat and potatoes is raised when selenite fertilizers are sprayed and transmitted through the soil. The selenium-enriched barley obtained as a result of spraying has been used in feed tests on chickens, pigs and horses (KÄÄNTEE andKURKELA 1980 a, and1980 b).
Materials and methods
The fertilizers used in the experiments were prepared at the small factory of Kemira Oy's research plant. Ordinary commercial products were used as raw material; the fertilizers were granulated by being sprinkled with NajSeOj-JHjO solute. The Se content aims for the fertilizers were 0.01 % Se, 0.05 % Se and 0.10 % Se and these levels were realized.
A tractor spray was used for carrying out the selenium spraying: a solution of sodium selenite in 400 litres of water was used over one hectare.
The field tests were carried out at the Kotkaniemi experimental farm at Vihti.
The fertilizers which were meant to be transmitted through the soil were placed with a combined drilling machine. The Kemira Oy Oulu research plant conducted the selenium analyses. The hydridemethod was used (SAARI and PAASO 1 980).
Spraying
Suvi barley (sown 17. 5. 1978) growing on sandy loam was sprayed on 21. 6. 1978. The barley was being grown for experimental fodder and was just beginning to sprout when the spraying was carried out. Follow-up samples of the Suvi barley were taken the next year.
The spraying treatment was repeated on another plot of sandy loam in the summer of 1979. The Aapo barley had been sown on 17-18. 5. and the spraying was carried out on 21. 6. 1979 when the barley was sprouting. The initial selenium content of the plot sprayed in 1979 was slightly higher than the soil sprayed the year before.
2 Fertilisers transmitted through the soil
Grain
The selenium fertilizer experiments conducted in 1978-79 were carried out on coarse mineral soil. Barley and spring wheat were treated with experimental fertilizers which were enriched with Na 2 Se0 3 . Half of the experiment site was limed with 5 t/ha of agricultural lime before the experiment was instigated. The mean analysis results of the soil samples taken on 1 1. 5. 1978 before liming and fertilizing were as follows; Selenium content was regulated to four levels with Na 2 Se0 3 : 0.00, 0.01, 0.5, and 0.10 % Se. The quantity of fertilizer with which the grain was treated was 500 kg/ha. The spring wheat (Tähti variety) harvest was very low both years. The mean 1978 wheat harvest was 1790 kg grain/ha. The 1979 figure was as low as 1310 kg grain/ha. The barley harvest was satisfactory: an average of 4975 kg/ha was obtained in 1978, and an average of 3255 kg/ha in 1979. Neither year's spring wheat crop was affected by the Se fertilizing. Halving with liming had no significant effect on the grain harvest either.
Soil samples were taken in the spring of 1979 after the first year of tests in order that selenium analyses might be made.
Following the first year of tests the experiment site was divided into two. One half of the site was re-treated with the same Se-enrichcd fertilizer, whereas the other half was treated with normal, seleniumless fertilizer. The type of fertilizer used was the same as that which had been employed the previous year. Liming was not repeated. Table 10. A few potatoes which had formed part of the 1979 experiment were boiled. This was done as a type of random test and the results obtained are presented in Table 11. (1150) ') repeat analyses were made of the flour used in the baking, and the figures in the column are not the same as those in Tables 4 and 8 for this reason.
2 ) the figures in paretheses were obtained by re-analyzing the unleavened loaves after they had been deep frozen for one year.
Discussion
The present study's findings agree with the results of earlier experiments (SYVÄLAHTI and KORKMAN 1978). Selenium may be given to crops by spraying or by transmitting it through the soil. The natural selenium content of Finnish crops is very low (cp. e.g. KOIVISTOINEN 1979) and this means that selenium fertilizers have a marked effect on both grain and potatoes. When 50 g Se/ha (500 kg/ha of fertilizer containing 0.01 % sodium selenite) were transmitted to crops through the soil, the selenium content of the crops rose by a mean of 56 n g/kg during the first year. When 2 50 g Se/ha were given, the selenium content increased by 404 u g/kg on an average, and the content rose by a mean of 925 ,u g/kg when 500 g Se/ha was used. GISSEL-NIELSEN (1977) found that a Se quantity corresponding to the smallest amount (60 g Se/ha) caused the Se content of barley to rise by 38 ,m g/kg, and the content of wheat by 64 M g/kg. The results of these experiments and the present study are thus of equivalent magnitude. On the other hand, the effect of the present study on the Se content of grain was less great than earlier studies might have led one to expect (cp. SYVÄLAHTI and KORKMAN 1978). Contrastingly, the natural content of spring wheat in particular was higher than normal (cp. YLARANTA 1980). The reason for this was not discovered. The very low harvest figures might have a bearing, but it would also appear that the soil of the experiment site had a greater than usual amount of selenium which could be used by plants.
The present study was unable to arrive at an unequivocal conclusion as to how liming increases selenium uptake (cp. GEERING et al. 1968). Conflicting results have been obtained in this field: in Sippola's study (SIPPOLA 1979) a high pH appeared to have some slight retarding effect on the Se uptake of plants in this timothy material.
The finding that selenium is comparatively evenly distributed over the various parts of wheat grains and does not become concentrated in the husk to the same extents as other elements was noteworthy. Whilst the most selenium is found in the husk and the least in the kernel, differences are not great.
The selenium content of the unleavened barley loaves did not fall during baking. The content remained constant or rose a little. The Se content of the loaves was not affected by being deep frozen for a year. Selenium was not lost when potatoes were boiled.
There have been very few studies made with selenium fertilizers and potatoes. The present study demonstrated that the amount of selenium transmitted to potatoes is proportional to the quantity of selenium given during fertilizing. An estimate was made of the total selenium content of the dry harvest and demonstrated that potato is unlikely to be a more efficient user of selenium than spring wheat or barley.
The same problem is inherent in selenium fertilizers as mineral micronutrient fertilizers: only a very small part of the fertilizing element is transmitted to the crop and the rest of the element is bound to the soil or placed beyond the reach of the crop in some other fashion, A rough estimate of the quantity of selenium transmitted to the edible part of the plant in the first year (spring wheat and barley 1978, potato 1978-79) The above figures demonstrate that a maximum of slightly over one per cent of the selenium given was transmitted to the edible part of the plant. There is a slight indication that a greater proportion of selenium is transmitted to the crop when the amount given is large than when it is small. The largest Se quantities increased the Se intake of the harvest by a factor of B-3B when compared to the zero level.
Both grain and potato showed a slight residual effect in the second year. This finding agrees with the findings of earlier Finnish studies (SYVÄLAHTI and KORK-MAN 1978). The study will be followed for a few more years in this respect.
A comparative study of the efficiency of selenite and selenate in practical fertilizing will be made in Finland in the near future (YLÄRANTA personal communication). It is hoped that this forthcoming study will enable the efficiency of selenium fertilizers to be improved.
Summary
Selenium fertilizer experiments were made in 1978 and 1979 at the Kemira Oy experimental farm. Sodium selenite, Na 2 Se0 3 5H 2 0, was employed as the selenium source. Barley was subjected to spraying tests, and fertilizers were transmitted through the soil for barley, spring wheat and potatoes. Two types of selenium fertilizer were prepared at the Kemira research factory for the fertilizer trials. The fertilizers contained 0.00, 0.01, 0.05 and 0.10 % of selenium.
When 0.1, 0.5 and 1.0 kg Se/ha was sprayed, selenium content figures were obtained which were clearly excess of the content which is often used as an objective, 50-100 ,u g Sc/kg of the absorbent parts of the plant. More field tests will have to be carried out to ascertain the right quantities to be used and the best spraying time before spraying may be used in order to boost the selenium content of crops.
The extents to which selenium fertilizers raised the selenium content of grain when the fertilizer was transmitted through the soil were as follows: 50 g Se/ha boosted the Se content by more than 50 /.i g/kg; 250 g Se/ha by approximately 400 ,« g/kg; and 500 g Se/ha by more than 900 ,u g/kg. Selenium was distributed in spring wheat in such a fashion that the content of the husk was the highest and the content of the kernel the lowest. However, the Se content of the husk was a mere mean 32 % higher than that of the kernel. The selenium content of potatoes rose to 100 fi g Se/kg dry matter when the soil was enriched with 500 g Se in the form of Na 2 SeO } per hectare. The selenium content of potatoes was found to be very low when no added selenium was given. The selenium content of potatoes did not fall when the potatoes were boiled. The present series of experiments was unable to increase selenium uptake of crops by liming. It should, however, be borne in mind that the pH value of the test plots was high for Finnish conditions -it stood at 6.0-6.2 whereas the mean pH value of Finnish arable soil is 5.6. | v3-fos |
2018-04-03T05:58:54.031Z | {
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} | s2 | A COMPARISON OF THE GASTROINTESTINAL TRACT IN GERM-FREE AND CONVENTIONAL MICE FED AN AMINO ACID MIXTURE OR PURIFIED WHOLE-EGG PROTEIN
An amino acid mixture diet(AA) simulated with purified whole-egg protein and a purified whole-egg protein diet(WE) were given to ICR strain male germ-free(GF) and conventional(CV) mice for three weeks from five to eight weeks of age. All mice were killed at eight weeks of age and the gastrointestinal tracts were removed. The lengths of the small intestine were measured and six parts, i.e., the stomach; upper, middle and lower parts of the small intestine; cecum; and colon and rectum were separated, and each part (with contents) was weighed immediately. The contents were removed from each part by washing with distilled water. For the gut without contents, only total nitrogen(TN) was estimated and for the gut contents, TN, protein nitrogen(PN) and water insoluble nitrogen(WIN) were estimated. The fresh weight of cecum with contents per 100g of body weight of GF mice fed on AA diet, 2.07•}0.11g (mean•}SE), was lighter than that of GF mice fed on WE diet, which was 4.51•}0.28g. The weight and length of the small intestine of AA diet groups were smaller than those of WE diet group. TN and PN(mg) in whole gut contents per 100g of body weight in the WE diet
It is commonly known that in germ-free(GF) rodents or rabbits, the cecum, in particular, becomes greatly enlarged. However, the reasons for this are still not understood. If GF rodents or rabbits are conventionalized, the cecum becomes almost the same size as that of conventional(CV) rodents or rabbits in a short period of time. Intestinal microbes are undoubtedly responsible for this phenomenon, but it is not yet clear what kinds of microbe play a role in this.
The authors have already reported on the relationship between intestinal microbes and nutrition of mice using GF, gnotobiotic and CV mice (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The purpose of this investigation was to make clear the influence of the absence of intestinal microbes or differences in nitrogen(N) sources of diets on the appearance of the gastrointestinal tract or N of gut contents in mice. Analyses. Total N (TN) of the gastrointestinal tract and contents was estimated, and protein N (PN) and water-insoluble N (WIN) of the gut contents were estimated. N was estimated by the semi-micro Kjeldahl method. PN estimation was made by the Barnstein method. For WIN estimation, the samples were filtered using filter paper (Toyo No 5A) and N of residual matter on the paper was taken as the WIN in the gut contents.
RESULTS
The body weights in the experimental period are shown in Table 2. The body weight gains of GF and CV mice in the initial week showed almost the same values for both diets, but thereafter WEGF and CV mice showed lower values than those of AA diet fed mice. The food intake was not measured in this experiment but the diets were given ad libitum.
The fresh weights of the gastrointestinal tract (with contents) per 100g of body weight are shown in Table 3. The weight and the length of the small intestine of WEGF mice were greater (p<0.02) than those of other mice except for the weight of the small intestine of WECV mice. The cecal weights per 100g of body weight and per g of small intestine of WEGF mice were higher (p<0.05) than those of the other mice. The weight of the whole intestine of WEGF mice was WIN (p<0.01). When the small intestine was divided into three parts equal in length, the highest levels of TN, PN, and WIN were generally found in the upper small intestinal contents. The lower part showed the lowest values, irrespective of diets or absence of intestinal microbes. The TN of the contents in the upper part of the small intestine of WEGF mice was high (p<0.01) and the WIN of WECV mice was higher than that of the other mice (p<0.02). The TN of the contents of the middle part of the small intestine from WEGF mice was high, but there were no differences between the WECV and WEGF mice. PN in mice fed on WE was higher than that of mice fed on AA (p<0.01) and the WIN of AAGF mice was the lowest (p<0.05). The TN and PN of the contents of the lower part of the small intestine from WEGF mice showed the highest values (p<0.001). The WIN values of all mice were almost zero, but the WEGF mice showed high values (p<0.01). The TN of the whole small intestinal contents of WEGF mice was the highest (p<0.001) and the PN of WEGF and CV mice was higher (p<0.001) than that of AAGF and CV mice, but there were no differences between GF and CV mice fed on the same diet. The WIN were not significant as in the case of PN.
The TN, PN, and WIN of cecal contents of WEGF mice showed the highest values (p<0.05). The TN of colon and rectal contents of WEGF mice was high, and the PN and WIN of WECV mice was higher. In all of the gut contents from the stomach to the rectum, the TN of WEGF mice was the highest (p<0.001) and showed significant differences in each group. The PN also had the same tendency as TN, but there were no differences between AAGF and CV mice. The WIN was the highest in WECV mice. Generally, the amount of N in gut contents of GF mice was higher than in those of CV mice, and the WE diet group had a high value, as seen in Table 3.
In the case of PN per 100mg of TN of gut contents (Table 5), WECV mice showed high values (p<0.02) in stomach contents. The values decreased in accordance with passage through the small intestine, and in the lower part, low values were shown in all of the mice. In the whole small intestinal contents, CV mice showed values higher than those of GF mice. Cecal and colon and rectal contents of WECV mice showed higher values (p<0.001). In the whole intestinal contents from the stomach to the rectum, WECV mice also had higher values (p<0.02) than the other mice and the absence of intestinal microflora exerted more of an influence than did the difference in diets.
In the case of WIN per 100mg of PN of gut contents (Table 6), the cecal contents of CV mice was higher (p<0.05) than that of GF mice and the same tendency was also seen in the colon and rectal contents. In the whole intestinal contents, higher values were observed for CV mice than for GF mice.
TN of the gastrointestinal tract without contents per 100g of body weight is shown in Table 7. CV mice showed higher values than GF mice in the stomach. TN of the small intestine when divided into three parts equal in length was the highest in the upper part and the lowest in the lower part in all of the mice. The values for TAKA Table 4 Total nitrogen, the cecum of WE diet-fed mice were higher than those of the AA diet-fed mice (p<0.01), but there were no differences between GF and CV mice fed the same diet. From the stomach to the rectum, WECV mice showed the highest values (p<0.01).
DISCUSSION
In this experiment, AA or WE diets were given ad libitum for three weeks to male ICR strain GF and CV mice. In the first week, the mice showed the same body weight gains, but after two weeks, WEGF and CV mice showed gains lower than with the AA diet group. The reasons for above were not clear, but the WE diet had a different mixing order as compared with the AA diet, i.e., in the WE diet, the salt mixture was added to corn oil initially but in the AA diet, the salt mixture was added after other materials were admixed. In the previous experiments (9,10), there were no such tendencies even though the WE diet had the same composition as in this experiment.
Individual fresh weights of the cecum of six GF mice fed the AA diet (Table 3) were as follows: 1.76, 1.86, 1.97, 2.07, 2.26, and 2.50g per 100g of body weight. The fresh weight of the small intestine and whole intestine from the stomach to the rectum, and the length of the small intestine in AA diet-fed mice were smaller than those in WE diet-fed mice and the rate of constriction in the above three cases from WE to AA was almost the same in GF and CV mice. However, the cecum of AAGF mice was constricted to about half of that of WEGF mice. Fresh weights of the stomach and colon and rectum might be influenced by food intake or fecal excretion just before sacrifice. Conversely, there might be no direct effect on the weight of the small intestine and cecum, as when the weight of cecum per g of small intestine was calculated, the values for AAGF mice were closer to those of CV mice than to those of WEGF mice. The TN and PN of gut contents per 100g of body weight showed the same tendencies as did the results of the fresh weight of the intestine including contents. For example, in a comparison of the results of fresh weights in Table 3 and TN of the gut contents in Table 4 for the cecum, AAGF mice showed about fresh weights lower by 54% and content TN lower by 66% as compared with WEGF mice. However, the CV mice showed lower values of only about 21% for fresh weights and 7% for content TN. Next, in a comparison between GF and CV mice fed the same diet, the fresh cecum weight of AAGF mice was 1.8times that of CV mice, but in WE diet-fed mice, the figure was 3.0times. The TN of cecal contents of AAGF mice was 1.3times that of CV mice and in the WE group, it was 3.6times. From these results, it can be considered that the intestine, especially with regard to the cecum, of AAGF mice was constricted more than that with WEGF mice. Moreover, fresh cecum weights or TN of cecal contents per 100g of body weight of WEGF and CV mice in these experiments were almost the same as in the previous report (6) when the mice were given the natural type of CL-2 diet.
In the experiment with GF mice and rats by Sacquet et al. (12), the cecal weight differed according to the kind of diet. The weight of the small intestine of GF and CV chicks fed on natural diet was greater than that of purified diet-fed chicks, and GF chicks showed larger values than CV chicks fed on the same diet (13). Hashimoto et al. (14) reported that the cecal weight of antigen-free diet fed GF mice was 2.79-2.89g per 100g of body weight compared with 4.74-5.73g for autoclaved CL-2 diet-fed GF mice. The TN of each part of the intestine after removal of contents showed no differences due to the absence of microbes or differences in the N source in diets in this experiment. These were similar to the results of the TN of the cecal sac (6) and the TN of each part of the intestine (7) of mice given the autoclaved CL-2 diet.
From these facts, the enlargement of the cecum due to the absence of intestinal microbes can be improved by giving the AA diet without the aid of intestinal microbes. Animals were utilized after degradation of dietary N from protein to amino acids by the action of several enzymes. Enzymes for proteolysis had no opportunity to act except on the secreted endogenous PN in the gut when given the AA diet. It is well known that in GF rodents and rabbits, the intestinal walls are longer and the contents accumulate so that the transit time is slower than that in CV animals. One possible reason which can be considered is that certain materials produced by the action of enzymes on the food protein in the small intestine cause the enlarged cecum in GF rodents and rabbits. In CV animals, the above materials might be transformed by the action of intestinal microbes.
With respect to amounts of N in gut contents, GF mice showed higher values than those of CV mice and the WE diet group had higher values than the AA diet group. PN(mg) per 100mg of TN and WIN(mg) per 100mg of PN of gut contents were calculated to clarify the influence of the absence of intestinal microbes or the difference in N source in diets on N composition of gut contents. WIN might contain something which cannot pass through filter paper, such as hair, fragments from the intestinal mucosa or undigestible food residue. However, in the AA diet group, there was no undigestible food residue present because the PN or WIN of the AA diet could not be analyzed by the same method as used in this work. PN(mg) per 100 mg of TN of cecal and colon and rectal contents of WECV mice showed high values. This tendency agreed well with the results of previous experiments using natural-type CL-2 diet (7,8) but the values of AACV mice were almost the same as those of AAGF or WEGF mice. Next, for WIN(mg) per 100mg of PN of cecal and colon and rectal contents, CV mice showed higher values than those of GF mice for both diets. In the experiments of Chen et al. (15) on the nature of the gut contents where casein, zein, amino acid, N-free and gelatin diets were given to CV rats, N in the insoluble residue of gut contents of zein diet fed rats was especially high and casein and amino acid diet-fed rats gave low values. Tricholoroacetic acid-precipitable N of gut contents showed no differences among the diets. The N aspects of gut contents in this experiment showed several tendencies in various parts of the intestine, but in general, the absence of intestinal microbes had more influence than did the difference in N source in the diets. | v3-fos |
2019-03-19T13:08:22.401Z | {
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} | s2 | Development and productivity of timothy (Phleum pratense L.)
. The research series on the growth and development of timothy was comprised of a comparison between the three most important timothy varieties in Finland, Tammisto, Nokka and Tarmo in different parts in Finland. The second part of the investigation involved the greenhouse study of the development of the same three varieties together with the northern type Norwegian variety Engmo and southern type Swedish line Sv. 0873. The development and growth of the three Finnish timothy varieties had similar dry matter and protein production capabilities. The Tammisto variety exhibited rather demanding requirements for growth factors and had regression coefficients greater than one for both dry matter and protein production. The Nokka variety can be clas-sified as a general variety on the basis of its dry matter production, and a well thriving variety with a low level of growth factors on the basis of its raw protein production. The Nokka regression coefficients were approximately one and less than one for dry matter and protein production respectively. The Tarmo variety in regard to energy and protein production is considered a modest variety which had the lowest regression coefficient of the three varieties. Tarmo In later growing stages, however, these same two varieties developed faster than the others. The regrowth capabilities of the Sv. 0873 line and the Engmo variety were weaker than those of the Finnish varieties. The Engmo variety, suited for northern conditions, had the largest root production whereas Sv. 0873, bred for southern conditions, had the weakest. The Engmo variety’s large root production promoted fast LAI development. Seeding depth had significance only in the first cutting. The size of the regrowth yield was primarily influenced by the size of the previous cutting. The yields of consecutive cuttings were negatively correlated.
Introduction
Finland is located on the outer edge of the marginal zone for plant production. A short growing season, relatively low average daily temperatures and frequently occurring late summer rains indicate that Finland does not belong to the actual cereal growing region. On the other hand, long days, sufficient amounts of light, the economical water utilization of forage crops and relatively favorable temperatures make Finland ideal for forage crop production.
The most important forage crop in Finland is timothy. Because of its good winter hardiness it is grown throughout the entire country, even in northern Lapland. The long days and abundant incoming radiation of the Finnish growing season speed up timothy's development. Timothy tolerates the early summer dryness rather well and is not too sensitive to the autumn rains.
Timothy has only modest requirements as to soil types, and its growth declines essentially only below the pH 5.0 level.
The dry matter and raw protein production capabilities of different timothy varieties have been published in innumerable studies. Because it is apparent that timothy, as the best wintering forage crop, would remain as Finland's most important forage hay, it is considered necessary to collect the research results of different institutes and compare the yield producing capabilities of the most important timothy varieties over long periods of time. At the same time, with greenhouse experiments, it will be attempted to determine the growth and development rhythms from seeding to the second cutting for the most important domestic and two foreign timothy varieties.
The optimum moisture level for timothy germination is 60 % of the field water holding capacity (Williams 1954). The emergence capability of timothy decreases noticeably when planted deeper than 2 cm. Ahlberg (1964) showed that at planting depths of 1 cm, 2 cm and 3 cm the reduction in emergence was 2%, 13 % and 22 % respectively as compared to surface seeding. In practice, however, surface seeding does not produce as good results as when seeding at a depth of 0.5 -1.5 cm (Moore 1943).
The vegetative development of timothy:
Timothy is divided into three categories based on its growing characteristics and means of utilization, a) pasture type, b) medium type and c) hay type. The pasture type plant is short and leafy with good shooting ability and vigorous regrowth. The hay type is tall and stiff with abundantstem formation. The medium type lies between the former two types. The Finnish varieties are closest to the hay type (Raininko and Juuti 1975).
Timothy produces shoots most abundantly at relatively low temperatures (15-25°C) with anbundant light. According to Simola (1962), raising the soil moisture from 20-60 % of the field capacity increased the number of shoots from 1.4 to 7.4 units. All main nutrients increase the number of shoots, nitrogen beeing the most effective. Shoot production of timothy is most pronounced in the spring. As the growing season progresses, shooting decreases while regrowth assumes a greater significance than shoot production.
Leaf area index, LAI, is defined to be the total surface area of all photosynthesizing surfaces per unit area. It reflexes the stand's actual production capacity and the dry matter yield of the canopy is primarily defined by LAI (Kvet et al. 1971). Raininko (1968) proved that timothy does not tolerate shade well. Consequently, irrigation produces only a slight raise in the LAI value. Raininko (1968) also showed that the maximum LAI for timothy was achieved at a nitrogen level of 100 kg N/ha, despite that the dry matter production increased with increasing height as additional N-fertilizer was applied.
The maximum LAI for timothy is 6.5 (Langer 1958). At the maximum LAI the prevention of light dispersion in the stand is 95 %.
The regrowth capability of timothy is less than that of other forage grasses (Valle and Virtanen 1932). Better regrowth is achieved when the cutting is done at an early stage of development. Also heavy applications of nitrogen can produce greater regrowth yields from timothy. In Finland, however, foreign timothy varieties have better regrowth capabilities than domestic varieties. On the other hand the regrowth of the domestic varieties Tammisto and Tarmo is less than that of the Nokka variety. The better regrowth ability of Nokka is also noticed at a high level of nitrogen (Raininko 1970). The greatest regrowth of timothy occurs in the first year stand (Ravantti and Ravantti 1955). As the crop ages, the regrowth capability decreases.
The yielding ability of timothy: In northern Finland timothy can be considered as the most reliable cultivated plant (Pohjakallio andSalonen 1956, Huokuna 1970).
In southern Finland red clover has shown to produce the best yields (Valle 1947). This fact is supported by the study of Ravantti (1955). According to Ravantti and Ravantti (1955) and Antila (1973) timothy, when compared to other forage crops has succeeded only to a relatively limited extent. In Antila's investigation (1973) At low levels of nitrogen timothy produces as much dry matter (DM) as meadow fescue and orchard grass (Laine 1966, Huokuna 1970. Anothe factor affecting the forage yield is the number of cuttings. Laine (1966) showed that the yielding ability of timothy, when harvested as hay, was better than that of fescue and orchard grass. However, if cut more frequently for silage use, the yielding capability of timothy was less than the ones for the species mentioned.
At low level of nitrogen timothy suffered heavily when the number of cuttings was increased from two to three (Raininko 1968 The highest yield can be achieved with the combined effect of nitrogen and irrigation (North 1961). During the dry summer of 1973 Mela (1975) obtained a 26 % increase in the DM yield of timothy by irrigation 2 x 30 mm. Elonen (1974) had even more significant gains with irrigation. In Viikki Raininko (1968) obtained the following increase in yields with the combined effect of N and irrigation. The best hay yields of timothy are harvested from the first and second years' stand. Keeping a timothy crop for more than three years is not worth while (Mukula 1973). The present day management techniques with heavy amounts of nitrogen and stressing cutting frequency reduce the wintering abilities of timothy and lower the yields as the stand ages. Earlier, timothy was considered as a long lived plant (Essen 1913) which could be maintained at full production condition for up to ten years (Valle 1929). In the 1960's at the experimental stations of the Agricultural Research Centre yields were rarely obtained under intensive silage production from stands older than three years (Mela and Järvi 1972).
The raw protein production of timothy: The raw protein content of timothy at the early stage of development is higher than that of meadow fescue or orchard grass at the same stage (Valle 1947). Ravantti and Ravantti (1955) ordered the most important forage species according to their levels of productivity based on protein yield for the first year under southern Finnish conditions: red clover, white clover, alsike clover, alfalfa, red fescue, rye grass, orchard grass, meadow fescue, timothy and Kentucky blue grass. For the second year crop the order of productivity was: red clover, alfalfa, orchard grass, meadow fescue, red fescue, rye grass, timothy, alsike clover, Kentucky blue grass and white clover.
Nitrogen fertilization changes the plants' raw protein productivity as Laine (1966) The nitrogen fertilization does not increase only the protein content but the total dry matter yield as well. In this way the protein yield continues to increase even after the increase in DM yield has stopped (Salonen 1963).
The protein yield of timothy can be raised 9 12 % by applying nitrogen 10 20 days before cutting (Antila 1976). On the basis of Raininko's study (1968) it is apparent that it is most advantageous for protein production to cut timothy three times during the growing season. As more cuts are made then the nitrogen level must be raised.
Irrigation has proved to have a slightly negative effect on forage yield protein content at a low level of nitrogen and an extremely negative one as the nitrogen level increases. Nevertheless, irrigation has a positive effect on protein yield per surface area unit, because watering increases the total dry matter yield. Raininko (1970) achieved the best increase in protein yield by irrigation and cutting three times.
In Ravantti's investigation (1965) the protein yield of timothy rose right up to the third production year. In Teittinen's study (1958) the protein yield rose in the second but then fell in the third production year. Raininko and Juuti (1975) experienced the second production year to be better than the first. Variations appeared also in three and four year old timothy stands.
The high nitrogen level of 300 kg N/ha produced abundant increases in the protein yields among stands kept in production 3 or 4 years.
Materials and methods
The investigation of the growth and development of timothy was divided into two parts, a productivity comparison of the three most important timothy varieties in different parts in Finland and two growth and development studies of five timothy varieties in greenhouse.
The investigated varieties were Tammisto, Tarmo and Nokka. The yielding ability of each variety was compared to the average yield level of the studied varieties in the way described by Finlay and Wilkinson (1963). For comparisons between the varieties data were collected from studies where the three varieties occured at the same time (Kivelä 1978). In variety comparisons conducted by the Hankkija Plant Breeding Institute the three varieties have appeared simultaneously 35 times in tests during 1957 7O. In the Plant Husbandry Institute's tests at Tikkurila the dry matter yields for the three varieties have been measured concurrently 27 times during 1949 76. The dry matter yield tests for the three varieties were carried out 62 times in southern (Hankkija and Tikkurila locations) and 29 times in northern (Lapland Experimental Station) Finland. The comparison of protein yields from the three varieties is based only on 37 tests conducted under southern Finnish conditions (Kivelä 1978).
The greenhouse study involving test Series I and II was carried out in summer 1976. In both series the three domestic timothy varieties Tammisto, Tarmo and Nokka were used as test material. The comparative test crops were a Norwegian variety, Engmo, bred for northern conditions and the Swedish timothy line Sv. 0873, which can be considered suitable for southern conditions.
Serie I was seeded in 0.2 m 2 plastic boxes with four rows per box. The seeding rate was 20 kg/ha and the distance between rows was 12.5 cm. The growing medium was clay with an anbundant amount of organic matter. Watering occurred automatically and the amount of fertilizer applied was 250 kg N/ha. The stands in all eight replications were equally thinned. From each variety one row (30 cm) was cut when the stand height reached 5, 10, 15, 20, 25, 30 and 35 cm respectively. The leaf area index (LAI) and shoot and root yield were measured for each stand. The 24 h average temperature was 18°C and the average relative humidity 50 %.
Serie II was planted in round, 0.08 m 2 plastic pots. The seeding rate was 20 kg/ha, and the planting depth was 2 cm in the first four replications and 4 cm in the second four. The stands were thinned to 20 plants per pot. The watering was automatic and the fertilization was the same as for Serie I. The stands' growth and development were followed at weekly intervals with variety, Tarmo, was developed from southern Finnish material and was put on the market in 1948. The longlivedness of both Tammisto and Tarmo has been rather good under Finnish growing conditions (Manner et al. 1966).
The local strain, Nokka, originates from the Nokka estate in southwest Finland and has been developed trough selection of the best material over the last 80 years.
In the Hankkija Plant Breeding Institute tests for 1927-56 the varieties which had been in tests at least 4-5 years behaved as follows (Ravantti 1960): the local strain Haukila and the timothy variety Tammisto have yielded equally as much as Nokka. The Tarmo variety remained 4 % units below the yield level of Nokka.
All of the foreign varieties in the tests were either at the same level as Tarmo or lower. During 1957-63 at Hankkija the best Danish varieties proved to equal Nokka and Tarmo, but were 4-9 % units more productive than Tammisto (Ravantti 1965). In the same study the Swedish varieties were 3-lB % units more productive than Tammisto. On the other hand, the Dutch, English and Canadian varieties that were studied turned out to be less productive than the best Finnish ones. Raininko (1970) pointed out that in regard to the productivity of Finnish timothy varieties, Tammisto, Tarmo and Nokka are relatively equal at a low level of nitrogen. At higher nitrogen levels Tammisto exceeds both Tarmo and Nokka in productivity by 11 -l3 % units.
In the Agricultural Research Center tests conducted on timothy in the 1960'5, Tammisto, Tarmo, Nokka, Bodin, Bottnia, Engmo and Sv. 0853 all gave equally high forage yields (Mela and Järvi 1972). Bottnia II and Engmo produced less yield at the southern experimental stations than at the northern one in Rovaniemi. The Canadian variety, Climax, had very poor succes in northern Finland.
The varieties of Nokka, Tarmo and Hja 1160 produced slightly higher yields than the control variety, Tammisto, in the tests carried out at experimental stations during 1968-75 (Mela 1976 (Mela 1976).
Changes occur in the productivity of timothy varieties as the crop ages.
The reasons for these changes are primarily because of different development rhythms and differences in wintering abilities (Mela and Järvi 1972). Under southern Finnish growing conditions particularly Nokka's productivity increases whereas the productivity of the Canadian variety Climax decreases as the stand ages. Under northern Finnish growing conditions the productivity of the Tarmo, Bodin, Bottnia 11, Nokka and Apukka timothy varieties has relatively improved in comparison to the Tammisto variety in regard to stand ageing (Järvi and Mela 1976).
Results:
The average yields of the three timothy varieties (Tammisto, Tarmo, Nokka), as studied by Hankkija during 1957 -7O in 35 different field tests, differed from each other by only 230 kg DM/ha (Table 1). The Agricultural Research Center tests covering the years 1949-76 came up with exactly the same difference. The average yield for all varieties in southern Finland was 7 000 kg DM/ha with the deviation between the varieties' means being only 190kg DM/ha. In northern Finland the varieties produced an average of 1 000 kg DM/ha less than in southern Finland (Table 1). The difference among the varieties' means in northern Finland was very small, only 50 kg DM/ha. The weighted average yields of the three varieties for all of Finland represent a yield level of 6 700 kg DM/ha. Between the varieties there were no significant differences in yield (Table 1). The fact that the standard deviation of the Tammisto yield was greater than those of the Tarmo and Nokka varieties, at all test sites, ( Table 1) points out that, Tammisto is more sensitive to changes in environmental conditions than either Tarmo or Nokka. The regression coefficients for southern and northern Finland and the entire country, based on the test material, are equal and near b = 1, describing almost the same sensitivities to different The coefficient of variation (V) was smallest for the northern Finland tests and largest for the Tikkurila tests. The coefficient of variation for the whole country was about V = 30 (Table 1). There were no significant differences in dry matter production between the varieties.
1.2. Protein production at different yield levels
On the basis of the Hankkija Plant Breeding Institute studies (Raininko 1970) it appeared that at a low level of nitrogen (50-150 kg N/ha) Nokka timothy gave as high or even better raw protein yields than Tammisto, but at a high level of nitrogen (300 kg N/ha) Nokka produced less. In the same way Tarmo timothy competes well with Tammisto at a low level of nitrogen (150 kg N/ha) but falls behind at a high level (300 kg N/ha) (Raininko and Juuti 1975).
Results:
Comparison of the raw protein production capabilities of the investigated timothy varieties was made difficult because the raw protein content was determined for very few tests. In addition, the tests were comparised of different varieties resulting in a limited amount of materialfor use in this particular comparison. The material used in the regression analysis for the comparison of protein productivities consisted of, however, 22 tests during 1958-68 and 15 tests during 1949 -64 from the Hankkija Plant Breeding Institute and the Agricultural Research Center. Plant Husbandry Institute respectively where the three timothy varieties occurred simultaneously.
The average yields for the timothy varieties. Hankkija 720 740 kg protein/ ha. Plant Husbandry Institute 450-470 kg protein/ha and average 610-620 kg protein/ha were indexed for all three of the investigated varieties ( Table 2).
The standard deviation and coefficient of variation for Tammisto were higher than ones for either Nokka or Tarmo, and indicate the variability of the Tammisto variety as well as its sensitivity to environmental changes. Differences in the regression coefficients for the certain variety between experimental sites can come from differences in the degrees of management intensity (Tables 1 and 2). The overall regression coefficients of varieties in southern Finland illustrate that Tarmo and Nokka varieties thrive well at different yield levels. The regression equations for raw protein productition between the three investigated varieties and the average of the varietis are as follows (Fig. 2): Fig. 1 The relationship between the dry matter productivity of a variety (kg DM ha -1 ) and the average yield of varieties in 91 timothy variety trials during 1949-76 in Finland. Fig. 2. The relationship between the protein productivity of a vareity (kg prot. ha' 1 ) and the average protein yield of varieties in 37 variety trials during 1958-64 in Southern Finland. Table 2. Protein production coefficient of correlation (r) between the timothy variety and the mean of the varieties, slope (b), intercept (a), standard deviation (SDy), mean yield of the variety (y) and variation coefficient of the mean of the variety (V) in different part of Finland in [1958][1959][1960][1961][1962][1963][1964] On the basis of the Finlay -Wilkinson categories the Tammisto timothy variety represents a demanding variety whose dry matter and raw protein production regression coefficient is greater than 1 and yield level is high. The Nokka variety can be considered general variety for dry matter production and a thriving variety for protein production, which already at a low level of nitrogen has good protein production. The Tarmo variety represents a thriving variety for both dry matter and protein production as its regression coefficient is less than 1 and has a relatively high yield level.
According to Rekunen (1979) Hari and Leikola's (1974) study the stand's height growth in the beginning was accelerated. In the second stage height growth is at a maximum and continues for a certain time. The third stage is one of a slowed rate ot growth and ends with the plant reaching its greatest height. Of the factors effecting height growth only temperature is of essential significance in Finnish conditions (Olofsson 1962, Pohjonen and Hari 1973, Hari et al. 1977. According to Hari and Leikola (1974) temperature accounts for 75-96 % of the height growth variation. Han et al. (1977) claim that the height growth of the forage increases until the point where the light intensity falls below 50 % of full sunlight. According to Heikinheimo (1960) long days shorten the period of vegatative growth and hasten the start of generative growth.
The stem of timothy increases in height evenly up until flowering (Salo 1975). In general, Finnish varieties have significantly longer stems than foreign varieties brought to Finland (Teittinen 1958). The Hankkija Plant Breeding Institute tests showed no large differences in stem height between domestic timothy varieties (Ravantti 1965 Results: Nokka and Engmo had the fastest primary development of the timothy varieties tested in the greenhouse (Fig. 3). The Tammisto variety had very vigorous growth initiation, but decreased relatively as growth progressed.
Growth initiation for the Svalöv line Sv. 0.873 was slower than the three test varieties (Tammisto, Tarmo, Nokka) but was noticeably faster than these three varieties in reaching a stand height of 30 cm (Fig. 4). The greatest height difference in favor of Svalov's Sv. 0873 can be seen in the first and second cuttings of stands planted at the shallow depth (Table 3). On the three test varieties Tarmo exhibits the most even growth and Tarmo and Tammisto are the shortest ones although the differences between the varieties were few Nokka had the best regrowth (Table 3). Fig. 4. The plant height development of five timothy varieties between emergence and the first cut and between the first and the second cut seeded at the depths of 2 cm and 4 cm. Table 3. Plant height of five timothy varieties (cm) in the first and second cuts in the greenhouse tests seeded at the depth of 2 cm and 4 cm. Brown and Blazer (1968) the maximu pasture and hay production is obtained by maintaining the optimal leaf area index (LAI) which is associated with the highest annual yield and the desired growth division rather than with the highest momentary rate of growth. Efficient and beneficial use of LAI in forage production presumes that, in regard to yielding, the optimum LAI is reached quickly and divided evenly throughout the entire stand. The optimum LAI for timothy as indicated by Langer (1972) is LAI = 6.5.
The LAI generally describes the formation of the crops dry matter yield rather well (Donald and Black 1958). The time of occurrence of the growing season and the growing time influence the realationship between the leaf area index and the dry matter yield. Raininko (1968) obtained the following equation between the spring yield (y) and LAI (x), log y = 0.1385 X -f-3.1218. The equation shows that the yield defined by the LAI occurs in a very narrow area. Results: From the start the Norwegian Engmo variety had the strongest LAI development and the Tarmo variety had the weakest. The LAl's of these two varieties differed significantly (LSD.OS = 0.68) from each other already 16 days after planting (Fig. 5). Also the Tammisto and Nokka varieties experienced more rapid LAI development in the beginning than Tarmo. However, by the first cutting (55 days after planting) the differences had evened out and showed how the varieties had different growth rhythms (Table 4). For the first cutting there were no significant differences between varieties at the deep seeding depth and at the normal depth only the development of the Engmo LAI was significantly faster than the others. During regrowth the intervariety differences at the shallow depth evened out and at the deep depth only Engmo showed that deep planting had retarded its shooting (Table 4). In both the 4. 2. 3. Development of dry matter production Shoot dry matter production: The shoot dry matter production of Tammisto, Nokka and Engmo are almost identical (Fig. 6) and follow the shape of the height growth curve. The development of the photosynthetic mechanism in the Tarmo variety and Svalöv line Sv. 0873 is slow, but continues into a later stage than in those varieties where it develops faster, right up to the first cutting (Table 5). The foreign varieties, Engmo and Svalöv, when compared to the domestic ones have noticeably weaker regrowth capabilities, as is reflected in their morphological and physiological characteristics. The development of shoot dry matter production follows the relationships LAI development has to planting depth and cuttings. For the two cuttings there were no significant differences between varieties as to the above ground part of the total dry matter production (Fig. 8).
The varieties functioned approximately the same at the shallow and deep planting depths. Fig. 6. The relationship between the growing time and the shoot dry matter production (kg DM ha -1 ) in the early development of five timothy varieties. Root dry matter production: Initial root system development was the same for all varieties with the exception of Tarmo, which, as for height, LAI and shoot development, experienced the slowest development (Fig. 7). However, only Engmo and Tammisto had larger root systems than Tarmo at the time of the second cutting (Fig. 8). The great above ground increase in dry matter experienced by the Svalöv line Sv. 0873 occurred at the expense of root system formation. Engmo's. vigorous root development fits the classification, that it is suited for northern climatic conditions. At the shallow planting depth (2 cm) both the Nokka variety and the Svalöv line Sv. 0873 had significantly less root dry matter production than the other varieties. There were no significant differences between the varieties at the deeper depth (4 cm) (Fig. 9).
Shoot-root ratio: The shoot-root ratio of timothy varieties provides a clear picture of plant development after seeding (Fig. 8). In general, it can be said that if the regression coefficient is greater than 0.45, the relative rate of root growth is greater than the relative rate of shoot growth. The Svalöv line Sv. 0873 and the Nokka variety develop their root systems the most vigorously after planting and Tarmo the least. Root growth for Tammisto is almost in the same class as for shoot development (Fig. 8). In later stages the relationship between varieties may change, as is shown in Fig. 9. Fig. 7. The relationship between the growing time and the root dry matter production (kg DM ha -1 ) in the early development of five timothy varieties. Fig. 8. The relationship between shoot and root dry matter production in the growing phase between 5 cm and 35 cm of height for five timothy varieties. Fig. 9. Shoot and root dry matter production (kg DM ha" 1 ) of the end of the developmental period of 89 days after seeding and cut twice during that time. When the above ground yield of the two cuts is compared to the yield of the root system, it is observed that the root system develops better at a shallow planting depth rather than at a deep one. In addition, the differences between above ground dry matter production are smaller (Fig. 9). At the shallow depth only two varieties had above ground production which surpass below ground production. All five of the varieties had greater shoot than root production at the 4 cm seeding depth (Table 6). Engmo and Tammisto have the strongest root system development.
2. Discussion
In the timothy variety tests conducted by the Agricultural Research Centre during the 1960's (Mela and Järvi 1972), the Tammisto, Nokka and Tarmo varieties succeeded equally as well throughout the entire country.
The Norwegian Engmo variety, tested by the Plant Husbandry Department in five tests at Tikkurila, was 6 % units less productive than Tammisto, out more productive than Tammisto under northern Finnish conditions. The Svalöv line Sv. 0873 has been in variety tests in Finland during 1975 77. It is considered as a southern Finnish variety. Antila (1979) stated that in southern Finland Sv. 0873 was 2-9 % units better than Tammisto, but 10-14 % units worse under mid and northern Finnish conditions. The biggest advantage of the Svalöv line Sv. 0873 has been its good regrowth for a second cutting.
The greenhouse tests show that, despite growth rhythm differences, the three most important domestic timothy varieties produce approximately the same amount of dry matter in the first and second cuts as well as in total yield.
These results correspond with those of field tests (Raininko 1970). The foreign Engmo variety and Svalöv line Sv. 0873, because of their classification as hay types, produced more in the first cut than the Finnish varieties did. A significant observation was that planting depth was of importance only in the first cutting. The second cut's yield level was determined primarily by the production level of former cut. The correlation was negative. Of the five varieties investigated Engmo had the greatest root production as reflected by its good wintering characteristics and suitability for northern climatic conditions. Also a low shoot/root ratio describes these same features. Engmo's good root growth most likely also stimulated a faster LAI development than was found for the other varieties.
3. Summary and conclusions
The tests dealing with the growth and development of timothy were divided into two parts, a literature study and a greenhouse study.
In the literature study the success of the three most important Finnish varieties, Tammisto, Nokka and Tarmo, under various growing conditions was tested according to the Finlay and Wilkinson procedure (1963). The test material comprised 91 Hankkija Plant Breeding Institute and Agricultural Research Centre comparisons of timothy varieties.
The greenhouse study included the aforementioned three domestic varieties as well as the northern hay type, Engmo, and the more southern hay type, Svalöv line Sv. 0873. This part of the study was divided into two series: Serie I dealt with the primary development of the varieties and Serie II also included regrowth capabilities.
The following conclusions can be drown from the study: 1. All of the three most important Finnish timothy varieties had the same dry matter and protein production capabilities.
2. Of the three varieties, Tammisto represents the most demanding variety as it had regression coefficients greater than 1 for both dry matter and protein production.
3. The Nokka variety can be considered a general dry matter producer and a thriving raw protein producer. The regression coefficients were approximately 1 and less than 1, respectively.
4. The Tarmo variety is the only one considered thriving for both dry matter and protein production because it had respective regression coefficients less than 1 and a relatively high yielding level.
5. The greenhouse experiment showed that the Tarmo variety had the slowest development rhythm of the domestics, and Sv. 0873 the slowest of the foreign ones. The differences in development rhytms evened out by the time of the first cut.
6. The regrowth capabilities of the hay types, Engmo and Svalöv line Sv.
0873, were less than those of the domestic varieties.
7. Engmo, being best suited for northern climatic conditions, had the best root growth and the southern variety, Sv. 0873, had the weakest. Large root production represented rapid development of LAI.
8. When germinating, the planting depth had significance only in the first cut. The regrowth yield was primarily effected by the size of the first yield. The yields were negatively correlated. | v3-fos |
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} | s2 | Exertional myopathy in Finnish Landrace pigs. A survey of the situation and evaluation of different control methods.
511
I Introduction
Low stress resistance in pigs causes economic losses in pig production; when pigs are exposed to unusual environmental strain, unexpected death may occur. The cause of death can be the result of both physical and psychic strain (LUDVIGSEN 1954, RYLCKER 1968, JÖNSSON 1978, and JOHANSSON and JÖNSSON 1979. Death can occur for example during the transport of pigs to the slaughterhouse, in the slaughterhouse or when moving breeding pigs from one piggery to another. The strain of a fight can kill a pig, boars may die in mounting and sows in delivery. The effect of a high ambient temperature is also important according to WENIGER ct al. (1970), ELIZONDO et al. (1976), ANDREN (1977) and MALMFORS and NILSSON (1979). Some pigs are unable to adjust to these conditions. Their muscle glycogenolysis or anaerobic metabolism increases, resulting in acidosis in which large amounts of lactic acid accumulate in their muscles. A tonic cramp is induced in muscles and the body temperature increases uncontrollably. This malignant hyperthermia (MH) and the whole complex of symptoms, called the porcine stress syndrome (PSS), is fatal, often leading to death within 10-30 minutes TOPEL et al. (1968). Physical and psychic strain causes necroses in heart muscle, which could be the final cause of death (JÖNSSON et al. 1974, JOHANSSON et al. 1974and JÖNSSON et al. 1980. The pathogenesis of both MH and PSS has so far not been fully elucidated. Numerous theories have been put forward, and the common denominator for the primary cause of death is believed to be difficulties in mitochondrial oxygenation. This probably stems from either a defect in the internal enzyme activity of cells or a deficiency in the external hormonal function. Low stress tolerance is related to a reduced aerobic capacity (STEINHARD ct al. 1974, OLLIVIER ct al. 1975 and many other researchers reviewed by BICKHARDT ct al. 1977 andLUDVIGSEN 1980). According to LISTER et al. (1976), the temperature rise was mainly induced by aerobic metabolism in the muscles and only to a lesser extent by anaerobic combustion.
The mitochondrial calcium metabolism was disturbed (BRITT ct al. 1975 and VAN DEN HENDE 1978). OLLIVIER ct al. 1975, SMITH and BAMPTON 1977, WEBB andSMITH 1977and SCHMITTEN and SCHEPERS 1979 reported that the cause of stress susceptibility was due to an autosomal rcccssivcly inherited gene, which had an almost complete penetrance. MINKEMA et al. (1976), ANDREN (1977), MABRY (1978) and SCHNEIDER et al. (1980) believed that the penetrance was complete. WEBB (1981) summarised the investigations to obtain an 0.89 penetrance. When death is caused by MH/PSS the muscles become pale, soft and watery. The result is degeneration of the stressed muscles or PSE meat (LUDVIGSEN 1954). According to LANNEK (1975), PSE is the postmortal state of PSS. RYLCKER (1968) showed that physical training tends to prevent the development of PSS. In the meat industry it has long been known that colour and water-holding capacity of meat vary considerably in relation to the prcslaughter treatment of the animal SYBESMA 1968, BUTTENSCHON 1980). A physically exhausted pig with depleted glycogen depots yields meat with a high pH, a dark colour and a dry, almost jelly-like structure (DFD meat) because of high water-holding capacity. If the pig has ample glycogen depots at the time of slaughter and is exposed to an overwhelming variety of stressors, a rapid postmortal pH decline may ensue. The result will be meat with a low pH and a pale colour and wet structure PSE meat (WISMER-PEDERSEN 1979).
The formation of PSE meat is a typical quality defect in the pig and depends on weak adjustability of muscle cells (VAN DEN HENDE ct al. 1978). The disposition of the pig to develop PSE might depend on low adenosine triphosphatase activity at pH 6.7 in comparison to that of other slaughter animals. Other animals have a more efficient aerobic metabolism than pigs (BENTLER 1972). This meat quality defect depended 40-50 per cent on inheritance. These results have been summa-rised by SCHEPER (1979).
If meat quality is not considered in breeding it will become poorer because of a positive correlation between the amount of meat and poor meat quality (LUNDSTRÖM 1975. This correlation, however, is not so strong that the quantity of meat cannot be increased without imparing the quality (STAUN and JENSEN 1971, LUNDSTRÖM 1975. In addition to genetic characteristics, physical and mental stress as well as external circumstances will affect the slaughter quality of meat (SCHEPER 1979). According to SCHEPER (1979), the weather on the day of slaughter or on the day before slaughter will produce a 10 per cent effect on meat. The transport distance and the handling of the animals at the slaughterhouse before slaughter account for 15-57 per cent of PSE/DFD occurrence (SCHEPER 1971, NIELSEN 1980. A short preslaughter treatment is followed by ample PSE and a long by DFD quantity, depending on the fluctuation in the meat energy storage at the time of slaughter (WISMER-PEDERSEN 1979 andLISTER 1979).
Pale soft exudative (PSE) muscle death during transport and other exertional stress situations and the porcine stress syndrome (PSS) arc all caused by the fact that in stress circumstances energy requirements in muscle arc primarily met by conversion of glycogen to lactate. Both phenomena should therefore be grouped under exertional myopathy as proposed by BICKHARDT et al. (1972). The frequency of exertional myopathy varies gready according to breed. The syndrome is most common in the Pictrain breed and occurs more often in the Landrace breeds than in the Yorkshire or Flampshire breeds (WEBB 1980 a). When the genotype component is large, the weakness can be diminished by breeding when suitable methods arc available, by which individuals with undesired hereditary characteristics can be recognized. A number of such methods have been developed and proposed for inclusion in breeding programmes (CHRISTIAN 1972, RICHTER et al. 1973, RASMUSSEN and CHRISTIAN 1976, BICKHARDT 1979b, MCGLOUGHLIN 1980, ALLEN et al. 1980.
II Purpose of the present study
This study endeavours to 1. test the validity of some different methods for identification of stress-susceptible pigs and pigs which produce meat of poor quality (PSE/DFD), 2. clarify the frequency of exertional myopathy in pigs at the top of the breeding pyramid of the Finnish Landrace pig using the most valid methods available, and 3. propose methods allowing improved stress resistance and meat quality in the breeding of the Finnish Landrace pig irrespective of other important breeding aims.
111 Literature review of methods used for the identification of stresssusceptible pigs and pigs with poor meat quality (PSE/DFD) 1. The halothane test HARRISON et al. (1969) showed that halothane anaesthesia produced hyperthermia and depletion of muscle adenosinephosphate in stress-susceptible pigs. The halothane reaction has subsequently been used to predict the porcine stress syndrome (PSS) and the associated condition of pale soft exudative meat (PSE) (HARRISON et al. 1969, SYBESMA and EIKELENBOOM 1969, HARRISON 1972, EIKELENBOOM and MINKEMA 1974. The MH/PSS symptoms usually begin within two minutes after administering anaesthesia. WEBB (1980b) concluded that narcosis lasting at least three minutes was sufficient for the test. The most common symptoms are very typical. The body temperature rises, lactic acid accumulates in the muscles followed by acidosis, the pig contacts hyperthermia and a strong, usually tonic cramp. Death results if the anaesthesia is not discontinued quickly after the onset of the symptoms (HARRISON et al. 1969).
When narcosis is interrupted bcforc.MH/PSS becomes irreversible the pig will recover rapidly from narcosis. As a result the halothane test can be used to identify stress-sensitive hogs. The halothane test is comparatively harmless. EIKELENBOOM (1977) established that only 0.68 % of all tested animals died in the course of the test. The dead test animals developed MH to such an extent that a threshold was probably passed beyond which the process was irreversible. ANDREN (1977) reported that in his tests on more than 2,000 Swedish Landracc pigs 1 5 % reacted to halothane, causing the death of five animals. According to SCHNEIDER et al. (1979) three per cent of the halothane-sensitivc pigs died in the test.
The iterancy of the halothane test is good. WEBB and JORDAN (1978) verified a 5 % error in repeated tests compared to 9 % by WEBB (1980b). ANDREN (1977) observed that the iterancy was also good in Sweden, although occasionally a repeated test produced results aberrant from previous tests. CHRISTIAN (1972) established that the halothane test seemed to be more than 90 % accurate in diagnosing PSE. ELDIK (1975) stated that the feeding of test pigs ad libidum or with a restricted diet did not affect the results of the test.
The specific mechanism of the action of halothane in triggering the syndrome is by no means clear (LISTER 1979). The site of action of halothane appears to be within the skeletal muscle itself KALOW 1970, MOULDS andDENBOROUGH 1974). According to KITTLER and DZAPO (1978), the halothane reaction docs not correlate with the general vitality of the animals, but does with meat quality. Smaller litters were in any ease borne to halothane-positive sows. WEBB and JORDAN (1978) observed that the piglets of halothanc-scnsitivc pigs were inferior to those of halothanc-resistant pigs. The halothane test has been applied by numerous researchers in many countries to different pig breeds (ALLEN et al. 1970, EIKELENBOOM and MINKEMA 1974,ANDR£N 1977, FROYSTEIN et al. 1977, WEBB and JORDAN 1978, MABRY 1978, GERWIG et al. 1979, MCGLOUGHLIN et al. 1979, GINDELE et al. 1980, SCHÖNNICHSEN et al. 1980, BAUMGARTNER et al. 1980and EIKELENBOOM 1981. WEBB (1981) has drawn up a table based on the results obtained by several investigators showing the fluctuation in halothane reactivity in different breeds (Table 1). Halothane-sensitive pigs differ from pigs not reacting to halothane in many traits related to performance. Pigs possessing the halothane gene (Hain) have more frequendy comparatively more meat on their carcases than do pigs lacking this gene (EIKELENBOOM ct al. 1978a, MABRY 1978, GERWIG et al. 1979, LUNDSTRÖM et ai. 1980, SCHNEIDER et ai. 1980. WEBB (1980b) has also summarised results obtained by a number of investigators regarding differences in performance traits in halothane-sensitive and halothane-resistant pigs (Table 2). WEBB (1980b) established that the halothane test should be conducted on pigs of 7 weeks of age or older. In younger pigs the test reveals a lack of halothane sensitivity. (1977); (21) Webb (1980a); (22) Webb and Jordan (1978). 1. Webb and Jordan (1978) 2. Eikelenboom (1977) 3. Christian (1977) 4. Wagner and Pasterling (1977) 5. Vcrstegen and others (1976( ) 6. Vdgeli (1978 7. Monin and others (1976) Several investigators have shown that halothane-scnsitive pigs also sustain other strains and that they more frequently develop PSE meat (EIKELENBOOM and MINKEMA 1974, ELDIK 1975, ANDREN and PERSSON 1977, WEBB and JORDAN 1978, EIKELENBOOM ct al. 1978b, JENSEN 1978a, MABRY 1978a, BRASCAMP et al. 1980. Mortality during the fattening period and the transport of the animals to the slaughterhouse was nearly ten times higher in hogs reacting to halothanc than in Dutch Landracc pigs with no reaction to halothane (5.27 % vs 0,56 %). Of those reacting to halothanc, 83 % developed more or less clearly PSE meat as opposed to only 36 % of the hogs not reacting (EIKELENBOOM et al. 1978 b). According to JENSEN (1978a), Danish Landracc halothanc-positive pigs had paler meat than halothanc-negative pigs, the meat quality measured in KK value being 1.49 units lower. EIKELENBOOM ct al. (1978a) proposed that the halothanc test was most effective for minimization of strcsssusccptibility and abnormal meat quality in the breeding and selection of Dutch Landracc pigs. This may not hold true for the Yorkshire breed, however. In the Yorkshire breed no correlation was found between halothanc sensitivity and poor meat quality. MARBY (1978) showed experimentally that the same conditions applied to the Yorkshire breed as to Landracc hogs. Halothane-scnsitive pigs produced smaller litters than the halothane-resistant pigs, and piglet mortality was greater for halothanesensitive than for halothane-resistant sows (SCHNEIDER et al. 1980).
Exercise tests
Poor stress resistance is most commonly revealed through the strain caused by transport. Many different forms of transport have been experimented with in investigating exertional myopathy. Test pigs have been transported over short and long distances and for different lengths of time in different temperatures, and have also been exercised on tread mills. The consequences of this strain has been analysed by determining blood enzymes which indicate the slaughter quality of the meat or by examining the meat after slaughter. Strain, its duration and temperature all act to affect the quality of meat to a great extent (RYLCKER 1968, RICHTER et al. 1976, ELIZONDO et al. 1976, LOVE et al. 1977, KALLWEIT and FEHRENTZ 1977, LUNDEHEIM 1977, BICKHARDT 1979, PERSSON ct al. 1979, MOSS 1979, BICKHARDT et al. 1980, BUTTENSCHON 1980. No single applicable exercise test specially designed for breeding programmes has been developed, but examinations have demonstrated that standardizing the strain before testing is essential in order to reveal the significance of pig genotype when using blood enzyme activities or meat quality as measurement tests (BICKHARDT 1970, BARTON et al. 1977b, PEDERSEN 1979, MOSS 1979, PFEIFFER et al. 1979).
Blood group systems
A survey of immunogcnetics and biochemical genetics as tools in pig breeding is presented by GAHNE (1979), and the blood groups of pigs and their determinations are summarised by ANDRESEN (1963). The association between genotypes of the H blood group system and the porcine stress syndrome as detected by the halothanc test was discovered by RASMUSSEN and CHRISTIAN (1976) and has been confirmed by many other workers (e.g. HOJNY ct al. 1979, IMLAH and THOMSON 1979b, ANDRESEN and JENSEN 1980, JÖRGENSEN 1980. Investigations by JENSEN ct al. (1976) andBARTON ct al. (1977) have indicated association between genotypes of the H blood system and the porcine meat colour score. The results show a significant correlation between inferior meat quality and the presence of the H a allele. However, since PSE and PSS do occur in H (a-) and H~H~individuals no direct causal relationship appears to exist between the H a allele and exertional myopathy. The frequency of the H a allele is lower in the Yorkshire breed that in the Landrace breed. The meat colour was also darker for the Yorkshire than for the Landrace breed according to IMLAH and THOMSON (1979).
There was a clear relationship between the H a allele and the colour index in Landrace pigs. Individuals with H a had a 13.4 % poorer meat colour compared to the mean value, but this correlation could not be significantly demonstrated in the Yorkshire breed (IMLAH and THOMSON 1979). MABRY (1978) on the other hand showed that a relationship between poor meat colour and the halothane reaction and H -blood groups existed in Yorkshire pigs. The relationship between halothane positivity and the H a allele also depended on the A blood group system (JORGENSEN et al. 1976, MABRY 1978, IMLAH and THOMSON 1979. In H a/a pigs, which were devoid of the A system factors A and O, and in H-/ pigs, which had cither Aor O, a clear relationship to PSS was defined by the halothane test. MABRY (1978) found that two blood types, (+, -/-) and (-, a/a), were consistendy stress susceptible, while three blood types, (+, a/a), (+, a/c) and (+, c/), were stress resistant. One blood type, (+, a/-) contained, however, both stress-susceptible and stress-resistant individuals, (+) indicating hemolysis for either A or O. MABRY (1978) also discovered that stress-susceptible animals were inferior in reproductive ability, mothering ability and preweaning growth, and that stress-positive animals were significandy more heavily muscled with larger eye muscle areas. In addition, stress susceptibility had a negative effect on muscle quality as positive pigs exhibited significandy paler colour, less marbling and greater transmission values compared to stressnegative animals. It was possible to predict correcdy 84.1 % of the stress-susceptible and 79.6 % of the stress-resistant pigs using blood group factors in the H and A blood group systems (IMLAH and THOMSON 1979). According to JENSEN et al. (1976), the H a allele could explain about a quarter of the PSE problems in Danish Landrace pigs. Results obtained with one Landrace breed cannot be direedy applied to another Landrace breed. MAJOR (1968) investigated blood relationships between different populations of Landrace pigs in Denmark, the Federal Republic of Germany, Holland, Hungary, Sweden, Czechoslovakia and the Soviet Union and found differences between all these populations. He distinguished between two different subgroups, one consisting of the Landrace of Germany, Holland and Hungary and the other of Danish, Swedish, Czechoslovakian and Soviet Landrace.
Glucosephosphate isomerase (Phi) types
The PSS detected by the halothane test seems to be associated with the polymorphic erytrocytic enzyme system glucosephosphate isomerase (EC 5. 3. I. 9.) (JORGENSEN et al. 1976). This enzyme is defined by two codominate alleles (A and B) which produce three different phenotypes (AA, AB and BB). JORGENSEN et al. (1976) showed that Phi®® was present in all halothanepositive Danish Landrace pigs. JORGENSEN (1977) additionally demonstrated the connection between the halothane reaction, the H blood group and Phi. ANDRESEN (1971), ANDRESEN and JENSEN 1977) and JORGENSEN (1980a) showed that all these loci are present in the same chromosome and arc closely linked to each other. The Hal locus is situated between the Phi and H a loci (ANDRESEN 1980 a). In breeding, these characteristics are uncomplementary, but in fact they measure the same trait from different dimensions (JENSEN 1978 b). It has been shown that in the Danish Landracc breed, pigs having the poorest meat quality were within the group of animals having both H a and at the same time JENSEN 1979). ANDRESEN (1980b) found that by using the H system and the Phi system parallelly in breeding, the meat quality in Danish Landrace pigs could be improved without needless loss of many H a pigs.
; s> however, so common in the Danish Landracc that it seems nearly impossible to eradicate it from this breed (ANDRESEN ct al. 1979 andANDRESEN 1980 c).
Creatine kinase (CK) test
Many serum enzymes such as creatine-kinase, lactic-dehydrogenase and glucosc-6-phosphate have been tested and summarised by PFEIFFER et al. (1979). Greatest interest has focused on creatine-kinase (CK) (EC 2. 7. 3.2.) (UNSHELM 1971, ADDIS ct al. 1974, BEERMAN ct al. 1975, BICKHARDT ct al. 1977, HWANG ct al. 1978, BICKHARDT 1981. The CK activity in blood has to indicate muscle injuries very specifically, because it is found in abundance just in muscle tissue (BICKHARDT 1970(BICKHARDT , 1979(BICKHARDT and 1981. Many investigators have discovered a negative correlation between CK activity in serum or in the plasma of unstressed pigs and meat quality parametres (UNSHELM 1971, SCHMIDT ct al. 1971, SCHMIDT et al. 1974, BEERMAN et al. 1975, WETTERMAN 1975and WAX et al. 1975), but after standardizing strain the correlations were even higher than before for the same animals (BICKHARDT 1970, ELIZONDO et al. 1976and HWANG et al. 1977. CK should be analysed about eight hours after standardized exercise (BICKHARDT and RICHTER 1980). The serum CK values achieved maximum levels 10-20 hours after physical strain or muscle injury STEINESS ct al. 1978). The CK activity after standard exercise follows an approximately logarithmic distribution. An increase in CK activity after exertion was higher in pigs predisposed to exertional myopathy than in stress-resistant pigs (MAXWELL ct al. 1976). It would seem that the increase in CK activity in plasma after physical exertion can be attributed to enzyme escaping from skeletal muscle, following metabolic disorders in the muscle fibers. There was no indication that increased CK activity in plasma was a result of intensified enzyme synthesis. CK activities found in serum were identical to those found in plasma ). In pigs with manifested exertional myopathy where morphological evidence of degeneration and necrosis of the muscle fibers was documented, serum CK activities up to 333 wkat/1 were observed for several days. Blood samples obtained from the vena cava were suitable for CK measurement, but the fact alone that the pig had to be held steady for the blood sample to be taken caused an increase in CK activity in the plasma on the order of 10 %, attributable to hacmoconcentration (BICKHARDT ct al. 1979). The CK activity determination and the reliability of the CK test in the investigation of pig exertional myopathy have been reviewed by BICKHARDT ct al. (1977). BICKHARDT et ai. (1980) experimented with many different exertion tests before measuring the CK activity and recommended the administration of 8 mg of neostigmine-atropine mixture parenterally before performing the CK test. Even this exertion test could cause the death of some stress-sensitive pigs. Earlier BICKHARDT (1970) considered that a walk of a distance of 100 metres and weighing the pigs 24 hours before the CK test represented suitable standardized strain.
The heritability of the CK activity in serum was about h 2 = 0.3. This was somewhat lower than the parametres measuring meat quality (FLOCK 1968). According to WATANABE et al. (1978), there existed only a small degree of correlation between scrum CK activity in resting pigs and meat quality measurements. BICKHARDT et al. (1977) demonstrated that the phenotypic correlation between scrum CK activities and meat quality parametres was r =0.4. As a negative phenotypic correlation between the CK activity of dams and the meat colour score of their daughters (r = 0.34) was apparent, this test was used in a breeding programme for four years. The growth rate in pigs improved and the amount of back fat thickness diminished during this time without impairing the quality of meat ).
The results of the CK activity determination from scrum obtained by automatic enzyme analyses or with the Luciferase method were very consistent (ANTONIK 1977). Occasionally observed extremely high values in pigs might anyhow complicate the use of automatic methods in determining CK activities . The heritability of the CK activity in serum was 0.73 for the Landrace pigs and 0.37 for the Yorkshire pigs, according to SCHWORER et al. (1980). BORGMAN et al. (1978) showed that the feeding level of the pig did not affect the results. When the test material was grouped so that the halothane positive and negative or PSE meat producers and good meat producers were separated into groups, a large deviation could be observed within all groups. At the same time however, a significant discrepancy was observed in the mean values for the different groups in that the mean values for halothane-reacting and PSE meat producing groups were larger than those for the halothane-resistant or groups producing good meat HWANG et al. 1978). The serum CK activity was dependent on the age of the pig and its physical activity. At the age of 1-3 months it was relatively stable . The applicability of the CK test in distinguishing stress-resistant and stress-susceptible pigs improved, however, when determinations were iteratively conducted for the same animals (THOREN-TOLLING 1980).
When the CK test is used in breeding, an elimination limit must be set. Because of the large fluctuation in CK values a constant problem will be the presence of both false positive and false negative results. The range of the elimination limit naturally depends on which of these results is more harmful. HWANG et al. (1978) established the elimination limit at 6.66 /tkat/1 in investigations of Pietrain hogs, because they obtained the mean value of 6.60 ixkat/1 for halothane-resistant Yorkshire pigs. BICKHARDT et al. (1979) set the elimination limit at 29.6 /tkat. SCHMIDT et al.
(1974) did not find clear relationships that would allow the CK test to be used as a predictor of meat quality in live pigs.
6. Meat quality measurements At least three different procedures are available for measuring stressed meat. It is possible to determine meat colour, water-holding capacity or pH. The meat colour determination is the one most frequently used. It has been technically the easiest to apply in routine analyses as compared to WHC and pH determinations. The determination of pH itself is relatively simple, but the pH has to be measured within a specified period of time after slaughter, which can create some difficulty. The colour measurement correlates well to other meat quality characteristics, such as pH and WHC. Its heritability is relatively good, and there are some reliable determination methods available (JENSEN 1978 a, LUNDSTRÖM et al. 1979).
Meat colour
The meat colour can be evaluated cither subjectively (CLAUSEN and THOMSEN 1956) or measured by reflectometres currently in use. The reflcctomctre measures the amount of light reflected by the meat surface; the higher the reading, the lighter is the colour of the meat. The meat colour correlated inversely to the carcase meat content, which is why one-sided breeding for more meat impairs meat quality . The correlation was, however, not so good that the meat content could not be improved without impairing meat quality (JENSEN ct al. 1967, STAUN and JENSEN 1971, LUNDSTRÖM 1975. Meat colour is dependent on two factors, namely the amount of meat pigment and the structure of the meat. Poor meat structure is mainly due to PSE meat (JENSEN 1978 a). According to the results obtained by many investigators, the heritability of meat colour varies between 0.05 (ALLEN ct. al. 1966) and 0.55 (PEASE and SCHMITH 1965), the average being 0.3 (reviewed by JENSEN, 1978 b). Danish researchers showed that the inheritability improved when meat colour was measured with pretreated meat. This pretreatment, or light curing, improves meat quality. This observation indicates that meat pigmentation depends more than meat structure on the genotype . Danish investigators have developed a meat quality index called the KK value (BARTON 1974, PEDERSEN 1979. The KK value consists of colour readings from both fresh and cured meat, corrected when necessary with the pH measured 24 hours after slaughter (pH 2 ). The inheritance of the KK value was calculated to be 0.5 (VESTERGÄRD 1977). The meat colour reading correlated well to other exertional myopathy meat quality measurements (LUNDSTRÖM et al. 1979). Because many external factors, such as transport and the whole preslaughtcr treatment, also affect meat colour to a great extent, these must be standardized in the best possible manner (LUNDSTRÖM et al. 1979, BARTON 1974. Even the stunning method affects the meat quality of stresssusceptible pigs (WAL 1971, HAMM 1972. The standardization of preslaughter treatment for Danish test pigs has been summarised by Barton (1974).
1, On the day of slaughter pigs are fed a reasonable amount of feed but are not weighed. 2. Loading utilises a hydraulic pig lift.
3. Transport, lasting about 40 min., uses a specially-designed lorry equipped with a non-slip floor, partitions and mechanical ventilation. 4. After transport the hogs are brought directly to the stunning room without using electric «whip or other means of force. 5. Stunning is performed with electricity on the floor.
In Sweden the meat colour reading was taken as a minimum value in breeding selection. Colour was measured at three different points from fresh cross-sections of the M. longissimus dorsi, and the mean reading value obtained was used as the meat colour reading. Since 1. 4. 1979 attempts have been made to eliminate the share of external factors affecting the meat colour readings by selectively using deviation instead of the total reading value. Sires of elite boars are allowed a maximum of three points deviation from the mean value, and sows seven points deviation from the mean colour value of animals slaughtered at the same time. The error caused by DFD meat is eliminated by evaluating the meat as PSE meat in the case that pH 2 (LUNDSTRÖM et al. 1980). Colour reading inheritability is high and it correlates excellently to the halothane test and blood group information. Parallel observations of the halothanc test and blood group determination in breeding contributes only very little when compared to selection based solely on colour reading (JENSEN 1978 b). In Denmark meat quality improvement has for this reason been based on the KK value alone since 1972 (PEDERSEN 1977 and.
Water-Holding capacity (WHC)
Watcriness is a characteristic property of PSE meat. This condition is not caused by large amounts of water but rather by reduced water-holding capacity (WISMER-PEDERSEN 1959). The poor water-holding capacity of PSE meat is a problematic quality property for the meat industry (PUOLANNE 1980). NIINIVAARA and POHJA (1953) observed that the meat water-holding capacity is dependent on the meat pH. The lower the pH from the isoelectric point of meat, the poorer is the water-holding capacity, and vice versa (VEIJOLA 1980). Several methods have been developed to measure the meat water-holding capacity. WEISS (1967) and RYLCKER (1968) centrifuged the bulk of meat. According to WEISS (1967), the WHC heritability was 0.54 among boars and 0.26 among sows. WENIGER et al. (1970) obtained a value of 0.57 for boars and 0.59 for sows by using a compression and filter paper method developed by HAMM (1953 and. NIINIVAARA and RYYNÄNEN (1953), GROSHE ct al. (1975) and LUNDSTRÖM et al. (1979) have also used the filter paper compression method.
According to ALLEN et al. (1966), the heritability value varied between 0.48 0.77 for the Duroc and Yorkshire breeds. STAUN and JENSEN (1971) obtained a heritability value of 0.14 for boars and 0.29 for sows when using the compression method. JENSEN ct al. (1967) obtained a value of 0.63. In breeding the colour reading is more widely applied than the WHC determination, although the measurement of WHC should constitute a suitable criterion for the selection of meat quality (LUNDSTRÖM et al. 1979, MALMFORS 1981.
Meat pH determination
The pH of PSE meat was significandy lower than the pH of normal meat when pH was measured 2 5-45 minutes after slaughter. When measuring pH 24 hours after slaughter, no difference was found (TAYLOR 1 966, JENSEN 1978 a). When the pH was determined 3/4 of an hour after slaughtering a strong correlation between the pH and meat colour was observed. JONSSON (1965) established this correlation to be about 0.7. It was discovered that the same difference exists in meat pH of halothane-positivc and halothane-negativc animals as exists between normal and PSE meat (EIKELENBOOM and MINKEMA 1974). WISMER-PEDERSEN (1959) has proposed the term pH, for the pH obtained 45 minutes after slaughter and pH 2 for the pH obtained 24 hours after slaughter. Adopting a meat colour reading for the identification of PSE meat is useful but not for recognizing DFD meat (JENSEN 1978 a). In DFD meat the pH drop is very small, even when compared to normal meat. The pH 2 of DFD meat is significantly higher than the pH 2 of both PSE and normal meat, i.c. 6. Inbreeding selection in general, pH, determination is substituted by meat colour measurements, but in addition to colour reading the pH 2 should be taken in order to recognize DFD meat. This is especially important when selection is based on mean values obtained from two to four test hogs slaughter estimations b, KANGASNIEMI 1980). According to WISMER-PEDERSEN (1980), it was not possible to state exact pH limits for detection of PSE, normal and DFD meat.
IV Conditions in Finland
In pig breeding in Finland, it has been purposefully endeavoured for more than two decades to reduce the amount of fat, mainly the back fat thickness, while increasing the amount of lean meat on the carcase with the intention to gain more back meat and ham. At the same time, feed efficiency and growth rate has been emphasized (HYVÄRINEN 1980). Until 1971 experimental hogs were slaughtered immediately after transport to the slaughterhouse, but when PSE meat emerged as a problem, this practice was changed, and the pigs were allowed to rest overnight before slaughtering. The PSE problem has since been reduced (KANGASNIEMI 1974). More attention has been paid to the quality of lean meat since the beginning of 1960. In progeny testing meat colour was first measured subjectively from a fresh surface cut from the M. longissimus dorsi. Then a supporting meat colour scale was obtained. In Finland the British colour slide was used. Since 1972 the colour reading was published in connection with test results, but the meat colour was not included in the official breeding programme (PARTANEN 1980). Since the beginning of 1977 the colour reading has been measured from a M. longissimus dorsi fresh cross-section surface by reflcctometrc. The results obtained from colour readings have ranged from 20-60 points, where 40 points indicates borderline meat and more than 45 points PSE meat (KANGASNIEMI 1978). Meat from Landracc hogs is lighter and the dispersion of the colour reading is wider than meat from Yorkshire pigs. During the summer higher readings are obtained in slaughtering than during the winter. Seasonal differences have been of the same order of magnitude than differences in breed (KANGASNIEMI 1978). The heritability based on metre readings was calculated to be 0.37. The genetic correlations between colour readings and carcase quality properties were verified from the same material to be -0.28 for the back fat thickness, 0.39 for the M. longissimus dorsi cross-section area and 0.36 for the percentage of meat on the carcase (KANGASNIEMI 1978).
The Finnish Animal Breeding Association's Section for Pig Breeding imposed a threshold limit for meat colour in the spring of 1977. At first the threshold was set at 41 points, but because colour readings obtained from groups produced during summer were remarkably higher than readings taken in the winter, the threshold was increased to 44 points. The threshold is currendy 44 points, but for groups produced during summer it is 46 points (Anon. 1977 and. From groups produced in 1977, 11,6 %of the Landrace and 0.7 %of the Yorkshire exceeded the colour reading point of 44 (KANGASNIEMI 1979). The test groups in progeny testing consist of four pigs, two castrated and two sows.
The colour readings for the test groups have deteriorated in both breeds during (KANGASNIEMI 1980. Sudden death of normal bacon hogs during transport and in slaughterhouses before slaughter is not infrequent. In 1978, 2,320 bacon bigs died at Finnish abbatours in this way. The same year 465 carcases were rejected because of PSE meat (Anon 1979 b). Readings obtained with the reflectometrc have not been corrected with pH 2 determinations. The importance of pH determinations has in any case been acknowledged, and investigations of including pH 2 in breeding programme judging was begun in 1980 (KANGASNIEMI 1980). According to Alho (1980), colour reading determinations from test pigs were occasionally taken on three hour old cross-cut meat surfaces. This delay might raise the colour reading because muscle myoglobin has probably pardy changed to lighter oxymyoglobin (PUOLANNE 1980). From the beginning of 1980 colour is measured immediately after the muscle has been cut, and from the beginning of 1981 measuring of pH 2 will be included in the programme (KANGASNIEMI 1980). Alho (1980) also verified that the transport distance for test hogs from progeny testing stations to slaughterhouses was roughly the same in five cases, but very short in one case (80 km versus 4 km). The pigs were kept in slaughterhouses overnight before slaughtering. The walking distance from the pen to the stunning area varied at different slaughterhouses. The greatest variations in pre slaughter treatment was due to the fact that in one slaughterhouse very many test animals were crammed together in the same pen, while in other slaughterhouses pens were more spacious.
Since 1977 the halothane test has been used at phenotype test stations (SALONEN 1980, PAÄLLYSAHOI9BO). After this boars which have reacted to halothane have not been approved for AI use.
V Material and methods
The material was selected so that all Finnish Landrace elite breeders were requested to test pigs in litters from which breeding animals were to be chosen. The elite units were asked to choose new breeding animals for their own use only from tested animals during 1979. In Finland pig breeding takes place principally in elite units ap-proved by the Finnish Animal Breeding Association. In 1979 there were 22 Landrace elite units. The breeding value of the animals is studied by performance testing at farms and by progeny testing at progeny testing stations. The AI boars are further performance-tested at special testing stations.
The halothane test was voluntary for pig breeders. The largest number of tested pigs in one herd consisted of 404 pigs from 7 5 litters, and the smallest of 16 pigs from three litters. With one exception, all elite units collaborated in the operation. The smallest elite unit was excluded on the grounds that in 1979 new sows were not chosen for its breeding purposes. In addition to the elite units, ten of the most important selected Landracc units participated. Of these, some sows left for the units' use were tested, but most were boars with a breeding value high enough to attract the commercial interest of elite units. Pigs from these same herds sent to testing stations for progeny testing were tested at the stations. In this way a direct comparison between results obtained in the field test and the slaughter quality properties of the same animals was obtained. Above all it was of great interest to compare the slaughter estimation values of meat colour readings and the percentage of lean meat on the carcase with the results of the field test. The final material consisted of 2,003 Finnish Landracc pigs selected from 21 Landracc elite units, ten Landracc selected units and three testing stations. In addition 63 Norwegian Landracc pigs from the Trondheim area were tested. This material was included for the reason that Norwegian Landracc pigs were brought from Norway with the intention to breed them with the Finnish Landracc breed. All pigs excluding those in progeny tests at pig testing stations were tested in the piggeries where they were born. The age of the pigs varied between three and 23 weeks, the mean age being nine weeks. Both sexes were represented, amounting to 761 males and 1,242 females. The whole material represents the top of Finnish Landracc breeding. The tested pigs originated from 525 litters and were siblings of 1 5 4 boars.
No special measures were taken in feeding or care before the test. The fodder was generally a dry commercial complete feed, but also partly a home-mixed dry feed was used. Test pigs were handled by their keepers and assistants.
Material processing and statistical methods
The results from the halothane test, H blood group typing, Phi-type determinations, measurement of CK activity in serum, meat colour and WHC measurements were compared to each other as well as to the K index and to the percentage of lean meat on the carcase. The data were analysed using the computer at the Department of Animal Breeding in the Finnish Agricultural Research Centre. In the presensation of the results, the number (n), the percentage of total material (%), the mean value (x) and standard deviation (S.D.) are given. Differences between means were tested for significance using the "t" test or the Chi-square method.
Calculation of method error was performed in replicates. The results of the repeated halothane test appear in table 3, and CK test results in tables 4, 5 and 6 and in appendix 1. The standard deviation of the method error from duplicate mcasurc-ments was calculated using the formula S= ± y -j~, where i is the difference between the two duplicates and n the number of samples. Method error is given as the coefficient of variation. In the statistical test, the degree of significance is stated as follows: n.s. = not significant p > 0.05 4-= significant at the 5 % level p < 0.05 4-4-= significant p < 0.01 4-4-4-= highly significant p < 0.001 Other abbreviations used are: Hal -= halothanc-scnsitivc pigs Hal-= halothane-resistant pigs Hallitter = litter where at least one Hal+ pig was discovered Hal-litter = litter where no Hal-pigs were discovered Halsire = sire from whose siblings at least one Halpig was discovered Halsire = sire from whose siblings no Halpigs were discovered 2. Production quality measurement The K index and the percentage of lean meat on the carcase were used as the dimensions for production quality. Growth rate, feed efficiency and meat colour deviations from test stations mean values were also included in tables 18 and 22. These data, along with the K index for the sires of tested pigs were obtained from nationwide progeny testing statistics (KANGASNIEMI 1979 and. In this official progeny testing the K index employed is calculated from the formula K = b[X, + b 2x 2 + b 3x 5 + b 4 x 4 + bjXj + 3.0, where Xj is the growth rate, x 2 is feed unit/kg, x} back fat thickness in millimetres, x 4 fat percentage and x 5 meat on the carcase expressed in percentages and bj-b 5 are the corresponding coefficients, while Xj-x, represent deviations from each quality calculated on the basis of a 12-month moving average. Growth rate and feed efficiency deviations are obtained from test stations, and slaughter quality characteristics arc represented by figures obtained from comparing individual breeds in the whole country. The percentage of lean meat on the carcase is arrived at when half of the carcase is stripped of fat, meat and bone, and the amount of meat is calculated in percentage terms for that half of the carcase. When at least three progeny groups of four pigs each have been tested a K index is calculated for their sire. 3. Halothane test AU 2,066 test pigs were anaesthetised with 4 % halothanc (2-bromo-2-chloro-l, 1trifluoro-cthane) in oxygen in a half-scaled system (Fluotcx Marc II manufactured by Cyprane Ltd., England). The oxygen flow throughout narcosis was 4-5 1/min. For the test, the pigs were lifted onto a table so as to lie on their right side. The animals were kept under narcosis for three minutes or until a typical halothane reaction was achieved. Typical symptoms chiefly appead as high muscle tonus in the extremi-ties and back muscles. Pigs developing a high muscle tonus were considered halothane positive (hal-l-), while animals still completely relaxed after three minutes of narcosis were classified as halothane negative (hal-). In some cases the reaction was immediately apparent. The normally narcosis-induced reduction in muscle tonus did not occur, but instead the pig began to stiffen immediately after it was lifted onto the table and the mask put on its snout. Some individuals displayed forced movements. In some of these hogs a typical hal-lreaction developed but not in all animals. Some intermediate form of hal-lor hal-reaction was confirmed. In these pigs a slight stiffness developed in the muscles but did not worsen even when narcosis continued more than five minutes. In other animals this muscle tonus gradually loosened and the animals peacefully went to sleep, while in other cases this slight muscle tonus persisted throughout narcosis. All of these twelve cases were considered to be hal-.
In calculating method error, the halothane test was repeated two weeks later with 30 hogs. In one litter consisting of 15 piglets, two hal-lpigs were detected in the first test and a third hal+ pig was found in the second. The age of the pigs was 5 3 days in the first test. In all other cases the repeated test yielded the same result as the first test (Table 3). One male tested at the age of nine weeks showed a hal-lreaction, and when retested at the age of 6 1/2 months reacted even then very strongly. At phenotype test stations some of the hal-males included in this study were retested. They all displayed a halreaction. Later a slaughter evaluation test was performed on all 86 pigs examined at test stations, and it was therefore possible in these cases to compare values obtained from live animals with the slaughter quality properties.
The potential hazard of halthane gas to investigators and their assistants was minimized by adequate ventilation and by directing the gas exhaled by pigs out of the room.
Blood sampling
About 5 cc of blood was withdrawn by a 1.40 X 60 mm needle from the vena cava (the needle might occasionally have been inserted into the jugular vein) immediately upon completion of the halothane test and while the pig was still under narcosis. Blood was additionally obtained immediately before narcosis from 1 30 pigs, and from 18 pigs 2 4 hours following the halothane test. Part of the blood sample was collected in tubes containing citrate for H blood group and Phi-type determinations, and another part of the samples was allowed to clot for serum CK activity determinations. The collected blood samples arrived at the laboratories the day after sampling and the serum was separated there. When the samples arrived later than this CK activity was not analysed. The Norwegian samples were processed for analysis one week after bleeding. Only H blood group and Phi-type enzymes were determined from these samples. In the experiment described in Table 6, CK activity was determined from plasma which was separated from the blood samples already in the piggeries.
Muscle sampling
From the first 307 halothane-tested pigs, muscle biopsies were taken from the M. longissimus dorsi to determine water-holding capacity (WHC). The biopsies were taken from the position of the last rib with a human biopsy needle (Tru-Cut Disposable needle 7.5 cm, Trevenol Lab, USA). The biopsy needle was driven 3-5 cm deep into the Musculus longissimus dorsi through a small incision. Biopsies were taken for WHC measurements immediately after the halothane test. Obvious bloody biopsies were discarded.
Blood group analysis
The H blood group systems Ha and He factors were determined from nearly all of the halothane-tcsted pigs, comprised of 1,991 out of the 2,003 Finnish pigs in addition to the 63 Norwegian pigs. Blood analysis was performed at the blood group laboratory of the Union of Finnish AI Associations. Ha was measured by indirect hemargglutination and He according to hemolytic proceedings, both described by ANDRESEN (1963).The genotypes are here described without the base letter H as follows: a/a, a/ , a/c, c/ and -/-. Because other H blood group system factors were not identified, (-) signifies only that a and c are not present. The a/a type is a homozygotc in relation to a, while a/ can be either a homoor heterozygote in relation to a, but docs not contain the c-factor. The designation c/ could mean either c/c or c/-. Other H system factors were not determined, since according to the literature they have no relation to exertional myopathy (JENSEN 1978 b).
Phi-type determinations
Phi-type determinations were performed on 1,349 pigs using electrophoresis. The tests were partly conducted at the blood group laboratory of the Union of Finnish AI Associations but mostly at the Department of Animal Breeding and Genetics at the Swedish University of Agricultural Sciences (GAHNE 1979), where Ph;AA ph;AB an j ph;BB were determined. Because of inadequate Finnish laboratory capacity, Phi determinations were not performed on all halothane-tcstcd pigs.
8. CK test CK activity in serum and in plasma was determined at the Department of Biochemistry of the College of Vetarinary Medicine in Helsinki using a computer directed analyser (The Gilford System 3500, Gilford Instrument Laboratories Inc., Ohio, USA). The determination was carried out according to the method recommended for determination of creatine kinase in blood (Anon, 1976). CK activity is given as microkatal/litre (ukat/1) and transformed to natural logarithms. The scrum samples were diluted 1:10 with 0.9 % NaCl before the test proper. As reagense CK NAC activated (Cat. no. 126357), Boehringer-Mannhcim GmbH, Federal Republic of Germany, was used. The variation coefficient of method error has been calculated from 30 analyses made from one sample with a coefficient of variation of 2.0.
Because it was not possible to bring all blood samples to the laboratory within 24 hours, CK activity was not carried out on all halothane-tested pigs. Nevertheless, CK determinations were made on at least 50 % of the pigs in all elite breeding herds.
Serum CK activity was determined in 1,711 pigs from serum samples taken immediately after the halothane test. The results are summarised in the tables and represent CK values obtained from serum samples taken just after the halothane test, if not otherwise stated.
In addition, CK activity was measured from serum samples obtained from 130 pigs just prior to the halothane test and from 18 pigs 24 hours after the halothane test. CK activity was also determined from the plasma of six pigs.
Meat colour measurements
In the present study meat colour was determined in 86 pigs, all of which were halothane tested at the testing stations. A light rcflectometrc (ELL smoke stain Reflectometre, model 43, manufactured by Evans Electroselenium Ltd., England) was used for determinations at the slaughterhouses. The metre reading indicated the meat colour point.
No steps were taken to affect the measurement technique nor the handling of pigs before slaughter. Investigations conducted by ALHO (1980) on progeny testing judgment technique at different slaughterhouses showed that more care should be devoted to uniform preslaughtcr treatment of test animals. The meat colour determinations ought to be always measured from a fresh meat surface. A delay in colour measurement of up to three hours was occasionally observed at one of the abbatours where pigs from progeny testing were slaughtered. In the present study no attempts were made to influence the lairing and stunning of the test animals and the judging of the carcase quality.
10. Water-holding capacity WHC measurements were made from muscle samples which were taken from the Musculus longissimus dorsi of the first 309 halothane-tested pigs immediately after the halothane test while the animals were still in a state of narcosis. The volume of the biopsy specimens varied between 2.8-25 mg. The biopsies were put on filter paper (room-dried, Schleicher & Schiill Nr 5893) and pressed in a glass compressor (trichina compressor). The glass plates were screwed firmly together for five minutes. The filter paper was then removed from the compressor and the biopsies taken off. The outlines of the moisture marks on the filter paper and the impression left on it by the biopsy specimens were drawn. Later the areas were calculated by a planimetre and the results substracted from each other. The compressed meat sample was weighed. WHC is given as cm 2 /g compressed meat. The three samples (one hal+ and two hal-) with moisture marks spreading to the edge of the glass were discarded. Attempts to transport the biopsies to the laboratory to perform the weighing of fresh samples and compression thereafter were unsuccessful because many of the specimens lost so much moisture that no wet marks were apparent on the filter paper.
Since the method mentioned above was difficult to carry out on a routine basis at piggeries in conjuction with the halothane test, WHC was also determined by another method. In these cases the biopsy specimens were put in previously weighed 2 ml glass tubes immediately after sampling, each containing a small piece of tin paper on top of a cotton plug. The tubes equipped with rubber stoppers were dried, weighed and scaled airtight at the laboratories. In the piggeries these tubes were unscrewed long enough to place the biopsy sample in the tube. The tubes were kept for 24 hours in thermos bottles containing ice. Before the tubes were weighed, they were wiped dry and the original weight of the biopsy specimen was calculated. The sample was then removed from the tube and separately weighed. The difference in weight was the amount of moisture evaporated from the specimen, where WHC was reported as the weight loss as a percentage of the original weight. Cases in which the moisture in the tubes exceeded 7 5 % of the original biopsy weight were omitted. According to the literature pork contains all in all 7 5 % water (GRAU and HAMM 1953). If the result exceeded 75 %, leakage was assumed to have occurred. Altogether 15 samples were discarded. None of these were from halothane-scnsitive pigs. In six cases the biopsy specimens had stuck to the glass surface and had to be discarded. This method sought to calculate drip loss. The variation coefficients of method error were calculated in four duplicate tests, yielding a coefficient of variation of 5.4 %.
Halothane test
During the test, the pulse rate of the hal+ pigs increased and exceeded 200 beats/min. already in the beginning of narcosis. The handling of the pigs before the test obviously affected the hal+ pigs very strongly. Many hal+ pigs were clearly in an excited state when carried to the table. Many halpigs also struggled, but they did not behave as excitedly as some hal+ pigs did.
At the end of the halothane test the pulse rate of the halpigs was x 184±20 beats/min. and the pulse of hal+ pigs was x 221 ±3B beats/min. This difference is statistically highly significant.
Six of the halothanc-testcd pigs died, all of them being hal+ pigs. The cause of death was the result of prolonged narcosis. In three cases narcosis was continued for one more minute after the onset of clear symptoms in order to obtain a satisfactory electrocardiogram. For the halothane test proper, such long-lasting narcosis was not required. In two cases forced movements and gradually increasing muscle rigidity occurred in the beginning, but a typical hal+ reaction developed slowly. The test was continued until the reaction was very distinct and apparent to the pig owner.
These pigs did not recuperate although narcosis was interrupted, but died ca 10 minutes later. One pig died the following night. The iterativeness of the hal-t-reaction was good; only a single halpig produced a hal+ reaction on a repeated test (Table 3).
CK test
When the material was sorted into hal+ and halgroups according to the results of the halothanc test, the mean value of the serum CK activity of hal + pigs was statistically significantly higher than the mean activity of halpigs. The most significant result was obtained when the serum was tested 24 hours after the halothanc test was administered (Table 4). The results obtained from serum CK activity determinations made just after the halothane test were usually of the same order of magnitude as those determinations made just before the test, although also large differences were apparent. In both cases, but namely for halpigs, some unexpectedly high CK activity values were obtained (Table 4 and appendix 1 group 2). Scrum CK activity determined from the same pig just before and immediately after the halothane test can be used to evaluate the results obtained in repetetive CK activity determinations in non-stressed pigs. The time difference between the two blood samplings is only about five minuses. The probable effect of the halothane test on serum CK activity could not possibly influence the CK values obtained in the latter serum sample. Twenty-four hours after the first halothane test, the CK activity was found to be significandy higher in the hal+ pigs. Scrum CK activity determined in samples obtained just after the test were on the average lower than in samples obtained just before the test, but the difference was not prominent (Table 4).
When the serum CK activity results obtained before and after the halothane test from the same individual were compared, the serum CK activity level itcrativeness was occasionally poor. According to correlation analysis, the correlation between these determinations was r=0.49, when the correlation was calculated for the same piggery. If herd space was not considered, the correlation between these two CK determinations was r=0.39. Large shifts both upwards and downwards from the mean values were observed (Table 4 and appendix 1).
To clarify the possible fluctuation in pig serum CK activities obtained from serum samples taken within short time intervals, serum samples were obtained from the same six pigs, four times every 1 5 minutes. This test also confirmed the poor repeatability of serum CK activity determinations (Table 5).
As the poor iterativeness of the test was suspected to be due to serum samples contaminated by muscle tissues in bleeding, the test was repeated with some pigs. In this case blood coagulation was inhibited with heparin, and the samples were immediately centrifuged after bleeding. In some blood samples small pieces of tissue could be seen floating on the plasma surface after centrifugation. Plasma CK activity was then determined in the usual way. The determination was made on separated plasma on the day of bleeding and on the following day using the plasma left in the blood tube. After centrifugation the iterativeness of the CK activity was a little better than (Tables 5 and 6). It was additionally demonstrated that the CK activity in the samples incubated overnight was higher than in the previous portion of the same sample (Table 6). These pigs were not included in the halothane test.
Pig blood hemolyses very easily. To elucidate the effect of hemolysis on serum CK activity determination in a single serum sample, equal amounts of hemolysatc diluted 1:11, 1:41 and 1:121 were added, whereafter the CK activity was determined. The hemolysis had no significant effect on the CK activity.
The 1:10 dilution of the serum before measurement only slightly affected the results. The coefficient of variation between results obtained from diluted and undiluted sera was 1.2 %. Repeated determinations from the same scrum sample yielded identical results. The coefficients of variation were 1.1 %. The handling of the pigs the day before the test caused large variations in serum CK activity determined at the same time from samples from different pigs at the same piggery. Pigs fought when animals from different litters were mixed in a pen on the day before the test. When testing these pigs fresh wounds such as skin scratches were apparent. The CK activity in these pigs was always high. Unexpectedly high values approaching 2 0 ukat/1 were, however, sometimes detected in hal-pigs, although these pigs were not handled before the test (Table , appendix 1 group 1).
Serum CK activities determined in hal+ male pigs at the end of the halothane test were slightly lower than the results obtained from hal+ female pigs. This difference was, however, not statistically significant (In 3.19 ± 0.42 contra In 3.36 ± 0.38. On the other hand, serum CK activity was of the same order of magnitude in halmale and female pigs x 19.0 ± 29.2 /tkat/1).
Small variations could be found in CK activity in serum from pigs with different H blood types. The differences relfectcd only the different numbers of hal+ pigs among the H blood types. According to statistical least-square analysis, the differences were not significant (Table 8). In all halpigs with different H blood types the CK activity was of the same order of magnitude (Table 8). At farms where the hal+ frequency in pigs was low, the CK activity in serum was also usually low but not always. At test stations tested halpigs with a meat colour point of St 44 showed CK activity that was not statistically significantly higher than in pigs with a meat colour point of < 44 (Table 9). The CK activity in scrum from the offspring of hal+ sires was not significandy higher than the CK activity in serum from the offpring of hal-sires (Table 10).
The CK activity in serum from pigs was comparatively low (Table 11).
Conclusion of the CK test evaluation
The CK activity was significantly higher in hal+ pigs as a group than in hal-hogs, although dispersion was great in both groups (Table 4). It was additionally (Tables 5 and 6 and appendix 1). These discrepancies could not be explained as technical analytical error. Some extremely high CK activity values can be justified as a result of animal handling on the day preceding the test, but this could not, however, explain poor test repeatability. Poor iterativeness was most probably caused by the contamination of the serum samples by muscle tissue as a result of bleeding from the vena cava. The immediate centrifugation and separation of the plasma increased the repeatability of CK activity determinations, but probably did not completely correct the error caused by muscle tissue contamination. If it were possible to expose the pigs to standardized strain the day before blood sampling for the serum CK activity detemination, it would probably yield more reliable results for breeding selection. This was demonstrated by results obtained with serum samples taken 24 hours after the halothanc test (Table 4).
Meat colour determinations
The meat colour points were higher for the hal+ than for the halpigs (Table 12 and appendices 2,3, 4 and 5). The percentage of lean meat on the carcase was the same in both groups. One hall pig had a very low meat colour point of 29 (Appendix table 3).
This was most probably a case of DFD meat, but it was not confirmed because no pH determinations were made. A meat colour reading of S 44 was obtained for 71.5 % of the hal+ and for 29.5 % of the halpigs (Table 12). In group one at the progeny testing station in southwestern Finland one hal+ pig was found. This group was given the highest meat colour reading, although this hal-Ipig was killed in a fight before slaughter and was therefore not included in the slaughter evaluation. The H blood group factor a/ was demonstrated for all pigs included in this group but not factor c. Group three, in which most of the animals had factor c, received a lower colour reading but also at the same time the lowest K index values (Appendix table 2). Group one tested at the progeny testing station in cast and central Finland included two hall pigs. All pigs in this group had the H blood group factor a. The group obtained a higher K index as well as a higher colour reading (Appendix table 3). It was also found among pigs tested at the swine research station in Hyvinkää that the hal+ pigs generally had a high meat colour reading (Appendix tables 4 and 5) and that hal+ and H a usually followed each other.
For some of the pigs tested on breeding farms, the meat colour points could be calculated as mean values from four litter mates raised as one group at one of the progeny testing stations. In these cases colour readings were obtained from normal progeny testing. Higher colour readings were in these cases demontrated among hal+ litters as compared to those among the hal-litters. The difference was, however, not statistically significant (Table 13). Large dispersion within and between groups was verified (Appendix tables 2,3, 4 and 5).
The haipigs from hal+ litters had a higher colour reading than pigs from hal-litters. The reading was 43.5 compared to 41.1, but the difference was not statistically significant. Determination of the meat water-holding capacity (WHC) WHC was the same in both hal+ and halpigs (Table 14). No significant differences in WHC could be demonstrated in pigs with different H blood types (Table 15).
Epidemiological results
The halothane test Totally 251 halothane sensitive (hal+) pigs were found. This means a hal+ frequency of 12.4 % for the Finnish Landrace and 3.2 % for the Norwegian material (Table 16). The hal n gene frequency in the Finnish Landracc breed is thus calculated to be 0.35, according to the Hardy-Weinberg law and assuming that halothane susceptibility is induced by one recessive autosomal gene which has a complete penetrance.
The Finnish material consisted of; 249 hal+ pigs 1 754 halpigs Of the Finnish pigs, 10.4 %of the males and 13.5 %of the females produced a positive halothane reaction. The hal+ frequency remained low when pigs less than 51 days old were tested in comparison to results obtained with pigs tested at an older age (Table 17). Halothane-scnsitivity remained below 20 % in the Finnish Landracc, but on some single farms, however, quite high values could be demonstrated (Appendix table 6). The animals included in this study represent the top of the Finnish Landrace breed. The growth rate of the sires exceeded the mean, measured in progeny testing during the same period of time. Hal+ pigs were discovered among the progeny of 64 sires (hal-l-sires). Only halpigs were found to spring from 90 sires (hal-sires). From hal+ sires an average of 20 offspring were tested and from hal-sires 17.2. The greatest number of hal+ sires were found among those with a high K index, when again hal-sires were more frequent among those with a low or negative index value (Tables 18 and 26 and appendix 7). Only few halsires had a high K index. The mean K index among hal+ sires was 7.46 compared to only 4.81 among halsires. The percentage of lean meat on the carcase was also greater for hal + sires than for hal-sires. The meat colour was lighter in hal-lsires than in halsires (Tables 18 and 26 and appendix 7), but this difference was not statistically significant. A better growth rate was also a more prominently inherited characteristic among hal+ sires than among halsires (Table 18 and 26.) H blood group factors The FI blood group factors were determined from 1,991 Finnish and 63 Norwegian pigs, 38 % being males and 62 % females. The Ha factor was present in 60.7 % of the Finnish Landracc and in 62 % of the Norwegian pigs (Table 19).
Of the Finnish Landracc hogs 38.0 % had the c factor as opposed to 5 5.6 % of the Norwegian animals. In the Finnish Landracc pigs the H blood group could be traced over two generations, and also especially in pigs which were selected by breeders as parents for the third generation. A slight decrease in the a factor was discovered in these selected animals, while a clear increase was seen in pigs with the c factor (Table 20).
Pigs with the a factor tested at test stations had a higher percentage of lean meat on the carcase than had pigs without the a factor. The meat colour reading did not deviate significandy among hogs with the a factor from the value determined in pigs without it (Table 21). Hal+ pigs had a tendency to produce lighter meat than hal-pigs.
Sires with factor a had a higher K index, a higher lean meat percentage on the carcase as well as higher colour readings than sires without factor a. Sires with a fac- tor also possessed a better growth rate and better feed efficiency than sires without it (Table 22). A high correlation between the H blood group factor a and the hal+ reaction was confirmed. Of the a/a pigs 39.6 % were halothane sensitive as were 20.7 % of the a/ pigs. Only 1.9 %of the a/c pigs and 0.4 %of the c/ pigs reacted to halothanc, compared to 6.1 %of the -/ pigs ( Table 23).
The correlation of H blood group factors and the hal+ frequency fluctuated greatly between different farms. In general a high Ha and a low He frequency was followed by a high hal+ frequency, but exceptions to this rule also occurred (Appendix table 6).
Phi enzyme types
All three Phi types were present in the Finnish Landracc, the AA being rarest and the BB most prevalent (Table 24). The BB also dominated in the Norvcgian material (Table 24). Hal+ pigs usually had the Phi-type 88, but some hal-F pigs with type AB were also discovered. In contrast no AA type was detected in hal+ pigs (Table 25). Of the hal+ pigs 96,6 % had the BB type and 3.4 % the AB type, whereas of halpigs 74 % had the BB type and 24 % the AB type. Of the hal+ pigs 90 % with the BB type had the H blood group factor a, but the H blood group factor a occurred in 5 2 % of halpigs with the BB type (Table 2 5).
The serum CK activity was markedly low in pigs with the AA type. This was also observed when only hal-hogs were compared (Table 11).
VII Discussion
Exertional myopathy frequency A knowledge of conditions at the top of the breeding pyramid gives some picture of the future of the breed as a whole. If the stock is to be changed by breeding, then action has naturally to be focused on the top of the pyramid, and for this reason it is important to know the prevailing situation.
The results of material consisting of more than 2,000 pigs presented in this study will reliably reflect the situation for exertional myopathy at the top of the Finnish Landrace breed in 1979. Breeding pigs from all elite herds excluding one were investigated. In addition, pigs from ten more breeding piggeries were studied where the breeding level of the animals was so high that they could be accepted as aspirant herds for elite breeding. The K index for the sires of these test pigs was 2.6 points higher than the mean value obtained for the whole Landrace breed in 1979. Of these sires about 70 % were used in the artificial insemination service. It was additionally verified that two-thirds of the breeding animals for the next generation in the elite herds were included in this study.
Halothane sensitivity Susceptibility to exertional myopathy as determined by the halothane test was confirmed in 12.4 % of the hogs tested, being so far the second highest hal+ frequency demonstrated in Scandinavia. The halothane sensitivity frequency could most probably have been still higher, in that case approaching the hal-l-frequency verified in the Swedish Landracc breed, if all the pigs used in the halothane test had been more than seven weeks old. The present investigation includes 421 pigs under 5 1 days of age at the time of testing, and among these pigs the halothane frequency was only about half of the value obtained for the 1,582 pigs tested at the age of 5 1 days or older. WEBB (1980 b) also showed that the hal-l-frequency was lower when pigs were exposed to the halothane test at an age of under seven weeks than when the same pigs were tested at an older age. VAN DEN HENDE ct al. (1976) demonstrated that the muscles of younger pigs had a more efficient aerobic metabolism than those of older pigs, and this might be the reason why younger individuals endure halothane better than older animals. The hal+ frequency among male pigs was slightly lower (10.4 %) than among female pigs (13.5 %). A contributing factor for this could be that male pigs were more often tested at the breeding piggeries while nearly all female pigs were tested in elite herds. Accordingly, the breeding level was most likely better among sows in general than among boars. (WEBB 1981). The two subgroups of Landracc breeds were characterized by MAJOR (1968) with the help of blood group analyses. The Finnish Landracc seems to be more closely related to the subgroup of Scandinavian Landracc breeds than to the Landracc breeds on the European continent. This is not surprising because of the active exchange of breeding animals between Finland, Sweden and Norway. Norway especially has exported a rather large number of Landracc hogs to Finland.
The Norwegian Landracc breed material included in this study revealed a halothane sensitivity of 3.2 %, which was considerably lower than the frequency demonstrated in the Finnish Landracc, and even lower than the figure of 5 % reported by WEBB and SMITH (1977). The present Norwegian material was tested in breeding herds in the Trondheim area and probably does not represent the top of the Norwegian Landracc breed as well as the material tested by WEBB and SMITH (1977). Halothane sensitivity is a characteristic inherited by the recessive Hal n gene, which has complete or nearly complete penetrance (SIMON 1980). The halothane test reveals about 90 % of the homozygotes, but hetcrozygotcs arc not revealed. When the hal-f-frequency was 12.4 %, it was possible to calculate using the Hardy-Wcinberg law that at least 45.6 %of the Landracc pigs carry the Hal n gene and that about 58 % of the Finnish Landracc pigs arc cither homozygotes or heterozygous carriers of this gene.
Carcase quality
In the present study the correlation between the Hal n gene and good production qualities was not clearly verified in the pigs from the test stations but was demonstrated in the much more extensive material from the breeding farms. The percentage of lean meat on the carcase of hal+ (Hal n Hal n ) pigs was not significantly higher than that of hal-and (Hal^Hal n ) pigs, but the K index and lean meat percentage on the carcase of hal+ (Hal n Hal n ) and (Hal^Hal n ) sires were higher than those of haland (Hal^Hal n ) sires.
It has been demonstrated that halothane-scnsitivc pigs usually have more meat on their carcases than pigs not sensitive to halothanc. Effective breeding for more meat on the carcase in a population where the Hal n gene is present will most probably increase the frequency. OLLIVIER et al. (1975) and MABRY (1978) have experimentally shown in selection tests that this calculated increase in fact occurs. Their selection tests also confirmed that the meat quality was poorer in hal+ pigs than in halpigs.
The reason why FROYSTEIN et al. (1978) were unable to demonstrate an increase in the number of hal+ pigs in the meaty line with low back fat compared to the high back fat line in the Norwegian selection experiment may result from the fact that the Hal n gene is rare in the Norwegian Landracc breed. The lack of an increased hal+ frequency could also partly arise from the fact that in the selection test in question more effort was devoted to thinning back fat than to increasing meatiness (VANGEN 1979). Selection for reduced back fat would affect meat quality less than selection for increased muscularity (LUNDSTRÖM 1975).
Meat quality
The poor meat quality of hal+ pigs compared to meat quality in halpigs has been verified by many but not all investigators (WEBB 1981), and this was also confirmed in the present study where it was shown that hal-Ipigs and the offspring of hal+ sires had a higher meat colour reading than the offspring of hal-sires. The difference in the meat colour readings of hal+ and halsires was not statistically significant, however. When meat quality is reliably determined and is taken comprehensively enough into the breeding programme, the spread of the Hal n gene can be prevented. These proceedings have been effectively practised in Denmark (JENSEN 1978 b) Norway (HEMMA 1978) and are now also employed in Sweden (LUNDSTRÖM et al. 1980). Crossbreeding may be used for improving meat quality but in crossbreeds meat quality appears to be intermediate between the two parent breeds, suggesting that no beneficial effect is to be gained from heterosis (WALSTRA et al. 1971, LEAN et al. 1972. Results by EIKELENBOOM et al. (1980) and indicated that carriers which were themselves stress-resistant were intermediate to the two homozygotes in meat quality and percentage of lean meat on the carcase.
H blood group system
The H blood group system factor a is more common in the Finnish Landrace breed than in the Finnish Yorkshire breed. According to the present study the frequency of the Ha blood factor in the Landrace was 60.7 %, while in Finnish Yorkshire boars it was 48.0 % (LINDSTRÖM 1980). A bigger difference was, however, seen in the frequency of the c factor, which was 38.0 % in the Landrace and 76.8 % in the Yorkshire breed. The c factor was present in 5 5.6 % of the Norwegian Landrace pigs tested in this study. Halothane sensitivity was shown to be very rare in Landrace pigs which had the c factor. Halothane sensitivity was also low in the populations where the c factor was common as was demonstrated both in the Finnish Yorkshire breed (0.2 %) and in the Norwegian Landrace breed (3.2 %).
The extremely low hal+ frequency in Finnish Landrace pigs having the H blood group factor c but not factor a provides no evidence that the same situation also prevails in other breeds nor that this positive stress-resistant situation in He pigs is permanent in the Finnish Landrace breed. JORGENSEN'S (1979) investigation including 5 31 Danish Landrace pigs showed a hal+ frequency of 5.5 % among He pigs.
Differences evident between breeds are probably an indication that Hal and H blood group loci arc not so closely linked that it should be impossible for a chromosomal crossover to occur between them. Evidence of this also shown by JORGENSEN (1981) reduces the usefulness of the role of H blood group factors as markers of stress resistance in breeding programmes, and requires that the halothane test is also used so that a change in the once established relationship between the marker and marking factors is not hidden from the breeders.
Exertional myopathy in breeding
The halothane sensitivity gene Poor meat quality caused by a poor stress resistance of genetical origin could be improved by eliminating the Hal n gene. Before such a procedure can be proposed, all properties, both good and bad, of the Hal n gene should be estimated and weighed against one another. In the pig population "ABRO" which WEBB investigated, the negative properties, or higher mortality and poorer rcproductivity connected to the Hal n gene, were so much more significant than the relatively greater meatiness of the carcase that the effect due to the Hal n gene was negative and created a loss of £ 3.60 for every bacon pig produced. It would have been feasible to eliminate the Hal n gene completely at least from the "ABRO" stock (WEBB 1980 a).
By removing the Hal n gene from the Finnish Landrace it is possible to reduce mortality due to the stress syndrome. The death rate of Landrace pigs at test stations and during transport from test stations to slaughterhouses could be reduced to at least half of the present death rate. The same positive development should take place at fattening piggeries and during transport from there to abbatoirs. showed that the death rate of hal+ pigs during transport and preslaughter treatment was nearly ten times higher than the death rate of halpigs. In the progeny testing of Finnish Landrace pigs, most probably more than 10 % (12.4 %) are hal+ pigs and the death rate of these pigs corresponds to at least 50 % of the total 'stress deaths" of Landrace pigs. A comparison of the death rate for Hal n hetcrozygotes and the death rate for pigs without the Hal n gene has not been performed, but it is most likely that death from stress is greater among heterozygote pigs than among pigs which have no Hal n gene at all. In Hal n gene homozygotes (hal+) and also in the group which had the greatest number of Hal n heterozygotes (hal+ litters halpigs), it was demonstrated that the meat colour reading was higher than in pigs from hal-litters and the offspring of halsires, both of which represent groups with few hal n gene carriers among them. Meat colour readings would thus certainly improve if fewer Hal n gene-carrying pigs were brought to the test stations.
When 33.2 % of all pigs at test stations included in this study were given a colour reading of more than 44 points, 71.5 % of the hal + pigs had the same high colour reading compared to only 29.5 % of the halpigs. If the hal+ pigs were excluded from breeding, all colour readings would improve so that only 29.5 %, or 3.7 percentage units less, of groups would receive a colour reading of more than 44 points. The improvement in meat quality should, however, be greater than 3.7 percentage units, as also the Hal n gene heterozygous pigs would begin to disappear. The meat quality of these pigs is an intermediate between the meat quality of the two previously mentioned groups. This has been pointed out by many researchers and summarised by JORGENSEN ( b and 1981( ) and SCHNEIDER ct al. (1980, and the results obtained in this study are also in agreement.
Carcase quality
Differences in the meatiness of hal+ and halpigs were not observed in the test station material. Due to this it could be assumed that elimination of the hal+ pigs would improve meat quality without reducing the amount of lean meat on the carcase. However, it has generally been demonstrated that hal+ pigs and pigs carrying the Hal n gene have more meat on their carcases than pigs without the Hal n gene (LUNDSTRÖM 1975a and EIKELENBOOMct al. 1978b and 1980. In this investigation it was also discovered in sires that the K index of hal+ sires and the percentage of carcase lean meat were higher than the K index and lean meat on the carcases of hal-sires. Although the eradication of the Hal n gene improves meat quality according to results obtained from test station material without reducing the amount of lean meat on the carcase, a reduction is to be expected. Improving meatiness will be possible, however, because even among halsires there are individuals with a high K index and a high percentage of lean meat on the carcase. Some of these sires in all probability do not have the Hal n gene, although certainly only a small number of such individuals exist.
The Hal n gene is present in approximately 58.0 %in all of the best breeding pigs. The complete destruction of the Hal n gene would therefore be problematic. The impediments caused by the Hal n gene, i.e. sensitivity to stress called ''stress death" and the formation of PSE/DFD meat, pose such large risks that the eradication of the gene becomes necessary. If nothing is done, the gene will be increasingly prevalent as long as the breeding programme for more meaty pigs is continued. Even the theoretical assumption of the total eradication of the gene might not be re-alistic because no method is available so far for direct identification of the heterozygotes. With the help of the halothane test and suitable test matings this becomes possible b, JORGENSEN 1981and SMITH 1981.
H blood group factors
The use of H blood groups, the Phi-type determination and the CK test have been proposed as methods to reveal Hal n gene heterozygous individuals. Results from the present study show that the Ha factor is closely linked to the hal+ reaction. Of the a/a pigs and a/ pigs, 39.6 % and 20.7 %, respectively, were hal+ hogs. The H blood group system factor a is not, however, casually associated with the halothane reaction, since all hal+ pigs do not have the a factor. This has been demonstrated by BARTON et al. (1977) andJORGENSEN (1977). In this study it was also shown that of the -/ pigs and the c/ pigs, 6.1% and 0.4 %, respectively, were hal+ individuals. If the H blood group factor a is completely removed from the Finnish Landracc, it would first of all be a very radical action, because 60.7 % of the best breeding animals possess this factor. This share is about the same as the percentage of Landrace pigs estimated to have the Hal n gene (58.0 %). The eradication of factor Ha should lower the hal+ frequency very effectively, since about 91 % of the hal+ pigs would disappear if the a factor is removed. The hal+ genotype frequency would be reduced from 12.4% to 1.12%. Most of the Hal n gene heterozygotes would also disappear, and the Hal n gene frequency would decline from 0.35 to 0.11. If Ha individuals having both a and c factors were preserved, it would be possible by selective eradication of 45.4 % but not 60.7 % of the breeding animals to diminish the number of hal+ pigs to nearly the same degree, or 88.7 %. This alternative should at any rate leave in breeding a little higher percentage of Hal n hctcrozygotes than the previous alternative. The eradication of Ha should lead to a 50 % reduction in stress death compared to the present situation. No statistically significant differences in meatiness or meat colour were demonstrated in pigs carrying the Ha factor or in pigs without the factor when these measurements were performed at test stations. However, Ha pigs tended to have more meat, but their meat was lighter than the meat from pigs without the Ha factor. Many investigators have shown that the amount of lean meat on the carcase is greater and the meat colour lighter in pigs with the factor a than in pigs without it (BARTON ct al. 1977and LUNDSTRÖM ct al. 1980and JORGENSEN 1981.
In this study a difference was discovered between sires with different H blood groups. Sires with the Ha factor had a K index 3.1 points higher than sires without the Ha factor. The dispersion within groups was large, however, and therefore the difference was not statistically significant. Both lean meat on the carcase, growth rate and ability to utilise fodder were above the mean average value in Ha sires. If Ha pigs were removed, the K index and meatiness would diminish, but the meat colour points would improve by about one index point. Breeding for meatiness and growth improvement could continue successfully because some individuals with a high K index are to be found among both He/ and H-/ sires. As their number is low, a momentary setback in breeding results would inevitably occur.
Halothane test
The halothane test was quite harmless to pigs. Of the 2,066 pigs tested only six died. These were all sensitive to halothane. Of these animals at least three could have been saved if the halothane test had not been conducted in conjunotion with other tests. The iterativeness of the halothane test was good but not 100 % successful. The interpretation of a positive result was in most cases easy, although some ambiguous cases appeared. The typical hal+ reaction in pigs is so dramatic on the basis of obtained results that there was no difficulty to convince the pig breeders to use the results of the halothane test in their breeding programmes. Because the nature of the material used in this investigation, the results obtained with the halothane test could only be repeated with few pigs. The test pigs were either the owners' best breeding animals or pigs from the progeny testing. In test repeatability, a less than 5 % error was confirmed. This corresponds to levels presented in the literature JORDAN 1978 and. When pigs at an age of 5 1 days or younger were tested, however, a lower hal-F frequency was found than in older pigs. The obtained hal+ frequency of 12.4 % is slightly underestimated to represent the situation for the whole breed. On the other hand, the hal+ frequency among individuals with lower breeding value is most probably somewhat lower than the situation apparent at the top of the breeding pyramid.
The possibility to reduce the Hal n gene frequency in a breed above all depends on how high the hal+ frequency is in the population in question. In the Finnish Landracc breed the hal+ frequency of 12.4 % fell below 20 %, considered by some researchers (LUNDSTRÖM et ai. 1980) to be a borderline above which the hal+ frequency must lie before the use of halothane test becomes profitable. Although the frequency for the whole breed remained below 20 %, some individual piggeries were observed to have a frequency above this borderline value. Conducting the halothane test in these piggeries would at least be profitable, and a rapid improvement in stress resistance is expected in the beginning, assuming that all breeding animals are tested and that all the reactors are rejected.
If all animals intended for breeding, including both males and females, are halothanc tested in a population where the Hal n frequency is 0.35 and all reactors are rejected, the gene frequency should decrease to 0.127 by the fifth generation and the share of hal+ pigs to 1.6 %. This change in gene frequency was calculated according to the formula where q is the gene frequency and s the coefficient of selection as introduced by FALCONER (1960).
If only male pigs were to be tested, the gene frequency in the fifth generation would be 0.2 and the share of hal+ animals 4 %. This calculation is based on the same formula. Taking into account more precisely the differences in gene frequencies between the male and female population does not yield a significantly different result.
In the calculations it has been assumed that halothanc sensitivity is caused by one recessive gene with the property of complete penetrance in the halothane test.
An additional assumption is that the effect of all males and females is of the same order of magnitude on building the next generation. No adjustment has been made for the fact that some boars are used in artificial insemination and can affect the whole population, while other boars on farms will affect only a small subpopulation. The possible weaker reproductivity of halothane-sensitive animals has also not been taken in consideration. If all breeding animals were tested and the reactors rejected, then the gene frequency in the first generation of selection would be reduced from 0.35 to 0.26. In the fifth generation of selection the reduction would be much smaller, or from 0.17 to 0.1 5. This would first correspond to a reduction of the hal+ frequency from 12.4 % to 6.7 % and then only from 2.92 to 2.12, respectively, and in this way indicates that the rate of progress decreases as selection continues.
Breeders who have the chance to use the halothane test on all breeding animals and choose only non-reacting females and males for breeding could under optimal circumstances expect a reduction in halothane-sensitive pigs in the manner calculated in table 27. All breeders do not have the opportunity to use the halothanc test on their pigs, but all breeders can use artificial insemination, and the boars used in the artificial insemination service arc halothane-tcsted. By using artificial insemination, it is conceivable that with an unselccted female population progress can be achieved as indicated in table 28.
In tables 27 and 28 improvement is calculated according to the same FALCONER (1960) formula used in the previous calculations. In table 28 the figure 0.5 is used as a coefficient of selection, and primarily corresponds to the selection of males only. The progress demonstrated above occurs only when the hetcrozygotcs are not selected for breeding more frequently than pigs completely free from this gene. However, these Hal n heterozygotes have been found to be meatier than individuals without this gene and SCHNEIDER et ai. 1980, and thus they are in fact in a better position to be more often chosen for breeding purposes (OLLIVIER ct al. 1976 and. This investigation showed that both the hal+ and hal-litters of hal+ sires had a better K index and more lean meat on the carcase than litters of halsires. The fall in halothane frequency shown in tables 27 and 28 should certainly not occur, because when selecting sires for artificial insemination the Hal n heterozygotes arc favoured although the Hal n homozygotes will be eliminated. Since females are not halothanc-tcstcd, both hetero-and homozygotes will be favoured among them. Actual progress towards better stress-resistance would then be slower. The toxicity of halothane Long-lasting (more than 20 minutes) or repeated halothane narcoses have been shown to be dangerous to both humans and several animal species, such as monkey, mouse, rat mink and dog. It can cause liver and kidney damage appearing as centrolobular necrosis in the liver and tubular necrosis in the kidneys (GREENHAM andWARE 1979 andCOUSINS 1980). It has been demonstrated that halothane produces a teratogenic as well as mutagenic effect ( V. BASFORD andFINK 1968, GRANT et al. 1 977 andFÖRSTER andBUTLER 1978). An above-normal abortion frequency has been reported in women working in operating rooms (COHEN et al. 1977). The halothane test when narcosis lasts 3-5 minutes is of no danger to pigs, and no analogous damage has been reported.
Malignant hypertermia (MH) can also be induced in sensitive pigs by other narcotic agents, as shown with chloroform by HARRISON et al. (1969) and with Suxamenthonium. MH can in addition be induced with caffein (VAN DEN HENDE ct al. 1978).
HaP 1 heterozygotes
Effective elimination assumes that in addition to Hal n homozygote (hal-l-)pigs it is possible to identify Hal n gene heterozygotes. These are not revealed by the halothanc test. If no other tests than the halothane test are available, elimination based on progeny testing as proposed by WEBB (1980b) and SMITH (1981) should be performed, and all sires and dams with even only one hal+ offspring eliminated.
A-O blood type system JORGENSEN (1977) and IMLAH and THOMSON (1979) demonstrated that halothane sensitivity is dependent not only on the H blood group system factor a but also on the A-O blood group system. When both systems were taken into account, it became possible to indicate hal+ pigs with 84.1 % certainty and halpigs with 79.6 % certainty. MABRY (1978) showed that hal+ individuals could be found among the H blood group system a/a pigs which did not react to the A-O system nor to Aor O, and without exception found among H-/ pigs which reacted to AorO. Of Ha/ pigs reacting in the A-O system to Aor O, some were hal-(-pigs and some halpigs. The A-O blood group factors were not investigated in any greater detail in the present study. Knowledge about these factors in relation to H system factors would have contributed further needed information to the elimination process, but according to results reported by MABRY (1978) proposing the A-O factors for consideration in the breeding programme further study was not practical. As for the elimination of hal+ pigs, both the addition and elimination of pigs with the A-O factors would be necessary.
Phi enzyme JORGENSEN (1977) demonstrated that all hal + pigs in the Danish Landrace breed had the Phi enzyme type Phi®®, while JENSEN (1978 b) reported that more hal+ pigs and poorer meat quality were found in pigs which simultaneously had both Ha and Phi®®. By considering the Phi-polymorphism, breeding against stress sensitivity and poor meat quality can be intensified (JORGENSEN 1980 a).
In this study it was demonstrated that the Phi®® is present more commonly in hal-lpigs, but also among PhA® pigs some hal-(-individuals were discovered. No hai -I-reaction was observed among PhiAA pig S> bin the PhiAA was ver y rare j n this material. Only 1.5 % of the pigs tested had the PhiAA On this basis, Phi typing in Finland at present only has small practical value for the breeding programme. As Phi®® is present in 76.9 % of all pigs tested, there exists in fact no reason to propose selection against it. Further studies indicate that eliminating Phi®® would not remove all Hal n gene-carrying individuals. ANDRESEN (1980a) substantiates on the same ground that elimination of the Phi®® genotype in the Danish Landrace breed is neither advisable nor adequate. This investigation showed that pigs with PhiAA had a markedly lower CK serum activity level. The CK activity of PhiA® pigs were also lower than those of the Phi®® pigs, and this difference persisted when pure halpigs were compared to each other.
CK test
Many investigators (reviewed by BICKHARDT et al. 1977) have emphasized the use of the serum CK enzyme activity determination (CK test) to recognize stresssensitive pigs, but only BICKHARDT et al. (1979) have applied it in a breeding programme. HWANG et al. (1978) demonstrated that the CK test separated hal+ and halpigs into groups but that at the same time the fluctuation in CK activity within the groups was large. Due to this fluctuation it was difficult to impose elimination limits needed in breeding programmes. Many other investigators have also shown this . The results obtained in this study were also in full agreement with these findings.
The fact that extremely high serum CK activity values arc sometimes demonstrated in pigs poses difficulties in using automatic analyses, as has been pointed out both by BICKHARDT et al. (1977) and KALLWEIT ct al (1977). The test error was also observed to increase along with a rise in the CK activity level. Test errors of the same type but much larger were demonstrated when the CK activity was determined in serum samples taken from the same pigs on different occasions. The reason for the poor iterativeness of the CK test probably mostly lay in the fact that very fre-quently serum samples were contaminated with muscle tissue, and these contaminations could cause false high CK activity values. False positive CK activity values have also caused difficulties for other investigators (HWANG et al. 1976, BICKHARDT 1979. THOREN-TOLLING (1980) showed that individual judgement improved as judgement was passed on repeated determinations.
From the breeding point of view, it would be of principal value to know if the CK test could be used to identify Hal n gene heterozygotes. Despite some technical drawbacks to the procedures, hal+ pigs or Hal n homozygotes clearly deviated from the halpig group in this investigation. But among halpigs it was not feasable to form a group representing the Hal n heterozygotes. It was expected that hal-offspring of hal+ sires would deviate from the offspring of halsires. The same CK activities were, however, obtained in both groups, and no remarkable differences in CK activity could be demonstrated in halpigs representing different H blood group types. The conclusion was accordingly that the CK test performed on "nonstressed" pigs was of no value for the demonstration of Hal n heterozygotes. LUECHER ct al. (1979) and SCHNEIDER et al. (1980) proposed, however, that the heterozygote CK activity fell between the values obtained for homozygotes and for the group lacking the Hal n gene. Perhaps they stressed the pigs before the serum CK activity was determined and were more careful in collecting blood samples. BICKHARDT et al. (1980) have emphasized the importance of standardized straining before the CK test determination. BICKHARDT et al. (1979) proposed that the serum CK activity be analysed about eight hours after standardized exercise.
It was also shown in this investigation that the activity of hal-tpigs increased radically 24 hours after the halothane test. In this study no attempt was made to elucidate the type of strain test necessary to cover the high CK fluctautions observed in resting pigs. BICKHARDT et al. (1980) previously used an exercise test and later drugs suited for parenteral administration.
Meat colour
The investigations conducted in this study also show that the colour of the meat correlates very well with other test results used to demonstrate exertional myopathy. Because of this high correlation and the relatively good inhcritability of meat colour, it has become the most important method to suppress exertional myopathy by using meat colour determination both in Denmark and Sweden (PEDERSEN 1979 andLUNDSTRÖM ct al. 1980). JENSEN and showed that although the other tests, primarily the halothane test, the H blood group system factor a and Phi enzyme typing, yield more information speeding up breeding progress, the contribution is, however, so small that it is most practical for Danish purposes to concentrate on meat colour. LUNDSTRÖM et ai. (1980) demonstrated that in the Swedish breeding programme too little attention has been paid so far to meat colour to make any progress. Both in Denmark and Sweden much importance has been attached to improving the reliability of the results obtained from meat colour determinations. The Danes have effectively standardized the transport of test pigs and the entire preslaughter treatment and have also developed the KK index as a measure for meat quality (BARTON 1974). In Sweden rejection is based on average deviations from numerical colour points measured from all test pigs slaughtered at the same ti-me (LUNDSTRÖM et ai. 1980). In both countries the colour readings are corrected with results obtained in pH 2 determinations so that DFD meat docs not disturb the evaluation principles. In this study problems related to meat colour determinations have not been investigated. The results obtained indicate, however, that criticism of meat colour readings in the Finnish progeny testing has been too severe, although deficiences which need rapid correction were found. Above all, the errors caused by DFD meat have to be rectified. The evaluation procedure should be modified so that DFD meat is evaluated as PSE meat. In order to reveal DFD meat the pH 2 should be determined. Perhaps a change in preslaughter treatment favouring PSE meat and not DFD meat should also be taken into consideration, as is the procedure in Denmark (BARTON 1974).
Water-holding capacity of meat
In this study, efforts were made to apply the meat water-holding capacity measurement to live animals, but without success. The obtained results did not correspond to results obtained in other tests determining exertional myopathy. The actual reason for unreliable results might be that too small biopsy specimens were taken. In the methods used to determine WHC, the processing of small samples magnified technical errors so that no real differences in WHC could be reliably measured. WALSTRA ct al. (1977) also failed in their efforts to use determinations on muscle biopsies from live animals as general criteria for meat quality characteristics. Results could, however, be improved with larger biopsy specimens (PFEIFFER 1981).
Implications for the breeding programme
The results obtained in this investigation indicate that the quickest and easiest means to reduce the hal+ frequency and improve the meat colour reading would be to favour the H blood group factor c. By increasing the number of pigs with the c factor, the hal+ frequency should decrease rapidly. In this group more than 88 % of the pigs were free of the Hal n gene. The meatiest individuals carrying the Ha blood group factor could be retained but should be mated to He individuals. In most piggeries using this method halothane-scnsitivc individuals would completely disappear as soon as the first generation of selection. A small number of Hal n carriers exist among He pigs, however, so the halothanc test should be used at phenotype stations to exclude the possibility of getting He Hal n Hal n pigs for artificial insemination. Additionally it would be necessary to expose the Hc/Hal n pigs with great degree of reliability. This is possible only if test matings arc carried out and a halothanc test is made on the offspring. This should be done at least to all sires carrying the He/ blood group and additionally having a high K index. When in test matings hal+ dams are mated to sires carrying the Hal n gene, half of the piglets born will be hal+ pigs. In a litter consisting of at least five piglets, the halothanc test will reveal if the sire used carries the Hal n gene or is free of the gene with 95 % confidence, and in a litter with seven piglets do so with 99 % confidence. If a test dam known to be a Hal n gene heterozygote or known to have at least one hal+ offspring is mated to a Hal n gene carrier boar, 25 %of the offspring bom will be homozygotes or hal+ pigs. In this case only one litter with 11 piglets has to be halothane-tested to reveal the sire genotype with 95 % confidence. To obtain a 99 % probability the litter should contain 16 piglets (PIRCHNER 1969). If the test is performed on the litters of daughters of the sire or on daughters of known heterozygous sires or dams, offspring from the litters of five dams should be tested before it can be confirmed whether or not the sire has the Hal n gene. VIII Conclusions and application of practical measures for the breeding programme As long as breeding for more meat is carried out in the Finnish Landrace breed where the Hal n gene and the drawbacks caused by it are present, the quantity of PSS and PSE/DFD will continue to increase. In order to prevent this it is necessary to include in the breeding programme clear procedures to restrain the dissemination of the Hal n gene. In a long term programme it seems to be profitable to try to eradicate the Hal n gene completely.
In 1979 the Hal n gene was present in at least 58 %of the most advanced Finnish Landrace pigs, and 12.4 % of them were homozygotes in regard to that gene. The halothane test has to be performed on pigs older than seven weeks to reliably reveal Hal n gene homozygotes. A fully satisfactory test identifying Hal n gene heterozygotes has to date not been demonstrated.
The iterativeness of the CK test was poor when the test was conducted without prior exertion. No real proof that the CK test performed on ''rested" pigs identified Hal n gene heterozygotes was demonstrated. Of the Phi enzyme types the Phi AA very seldom follows the Hal n gene, but the Phi AA is rare. Only 1.5 %of all pigs tested had this Phi type. The determination of the H blood group system factor a and c provides in addition to the meat colour measurement and the halothane test the most advanced information to identify Hal n gene-carrying individuals. Of the Ha-carrying pigs most probably 91 % have the Hal n gene. No casual relationship exists, however, between the H blood group factor a and the Hal n gene. Of He pigs 0.4 %, of -/ pigs 6,1 % and of Ha/c pigs 1.9 % reacted to the halothane test.
The Hal n gene-carrying pigs both homo-and heterozygotes generally have lighter meat than pigs free of this gene. The meat colour reading follows the results obtained in determining exertional myopathy. The mean value of the meat colour from four test pigs cannot be a reliable basis for selection if the probable presence of DFD meat is not observed. DFD meat has to be judged as PSE meat. Hal n genecarrying pigs were not identified with water-holding capacity determinations performed in muscle biopsies from living pigs.
A breeding programme for the Finnish Landrace breed should according to the results of this study contain the following measures.
1. In progeny testing the meat colour should be correlated to the results obtained in pH 2 determinations and strongly pointed out in order not to aggravate the current exertional myopathy situation. 2. The breeding herds could resist exertional myopathy by employing the H blood group system factor c. The meatiest a/a and a/ pigs could be saved, but they should not be mated to each other. The Ha (a/a and a/-) pigs should be mated to c pigs. If c pigs are not available, a/c or -/ pigs should be used, and of these a/c pigs are more suitable.
3. The halothane test should be used at phenotype testing stations when selecting sires for artificial insemination when selecting breeding animals in piggeries where the hal+ frequency is higher than 15 % as a progeny test on all c sires having a high K index It is enough to test only one litter of seven pigs if the dam is a hal-ldam, or 11 pigs if the dam is known to give birth to hal+ offspring. If such dams are not available, the test should be conducted on daughters of hal+ dams or sires, or on the boars' own daughters. In this case whole litters from five dams have to be halothanetested before it is reliably guaranteed that the sire used is free of the Hal n gene. IX References rodussa. Parhaat H a/a ja H a/ siat voitaisiin pitää kunhan ne aina paritettaisiin H c sikojen kanssa.
XI Appendix
Appendix | v3-fos |
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} | s2 | PLANTING WHEAT SEED DAMAGED BY FROST BEFORE HARVEST
This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1997 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.
Frosts associated with cold fronts during late spring damage winter wheat in Kansas in many years. Damage ranges from light to severe depending on the stage of plant growth, the growing conditions, and the intensity and duration of the frost. Genetic resistance to the problem is not available, and varieties of wheat differ little in susceptibility at similar stages. The symptoms of injury to plants and effects on yield from late frosts are described in Spring Freeze Injury to Kansas Wheat (C646, Kansas Cooperative Extension Service).
The developing kernels of wheat are affected directly by frost during maturation of the crop. However, little information from research is available regarding the effect of injury on the quality of the grain for seed.
Temperatures of 25 to 28°F on May 25-29 caused considerable damage to wheat in northwestern Kansas in 1992. The developmental stage of the kernels ranged from milky ripe to soft dough when the frost occurred, and the reduction in grain yield at harvest varied from slight to severe. Growers were concerned about the suitability of the grain as seed for the next year's crop. They were given general recommendations to test the germination of the seed and to not use grain that was shriveled or had low test weight, because specific advice based on research with frost-damaged seed was unavailable.
The frost in 1992 enabled extensive tests of the suitability of damaged wheat for seed. Samples of hard red winter wheat were obtained from certified seed producers, and their kernel characteristics and performance as seed were determined.
This publication from the Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information is available from http://www.ksre.ksu.edu.
Procedures
The variety TAM 107 was used because it is the most widely grown in northwestern Kansas. Eleven seedlots were evaluated, and results are presented for four lots that represent a range of injury. A sound seedlot of TAM 107 from the area was the control.
Test weights of the uncleaned seedlots containing 12% moisture were measured by Federal Grain Inspection Service methods. All seedlots, particularly those that were damaged most, contained a large range of kernel sizes. The distribution of sizes was determined by sieving the kernels with 6/64 x 3/4-inch and 5/64 x 3/4-inch slotted screens and calculating the percentages by weight above and below the screens. One thousand kernels in each fraction were weighed to determine their average weight.
Germination of 100 kernels in each of the three size fractions of all seedlots was measured after they were prechilled at 41°F for 5 days and incubated on moistened, heavy paper at 59°F for 7 days. After 15 months of storage at room temperature, germination was measured again without the prechilling treatment. Samples of the seedlots also were subjected to accelerated aging at 104°F for 72 hours, prechilled, and tested for germination.
Ability of the seedlots to form a stand of wheat was estimated by planting the seeds in a greenhouse at different depths. Twenty-five seeds in each size fraction of all seedlots were planted 1, 2, 3, or 4 inches deep in masonry sand, which was moistened as needed, and emerged seedlings were counted twice weekly.
All experiments were in randomized complete block designs with four replications of treatments. Data were analyzed statistically by the general linear model method.
Results
Test weight of the wheat decreased steadily as the level of frost damage increased (Table 1). The nondamaged seedlot had a high percentage of large kernels and few small kernels, whereas the severely damaged seedlot had fewer large kernels and more small kernels. The kernel weights of all size fractions fell as the severity of injury increased.
Germination of large kernels was high after harvest, after storage, and after accelerated aging, regardless of the level of frost injury (Table 2). Medium kernels also germinated well after no or slight injury, but viability dropped after moderate or severe injury. Frost was most damaging to small kernels, which germinated poorly in all tests after moderate or severe injury.
Seedling emergence from different planting depths followed the same pattern as germination (Table 3). Emergence was high from all planting depths for large seeds after all levels of injury. Moderate or severe injury decreased emergence at all planting depths for medium kernels. Emergence was always poorer from small kernels than from large or medium kernels, particularly at the greater levels of frost injury.
Discussion
Frost damage was obviously selective, affecting some kernels and leaving others in sound condition. This selectivity. which probably was associated with differences in maturity among tillers or among kernels on the same spike. has important implications for the use of frost-damaged wheat for seed.
Deficiencies in test weight, germination, and emergence were caused by the increased content of small kernels in frost-damaged seedlots. Moderate or severe This publication from the Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information is available from http://www.ksre.ksu.edu. | v3-fos |
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} | s2 | Progesterone receptors in normal mammary gland: receptor modulations in relation to differentiation.
The biological basis for the observed modulation in cytoplasmic progesterone receptors (PgR) of normal mammary gland occurring during mammary development was investigated. Specifically, the relative roles of hormones vs. differentiation on (a) the decrease in PgR concentration during pregnancy and lactation and (b) the loss of mammary responsiveness to estrogen during lactation were examined. PgR were measured using the synthetic progestin, R5020, as the ligand. The hormones estrogen and progesterone were tested in vivo for their effect of PgR concentration. Mammary gland differentiation was assessed morphologically and by measuring enzymatically active alpha- lactalbumin. These studies show that there is a stepwise decrease in PgR that occurs in two stages. The first decrease is completed by day 12 of pregnancy and the second decrease occurs only after parturition. There appears to be a hormonal basis for the first decrease and it appears to be caused by the negative effect of progesterone on estrogen- mediated increase in PgR. In direct contrast, the absence of PgR during lactation and the mammary tissue insensitivity to estrogenic stimulation of PgR were not related to the hormonal milieu of lactation but were directly related to the secretory state of the mammary gland and lactation per se.
For steroid hormones to produce a biological response, it is believed that they must initially interact with their macromolecular cytoplasmic receptors (l7) . In target tissues for estrogen and progesterone such as the uterus and certain normal and neoplastic mammary tissues, cytoplasmic estrogen receptors (ER) and progesterone receptors (PgR) are present. Furthermore, PgR synthesis in these tissues appears to be under estrogenic control and thus PgR can also serve importantly as markers of estrogen action (13-15, 20, 26, 28, 37, 38, 41, 43). We have previously reported that PgR concentration per cell varies with the developmental state of the mammary gland; PgR are present in virgin gland, decrease during pregnancy, are undetectable during lactation, and reappear during lactational involution (14) . Most striking is the observation that not only are PgR absent during lactation but also that estrogen administration to lactators fails to result in the increased concentration of mammary PgR. This inability to respond to estrogen is specific to lactating mammary tissue because (a) uteri of lactating mice respond to exogenous estrogen with increased PgR levels and (b) in virgin mammary gland the level of PgR is augmented by estrogen administration in a manner similar to that for the uterus (13) . The present studies were undertaken to determine the basis for (a) the observed modulation in PgR concentration during mammary development and (b) the loss of mammary responsiveness to estrogen during lactation. We found that there was a hormonal basis for the decrease in PgR concentration that occurs during pregnancy and that this decrease was most likely attributable to progesterone. In contrast, the absence of PgR during lactation was not related to the hormonal milieu of lactation but was directly related to the secretory state of the gland and lactation per se .'
Animals
Female BALB/c mice were used at 2-5 mo of age and were from our own colony, Intact or ovariectomized virgin, pregnant, or lactating mice were obtained and used as described previously; ovariectomized virgin mice were used 14 d after ovariectomy (13,14). In certain experiments, animals were ovariectomized and hysterectomized on day 14-16 of pregnancy, and hormone injections were initiated 24 h later . Unilateral thelectomy (nipple removal) was accomplished by cauterization before mating ; litter sizes were adjusted to six pups .
Hormones
All hormones used for injections were purchased from Sigma Chemical Co ., St . Louis, Mo . Unlabeled R5020 was purchased from New EnglandNuclear. The hormones were administered by subcutaneous injection as a solution in 1% ethanol in saline or in sesame oil.
Steroid Receptor Assay
Tissue homogenates were prepared in a phosphate-glycerol buffer (5 mM sodium phosphate, 10 mM thioglycerol, 10% glycerol, pH 7.4) and centrifuged at 12.350 g for 1 h. Mammary glands were homogenized at a concentration of I gm/ml. Unless otherwise specified, the superriates designated as cytoplasmic extracts were used for steroid receptor assays. For measurements of PgR, aliquots of cytoplasmic extracts were incubated with 20 nM of f`H]R5020 alone or in the presence of a 100-fold excess of unlabeled 85020. All cytoplasmic extracts were incubated with hormones at 4°C for 4 h before assay. The bound readioactive steroid in all the incubations was estimated using the dextran-coated charcoal assay of Korenman (18) as described previously (42) for mammary glands .
Some further comments on the estimation of PgR concentration in the present studies using R5020 as the ligand are necessary. Previously, we have reported that dexamethasone can significantly compete for certain specific 85020-binding sites; however, these binding sites did not appear to be high-affinity PgR (42) . Subsequent studies have revealed that inclusion of dithiothreitol (DTT) in the buffer augments the degree of competition by dexamethasone to specific R5020binding sites, and this is compatible with recent observations in our laboratory that the glucocorticoid-binding sites in mammary tissues are stabilized by DTT (24). Therefore, in the present studies, DTT was not included in the homogenizing buffer; however, it should be mentioned that even when DTT was excluded, some dexamethasone competition was still observed. In all cases, even if the binding data are corrected for dexamethasone competition, the relative results remain unchanged. For these reasons and because the precise identity of these dexamethasone-competible sites is as yet unclear, the specific binding data reported in these studies do not include any correction for competition by dexamethasone to specific 85020-binding sites. We have also determined that endogenously bound progesterone would be exchanged 100% by 85020 under the present assay conditions' in a manner similar to that reported for mouse uterus (36) ; thus, values for specific 85020 binding represent the total number of cytoplasmic PgR. a-Lactalbumin Measurement a-Lactalbumin activity was assayed by the method of Ip and Dao (16), based on that of Ebner et al . (10), with modifications as follows. Mammary tissue was homogenized with one 15-s burst of a Polytron PT 10-ST (Brinkmann Instruments, Inc., Westbury, N. Y.) . Homogenates were not centrifuged but used after filtration through organza . The reaction mixture contained either 20 or 50 JAI of homogenate, 2 paint Tris-HCI buffer (pH 7.4), 1 pmol MnClt, 60 nmol UDPgalactose (supplemented with --15,000 cpm UDP-["C]galactose [New England Nuclear]). The total volume of the reaction mixture was 100 pl. a-Lactalbumin activity was estimated in the presence of excess bovine milk galactosyl-transferree (5 mU ; Sigma Chemical Co.) and 2 iumol of r)-glucose acceptor to form ("C]lactose. A standard curve was generated with increasing amounts of bovine a-lactalbumin to ensure that the reaction was in the linear portion of the curve with respect to a-lactalbumin concentration. To correct for the nonspecific production of ["C]galactose resulting from endogenous hydrolysis of UDPz S. Z. Haslam and G. Shyamala, unpublished observations. ['"C]galactose, a control reaction was included for each sample, using distilled water in place of glucose as theacceptor . The incubations of thereaction mixture were carried out at 37°C for 30 min in a shaking water bath ; the reaction was stopped by cooling in ice and the addition of I00,ul of cold water. The content of each tube was passed through a column (0.5 x 4 cm) of Bio-Rad AG 1-X2 anion exchange resin (Bin-Rad Laboratories, Richmond, Calif.) in the chloride cycle. Reaction tubeswere washed with 0.5 ml of water that was then transferred to the columns. Neutral sugars on the column were eluted with another I ml of water directly into scintillation vials. Radioactivity in the eluate was measured with 10 ml of formula 950A (New England Nuclear) by liquid scintillation counting. Recovery of neutral sugars from the column was >80% with no elution of UDPgalactose. The product of the enzymatic reaction was identified as lactose by paper chromatography (34) .
To assess mammary gland morphology and extent of LAD, mammary tissue samples were prepared for histological examination as described previously (14) . The extent of LAD was evaluated by estimating the percentage of mammary tissue that was represented by epithelial cells in lobuloalveolar organization . Sections of mammary tissue in which --50% of the epithelial cells were lobuloalveolar were scored as ++, whereas sections in which close to 100% of the epithelial cells were lobuloalveolar were assigned a score of ++++ . An average of 10 pieces of tissue were examined microscopically for each experimental condition that was evaluated for LAD.
DNA content of the tissues was estimated according to Ceriotti (3), and protein concentration was assayed according to Lowry et al. (22).
The Pattern of PgR Modulation in Relation to Mammary Gland Differentiation in Intact Mice
Our earlier studies indicated that mammary PgR concentration was modulated as a function of mammary gland development . Namely, we found that PgR were abundant in virgin gland, were decreased during pregnancy, and were totally undetectable during lactation (14). To identify the physiological basis for these observed modulations in mammary PgR, in the following studies we examined the relationship between mammary PgR and mammary differentiation. There are a number of morphological and biochemical criteria that can be used to measure the progression of mammary gland differentiation from the undifferentiated ductal epithelium of virgin mice to the fully differentiated secretory lobuloalveolar epithelium of lactating mice . In the present studies one morphological criterion and one biochemical criterion were used, namely degree of lobuloalveolar differentiation (LAD) and amount of a-lactalbumin activity . In mice, LAD occurs mainly during pregnancy, whereas a-lactalbumin activity, which is barely detectable during pregnancy, increases dramatically around parturition and reaches peak levels during lactation (25).
The results of the studies on the relationship between mammarygland differentiation and PgR levels during normal mammary gland development in intact untreated mice as summarized in Table I reveal two stages of PgR modulation. The first 80% reduction in mammary PgR is completed by day 12 of pregnancy and the second reduction, resulting in a total loss of PgR, occurs after parturition . It is of interest that after day 12 of pregnancy, although there is no further decrease in mammary PgR, the gland continues to become progressively more differentiated as indicated by the increases in a-lactalbumin activity and LAD (Fig. I a _f ). Thus these data suggested that there is not a causal relationship between mammary PgR and differentiation . This raised the possibility that the factors responsible for the decrease in PgR were also simultaneously leading to mammary differentiation .
The Effects of Estrogen and Progesterone on Mammary PgR and Differentiation
The two major hormones of pregnancy, estrogen (E) and progesterone (P), are known to influence that PgR level in various target tissues (20,25,38,43) and are also known to cause epithelial cell proliferation and LAD in mammary glands of ovariectomized virgin mice (2,29) . For these reasons, in the following experiments the simultaneous effect of E and P on mammary PgR levels and differentiation was examined. To distinguish the direct effects of the hormones on PgR levels The morphology of mammary glands of ovariectomized virgin mice after estrogen and progesterone treatment is shown in Fig . 2 a-e . After treatment with oil, E or P alone for 7 or 14 d or E and P for 7 d, mammary glands were predominantly ductal and thus undifferentiated . In contrast, the mammary glands of mice treated with E and P for 14 d had begun differentiation as indicated by the presence of alveoli .
The effect of E and P on mammary PgR concentration in ovariectomized virgin mice is shown in Fig. 3 . Administration of E alone for either 7 or 14 d resulted in a significant increase in mammary PgR . Administration of P alone for either 7 or 14 d resulted in PgR levels similar to those of vehicle control, whereas P in combination with E for 7 d resulted in PgR values not significantly different from those obtained with E alone . However, administration of P in combination with E for 14 d resulted in a 20% decrease of mammary PgR as compared to treatment with E alone . Although it was clear that E could cause an increase in mammary PgR and that P can cause a decrease in PgR, the 20% reduction in PgR caused by progesterone under the above experimental conditions could not account for the 80010 reduction in PgR that is observed during pregnancy . This raised the possibility that during pregnancy the loss of PgR might have resulted from either a decreased sensitivity of mammary tissue to estrogen or an increased ability of progesterone to decrease PgR. The following experi-ments were carried out to distinguish between these two possibilities.
In these studies, 24 h before the initiation of hormone treatment, pregnant mice (14-16 d) were ovariectomized and hysterectomized to remove the major sources of endogenous hormones, and then the effects of E and P on mammary PgR were tested. The results of these experiments are presented in Fig . 4 . Withdrawal of hormones for 24 h (time zero control) or 5 d (vehicle group) resulted in a decrease in PgR, whereas administration of E alone produced a significant increase in PgR . In contrast to its effect in mammary glands of ovariectomized virgins, P in combination with E produced a greater decrease in PgR (6001o vs. 20%) and the PgR concentration was similar to that observed in intact pregnant mice. Thus the data in Fig . 4 clearly indicated that the decrease in mammary PgR during pregnancy was not the result of a decrease in estrogenic sensitivity of the tissue with respect to PgR increase but rather was the result of an enhanced ability of progesterone to decrease PgR . Furthermore, it also demonstrated that the differentiation of mammary gland in itself did not alter the mammary tissue responsiveness to estrogen . Consequently, it was also clear that factors other than mammary gland differentiation that occurs during pregnancy must have been responsible for the total loss of PgR and the loss of responsiveness to estrogen that is observed after parturition . This led us to consider that lactation and secretion per se might have an independent effect on PgR concentration and responsiveness to estrogen .
Relationship of Lactation to PgR Modulation
To identify the precise effect of lactation on PgR, we felt that it was necessary to dissociate the hormonal milieu of lactation from the secretory state of the tissue. It is well known (7) that, in addition to the appropriate hormonal milieu, initiation and maintenance of copious milk secretion requires the physical removal of milk; this is accomplished in nature by 73 4 THE JOURNAL Or CELL BIOLOGY " VOLUME fib, 1980 suckling . By preventing suckling on one side of a lactating mouse, using the technique of unilateral thelectomy (nipple removal), we were able to obtain fully lactating mammary tissue from nipple-intact glands and nonlactating mammary tissue from the thelectomized glands from the same postpartum mouse (40) . As can be seen from Table II, thelectomy in itself had no effect on mammary PgR in virgin and pregnant animals and it also did not prevent the decrease in PgR observed during pregnancy. A significant difference between PgR levels of intact and thelectomized glands was detectable only in postpartum mice ; in this case, PgR were always detectable in the thelectomized, nonlactating glands but were absent in the contralateral intact lactating glands . Table II also shows that although thelectomy had no effect on mammary differentiation during pregnancy as assessed by a-lactalbumin activity, in postpartum mice the thelectomized glands had less a-lactalbumin activity compared to the intact lactating glands . The low a-lactalbumin activity of thelectomized glands was attributable to the non-lactational state of the gland and was corroborated histologically (Fig . 5a-d) .
We next examined the effect of estradiol on PgR of mammary glands in unilaterally thelectomized mice . These results are presented in Table III . We were consistently able to increase PgR concentration by exogenous administration of E in thelectomized, nonlactating mammary tissue, whereas PgR failed to be augmented by E in lactating nonthelectomized mammary tissue; the uteri of these animals also responded to E with increased level of PgR .
DISCUSSION
The results of our present studies indicate that as mammary glands of virgin mice differentiate there is a stepwise loss of PgR that occurs in two stages. The first decrease in PgR is completed by day 12 of pregnancy, whereas the second decrease occurs after parturition and results in a total loss of PgR in mammary tissue . Estrogen and progesterone appear to be the principal regulators of PgR concentration in mammary tissue ; estrogen increases PgR concentration, whereas progesterone reduces the concentration of PgR. In view of these hormonal effects on PgR concentration, and because estrogen and pro- gesterone are also major hormones of pregnancy, we believe that the decrease in PgR occurring during pregnancy is attributable to the negative effect of progesterone on PgR . Because estrogen and progesterone also cause differentiation of the mammary gland, the net effect of these hormones on both processes leads to the previously observed apparent inverse relationship between mammary PgR and differentiation (14). However, it does not appear that the decrease in PgR and differentiation are causally related, because during pregnancy the progression of differentiation as determined by LAD and a-lactalbumin activity did not result in a progressive loss of PgR . The second decrease in PgR, which occurs at postpartum, appears to be specifically related to the secretory state of the gland rather than to a negative effect of the hormonal milieu of lactation on PgR .
The mechanisms by which cytoplasmic mammary PgR are decreased either by hormones or during lactation are not known. As mentioned earlier, under our present assay conditions we are measuring total cytoplasmic PgR and thus it is not likely that the reduction of or lack of PgR is attributable to endogenously filled sites . But because we only measured cytoplasmic PgR, it is conceivable that reduction of PgR could be the result of a high concentration of nuclear bound PgR . However, studies on the nuclear translocation and retention of PgR in uteri of a number of mammalian species indicate that a relatively small fraction of total cytoplasmic PgR are ever translocated to the nucleus (4,45) . An alternative explanation for reduced level of PgR may be the degradation/deactivation of existing PgR and the failure of new PgR to be synthesized . In view of a number of reports on the ability of molybdate to stabilize glucocorticoid and progesterone receptors (26,(30)(31)(32) in various target tissues, we tested its effect on mammary PgR . We found that 20 mM molybdate added to homogenization buffer did not enhance PgR binding in virgin mammary tissues and, most importantly, it did not reveal masked or unapparent PgR in lactating mammary tissue .' However, we cannot rule out the possibility that PgR are indeed resynthesized but remain sequestered in the nucleus in a form not bound to hormone . Although examples of this latter phenomenon are lacking in normal target tissues, certain human mammary ' S. Z. Haslam and G. Shyamala, unpublished observations . carcinoma cell culture lines have been demonstrated to possess unoccupied nuclear receptor sites for estrogen (12,47). Although further studies are obviously required to determine what mechanisms are operating to decrease PgR during lactation in normal mammary tissues, it should be emphasized that whatever mechanisms are operative they are specific to the secretory state of the gland.
The present studies have demonstrated for the first time that progesterone can decrease the concentration of its own receptor in mammary tissues, which agrees with similar findings in uterine tissue (4,21,27,44,45); this lends further credence to the concept that progesterone's effects may also be receptormediated in mammary tissue . The present studies also revealed some important information about the ability of progesterone to modulate estrogen action in mammary tissue . Progesterone when administered in combination with estrogen for 7 d failed to affect the estrogen-mediated increase in mammary PgR and, during this period, the mammary glands were predominantly ductal and thus undifferentiated . In contrast, when progesterone was administered with estrogen to pregnant animals whose mammary glands were extensively lobuloalveolar and thus differentiated, it significantly decreased mammary PgR when administered with estrogen . Thus, it is tempting to speculate that the ability of progesterone to decrease PgR may depend on the state of mammary differentiation and also that this ability may be acquired during lobuloalveolar differentiation. This speculation might explain the results of recent studies on the transplantable urethan-induced mouse mammary carcinoma MXT-3590, which is of ductal origin . In this tumor, estrogen can augment both tumor growth and PgR concentration but progesterone fails to antagonize tumor growth (46) . It is possible that the inability of progesterone to antagonize estrogen-mediated tumor growth is a reflection of the ductal origin of the MXT-3590 tumor. The effects of estrogen and progesterone on PgR have also been examined in dimethylbenzanthracene-induced primary rat mammary tumors, and from this study it appears that there can be a dissociation between estrogenic regulation of mammary growth and PgR (15) . However, that PgR, in both hormone-dependent mouse and rat mammary tumors, are under acute estrogenic regulation is most comparable to the situation present in mammary tissue of virgin and pregnant mice, but distinct from the estrogen-insensitive state of lactating mammary tissue .
The effect of estrogen to increase and progesterone to decrease PgR has also been reported for uterine tissue (1, 6,20,28,38,43). However, it is not known whether uterine cytodifferentiation acts to modify the response of the uterus to hormones, as occurs in the mammary gland. In this regard, differential responsiveness of cells to P or E and P have been described in oviduct development and function, and such differences appear to be determined by the types of cells present and by the stage of oviduct development (32,33,35). Also, in recent studies of estrogen action and estrogen antagonists in the rat uterus, it has been proposed that the cell type (endometrial vs . myometrial cells) may determine that nature of the biological response to the hormone antagonists (5).
The mechanisms by which lactation results in mammary gland estrogen insensitivity, and how this might be reversed, are currently being investigated . Understanding how cells modify their requirement for, or response to, growth regulatory molecules such as hormones is critical to our understanding of the basis of the loss of regulation that occurs in certain disease states such as neoplasia. | v3-fos |
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} | s2 | 1979 Research Results Southeast Kansas Branch Agricultural Experiment Station
Southeast Kansas Experiment Station is in its 30th year of operation. The emphasis has changed over this period to reflect changes in agricultural emphases of the area. The professional research staff consists of four scientists, each with a broad area of research responsibility. Together they emphasize improvement in crop production, forage production, beef cattle production and soil and water management.
INTRODUCTION
Southeast Kansas Experiment Station is in its 30th year of operation. The emphasis has changed over this period to reflect changes in agricultural emphases of the area. The professional research staff consists of four scientists, each with a broad area of research responsibility. Together they emphasize improvement in crop production, forage production, beef cattle production and soil and water management.
The criterion for undertaking a research project is its expected contribution to improving agriculture in southeastern Kansas. Research at the Station contributes beyond the 15 counties of responsibility but the Branch Station scientists put priority on area needs.
The purpose of our research and this publication is to serve Southeast Kansas Agriculture. We hope these reports will interest and benefit our readers. We welcome suggestions to improve our efforts to improve Agriculture and, thus, improve life for all. The small-grain-variety test is to help southeastern Kansas growers select winter wheat, barley, and spring oat varieties best suited for the area.
Gains of Yearling Steers on
Procedure: In 1979, 22 wheat varieties, six barley varieties, and eight spring oat varieties were compared.
Results: Wheat yields in 1979 were a record after unusually cool spring delayed heading and was followed by ideal cool weather and adequate rainfall during flowering and grain filling. Hart, a soft wheat, was the top yielder at 80 bushels per acre. The average for all varieties was 71 bushels per acre, with a range from 55 to 80. Complete wheat variety results are compiled in Kansas Agric. Experiment Station Report of Progress 370, available at county extension offices.
Barley varieties averaged 65 bushels per acre, with Paoli the high yielder at 82 bushels.
Spring oat varieties also set record yields, ranging from 131 to 152 bushels per acre. Barley and spring oat variety results appear in Tables 1 and 2. Wheat seeding rates in southeastern Kansas have gradually increased the past several years, as more semi-dwarf and soft wheat varieties are grown. But the effect of seeding rates on semi-dwarf and soft wheat varieties has not been evaluated in southeastern Kansas.
Procedure: Beginning in 1978 four varieties (Trison, Newton, Centurk, and Abe) were seeded at 60, 90, and 120 pounds per acre. In 1979 Hart, a more popular soft wheat variety, was substituted for Abe.
Results: Wheat yields were outstanding in 1979 with the semi-dwarf (Newton) and the soft variety (Hart) yielding 68 bushels per acre. The 90and 120pound seeding rates were significantly better than the 50pound (1 bu.) rate. In 1978, when wheat was seeded in late November, the 120-pound rate was best for all varieties.
Effect of Fungicide Seed Treatments on Wheat Yields
Many fanners no longer treat seed wheat with a fungicide at planting time; however, where smutty wheat has been a problem, treating seed wheat with the proper fungicide is good managemen t.
Procedure: Since 1976 wheat has been grown continuous ly on the same soil site to evaluate fungicide seed treatments applied as planter-bo x formulatio ns. In 1979, Vitavax-25 08, Granox N-M and a control (no treatment) were compared.
Results: There was no yield advantage from fungicide seed treatments in 1979, although a year earlier fungicide treatments gave a slight yield benefit when wheat was planted in late November. Neither bunt nor loose smut has been a problem during the 4-year evaluation . Where either disease is present, fungicides generally increase yields.
6 EJfect of Ice Cover on Soil Surface Where Nitrogen is Applied to Winter viheat It is recommended that nitrogen be applied to winter wheat in late winter when there is no frozen ice cover on the soil surface. But the amount of nitrogen loss under these conditions has not been fully determined. Procedure: In 1979 nitrogen, as urea, was applied (l) in February when there was a 2 to 3-inch ice layer and (2) when the ice cover was melted.
Results: Yields were about 5 bushels per acre less when N was applied on the frozen ice cover than when applied after the ice cover had melted. However, nitrogen applied on the frozen ice increased yields 5 bu/acre over the control (no nitrogen).
Fall and Spring Nitrogen Applications Compared for Wheat
When winter precipitation is above normal, farmers in southeastern Kansas wonder if much nitrogen is lost after being applied to wheat in the fall. They also wonder if semi-dwarf wheat varieties tolerate higher N rates without lodging.
Procedure: In 1979 we compared fall and spring N applications at 30, 60, and 90 N pounds per acre on Trison, a standard height variety, and Newton, a semi-dwarf.
Results: Fall and spring N applications showed no significant differences in yield or grain protein. Lodging from high N rates was not a problem with either variety. Optimum yields were obtained with 60 pounds of N per acre, although grain protein increases paralled applied nitrogen increases.
There was no significant yield difference between Trison and Newton in 1979 (Table 4), nor was there any interaction between variety and N rate. Results: Grain yields averaged 126 bushels per acre for all hybrids, with a range of 86 to 159 bushels per acre. Good growing conditons early in the season and adequate moisture during the critical grain-filling period led to high yields. Complete grain sorghum yield results are compiled in Agric. Expt. Station Report of Progress 375, available at county extension offices.
Grain Sorghum Hybrids and Planting Dates Compared
Grain sorghum hybrids with different plant maturities are planted from late April through early June in southeastern Kansas. Hybrid maturity and planting date are chosen to avoid flowering during the normal hot, dry period of late July and early August. More information is needed, however, to determine optimum planting dates with respect to hybrid maturities.
Procedure: Beginning in 1979 six grain sorghum hybrids -representing early (Pioneer 8790), medium (Acco lOlGR, Acco 1089, and Pioneer 8585), and medium-late (Pioneer 8272 and Prairie Valley 708 GR) maturities, were planted on four dates (May 16 and 31, June 13, and July 3). 8 Res.ults: In 1979 the highest yields came from the mid-June planting, regardless of hybrid maturity. The early July planting was too late for the medium and 1onger season hybrids, as they did not reach physiological maturity befQre the first killing frost. * Some bird damage early.
Fall and Spring Nitrog en Applic ation Compa red on Grain Sorghu ms Nitrog en norma lly is applie d to grain sorghum in the spring in southe astern Kansas . Depend ing on labor and weath er, late fall might be advant ageous some years, so resear ch is needed to determ ine if fall applic ations result in signif icant N losses in clay-p an soils.
Proced ure: For the past three years, fall and spring N applic ations on grain sorghum were compa red at four rates (40,80 , 120 and 160 pounds per acre). The N source s, urea and anhydr ous ammon ia, were also compar ed in this study.
Result s: Small , but signif icant, yield benef its favor spring N applic ations. Yield differ ences betwee n urea and anhydr ous ammon ia were not signif icant. Nitrog en rates exceed ing 120 pounds per acre have not increa sed yields under drylan d condi tions. Lower N rates, 80 to 100 lbs/A, result ed in greate r fertil izer effici ency (Table 6). Table 6. Effects of nitrogen rates, nitrogen carriers and application times on grain sorghum yields, Parsons, 1979 and1977-79 Though more sensitive to herbicide injury than corn, grain sorghum is an important cash and feed crop in southeastern Kansas. The main concern with grain sorghum herbicides is to select a combination that control weeds without excessively injuring the crop.
Procedure: In 1979 we compared grain sorghum herbicides in conventional tillage and no-till systems.
Results: Where velvetleaf was a major problem, AAtrex and/or Bladex in several combinations with Sexton, and Ramrod/atrazine gave good control (Table 7). Incorporating Igran + AAtrex shallow reduced crop injury but also reduced velvetleaf control somewhat. AAtrex, applied after sorghum emerged, gave good control of small velvetleaf (less than 2 inches tall).
Modown, a newer broadleaf herbicide, gave fair control of velvetleaf.
Bicep at 2.7 lbs active ingredient per acre -incorporated lightly before planting or applied shortly after planting -did not control velvetleaf adequately. Even though the seed (Funk's 623GBR) had been treated with Concept, Bicep caused some early stunting injury and delayed maturity somewhat.
In the no-till study (Table 8 ), grain sorghum was planted no-till with a Buffalo slot-shoe planter into the previous years' grain sorghum residue that had been mowed. Most herbicides except Bicep, gave poor crabgrass control. Bicep controlled crabgrass longer than did tank mixtures containing Bexton or Igran. · Paraquat effectively controlled winter annual species and small annual weeds present in late April. All Paraquat treatments, along with some of the residual tank mixes, were applied in late April when weeds were less than 4 inches tall. The remaining residual herbicide treatments were applied after planting and before sorghum emerged, or 10 days after the initial Paraquat application. Although corn acreage is limited in southeastern Kansas, it is an important cash and feed crop for many farmers. Keeping the crop clean of troublesome weeds is highly important in achieving optimum yields.
Procedure: In 1979 we evaluated several different herbicide combinations on three different soil sites harboring various weed species.
Results: Preplant incorporated herbicides (Eradicane and Sutan+) in combination with AAtrex and/or Bladex effectively controlled crabgrass, giant foxtail, velvetleaf and smooth pigweed.
At another site AAtrex and/or Bladex in a tank-mix with an anr1~al n,rass herbicide (Lasso, Dual, or Prowl) applied before corn emerged effectively controlled smooth pigweed and crabgrass. AAtrex controlled pigweed better than Bladex did, and Bicep controlled pigweed and crabgrass.
At one location where yellow nutsedge was a problem, Dual seemed to be more effective than Lasso.
None of the herbicides tested suppressed perennial problem weeds like climbing milkweed.
Soybean Variety Performance Test
Southeastern Kansas is the leading soybean producing area in the state, so extensive research is devoted to variety testing.
Procedure: In 1979, 42 soybean varieties, of private and university origin, were compared at the Columbus field in Cherokee county.
Results: Extremely dry weather during September and early October severely depressed soybean yields of later-maturing varieties. Varieties of Group III and early Group IV maturity yielded 30 to 35 bushels per acre, while later-maturing (Group IV and Group V) varieties yielded 20 to 30 bushels per acre. Complete soybean variety results are compiled in Agric. Expt. Station Report of Progress 376, available at county extension offices.
Soybean Varieties and Planting Dates Compared
The growing season in southeastern Kansas permits farmers to plant soybean varieties of various maturities over a wide range of dates. In general, full season varieties planted in June have yielded best, however, some of the newer varieties have not been evaluated over a wide range of planting dates.
Procedure: Since 1976 soybean varieties representing a wide maturity range have been planted from May through July. In 1976 varieties planted included Williams, Cutler 71, Crawford, Essex, and Forrest. In previous years, later maturing varieties (Tracy and Bragg) had been planted.
Results: Soybeans yields in 1979 were not normal for southeastern Kansas ·because September and early October were unusually dry. As a result, later-m~turing varieties, caught in the pod-filling stage during the dry period, yielded below normal.
Four years of testing indicate that varieties of Essex maturity and earlier can be planted as late as July 10 in extreme southeastern Kansas, while Forrest normally should not be planted later than June 25. Varieties later in maturity than Forrest, like Tracy and Bragg, do not mature soon enough for southeastern Kansas.
Effects of Row Spacing on Soybean Yields with Full Season Varieties.
Narrower rows have been advocated as a way to boost soybean yields. How narrow rows affect longer season varieties grown in southeastern Kansas has not been studied.
Procedure: For the past three years, we planted Essex and Forrest at 7-, 14-, 21and 30-inch row spacings, with seeding rates adjusted for the different row spacings, with the per-acre rate nearly the same regardless of row spacing.
Results: With full season Group V varieties, narrower rows have not increased soybean yields. In 1979, the 21-inch spacing gave a slight but not significant advantage. Full season varieties seem to react differently to narrow row spacings than the shorter season varieties do north of here where narrow rows have increased yields substantially.
Effects of Cropping Sequence on Soybean Yields
Soybeans are the number one cash crop in southeastern Kansas, and they are grown in several cropping sequences. More information is needed, however, to determine how different cropping rotations influence soybean yields, residual fertility, crop residues, and results from double cropping.
Fluid Lime and Ag Limestone Compared
Interest in fluid lime developed several years ago when commercial, liquid-fertilizer vendors promoted applying a lime suspension with sprayer equipment.
Procedur e: Since 1977, we have been comparin g a liquid lime suspensio n with agricultu ral limeston e on an acid soil producing soybeans . An initial rate of 5,000 pound effectiv e calcium carbonat e (ECC) per acre has been compared with annual ECC rates of 500 and l ,000 pounds per acre applied each spring before spring tillage.
Results: After three years, results indicate that liquid lime suspensi ons and agricultu ral limeston e cause similar changes in pH. Low rates of lime (500-1000 pounds ECC per acre) require yearly applicat ions to raise the soil pH to an optimum level.
Residual Effects of Phosphor us on Soybean Yields
Many soils in southeas tern Kansas are low in availabl e phosphor us. When phosphor us fertilize r is applied, part of it becomes unavaila ble over time and cannot be taken up by the plant-ro ot system. How much phosphor us fixation results from residual P applicat ions is not fully known for our clay-pan acid soils.
Procedur e: Beginnin g in 1978, we initiated comparis ons to see if heavy, first-ye ar applicat ions (200 pounds P 2 o 5 per acre) would be as effectiv e for soybeans as 100 pounds P 2 o 5 per acre applied every other year, or as effectiv e as annual applicat ions of 50 pounds per acre. After 4 years, all plots will have received the same amount of P 2 o 5 . The two P sources used were diammoniu m orthopho sphate (AOP, 18-46-0) and ammonium polyphos phate (APP, 15-62-0) .
Results: Yields have increased 2 to 5 bushels per acre as a result of the applied phosphor us on a silt loam soil testing 10 pounds P/A. Two years after the 100-and 200-pound rates were applied, phosphor us responses are still good. Likewise , the annual 50 pounds P 2 o 5 per acre increased yields signific antly. Our recent results support earlier work that showed soybean yield response s from P fertiliza tion are normally signific ant on soils testing less than 15 pounds of availabl e P per acre.
Effects of Phosphor us and Potassium Fertiliza tion on Soybean Yields
Soybeans have not responded consiste ntly to fertilize r applicati ons in southeas tern Kansas. More research is needed to determin e under what soil condition s a fertiliz er response is likely.
Procedur e: In 1979 three location s in Cherokee county were fertilize d with various rates of phosphor us and potassium according to soil test. Fertiliz er was broadcas t and incorpor ated before planting .
Results: 1979 results confirm earlier work, which indicated that soybean response to phosphor us and potassium likely will be small where soil P tests in the 20's or more and soil K exceeds 100 pounds per acre. However, under some medium testing soil conditio ns a 3-to 5-bushel response from phosphat e and potash has been observed , though not consista ntly. Yield benefits from added fertilize r have been more positive where both phosphat e and potassium were applied to low-test ing soils. Soybean Response to Fertilizer Applied Ahead of Wheat in a Double-cropping f<otation Double-cropping wheat and soybeans is common in southeastern Kansas, but farmers seldom apply more phosphorus and potassium to the wheat when soybeans follow in a doublecropping rotation.
Procedure: In 1976 we established a study to determine how applying phosphorus and potassium to wheat would influence soybeans that follow the same year. We also included lime as a variable. The study was on a site that tested medium in soil phosphate and low in potassium; pH was 5.8.
Results: Wheat yields the past three years have increased substantially from the additional phosphorus, but lime and potassium have had little effect. Soybeans have benefited from the lime, but the residual phosphate and potassium from the preceding wheat crop have not increased soybean yields significantly. (Table 10) Table 10. Soybean response to fertilizer applied to preceding wheat crop in a double-cropping rotation, Parsons, 1979Parsons, . 1977Parsons, -79 1977 Fertilizer, lbs/a 1' Procedure: In 1979 the main emphasis was evaluating soybean herbicides to control broadleaf weeds common in many southeastern Kansas fields. Herbicide studies were on sites heavily infested with velvetleaf, cocklebur, and annual morningglory.
Results: Sencor and/or Lexone at 0.25 a.i./acre effectively controlled velvetleaf, regardless of soil type. However, a near maximum labelled rate (0.38 to 0.50 a.i./acre) Sencor or Lexone was needed to control cocklebur in this silty clay loam soil, even then control was erratic. Velvetleaf and cocklebur control appeared to be improved when herbicides are applied after planting but before crop emergence rather than incorporated before planting.
Control of velvetleaf by Lorox on a medium textured soil required 0.75 lb a.i./acre. Higher rates severly damaged soybean germination, although 1.0 lb a.i./acre caused no injury on soils with more clay content. But cocklebur control was only fair even at the higher rates.
Goal, a newer broadleaf herbicide, controlled velvetleaf at 0.38 lb a.i./acre, but that rate severly reduced soybean germination on a medium textured soil. Likewise, Modown at l .5 lbs a.i ./acre reduced germination and gave poor velvetleaf control. Incorporating Modown before planting reduced damage, but the incorporation must be shallow for acceptable broadleaf control.
Basagran, applied after soybeans emerge gave excellent velvetleaf and cocklebur control. Morningglory control was fair. Blazer, another postemergent, broadleaf herbicide, did not control velvetleaf so well as Basagran did, but it controlled cocklebur and morningglory as well as Basagran. Blazer appeared to cause slightly more leaf burning than Basagran, but plants recovered within 10 days.
In a no-till study where soybeans were planted into existing wheat stubble, pigweed was satisfactorily controlled by all herbicides tested. More research is needed, however, to evaluate herbicides and rates in no-till wheat stubble. l/ Applied after planting and before soybeans emerged.
2/ Applied after soybeans emerged; cocklebur plants were less than 6 11 tall, velvetleaf plants were less than 2" tall, and morningglory had not started to vine.
Heavy weed pressure from cocklebur, velvetleaf, and annual morningglory.
Basagran and Blazer treatments resulted in temporary leaf burning, but plants recovered within 10 days.
There were no noticeable crop injury from the pre-emergent treatments.
Grain yields, not taken, were considered average.
LIVESTOCK INVESTIGATIONS, 1979
Richard J. Johnson and Lyle W. Lomas
General Information
Experimental cattle were from the Station herd or purchased locally to provide as much uniformity of genetic and environmental background as possible. All were individually identified and provided preventive health measures as needed.
Feed was grown on the Station, purchased locally, or from Kansas State University Department of Grain Science feed plant. All pastures were fertilized according to soil test information.
To minimize differences due to fill, beginning and final weights of all cattle on test were taken after an overnight shrink.
Where statistical significance is indicated, the difference between treatments would occur by chance alone no more than 5 in 100 times.
Because results reported are based on one year's study, they must be regarded as tentative. Further study may strengthen or weaken the apparent conclusions of any one-year trial.
The use of any brand names is for better communication only and not to be taken as an endoresement.
Gains of Yearling Steers on Brome Pasture with Energy Supplementation
Cool season grasses like fescue and smooth brome produce well during spring and again in fall, but not during the summer grazing period. So energy supplementation might economically maintain and improve summer gains of stocker cattle. This study compared supplementation at three levels with none and estimated possible returns from each level of supplementation.
Sixty yearling Hereford steers weighing approximately 525 pounds were innoculated for bovine viral diarrhea and blackleg and implanted with 36 mg Ralgro. Three days later they were allotted by weight to four groups of seven and four groups of eight each. On May 7 steers were placed one group to each of eight five-acre brome pastures. One group of seven head and one of eight were assigned to each of these treatments: l. .!.! Based on gain increment due to supplement.
Assuming the cost of the grain at six cents per pound, daily cost was 12, 24, 36 cents for treatments two, three, and four. There was the additional cost of labor to feed the supplement daily. However, the 12, 24 and 36 cents added cost resulted in 0.4, 0.8 and 1.0 pounds gain.
The pastures where steers were supplemented maintained grass growth much better than those with no supplement.
In a similar study over a shorter period, 32 steers averaging about 500 lbs were allotted by weight to four groups and each group placed on a ten-acre brome pasture. Two groups received no additional feed and two an energy concentrate feed (primarily ground grain sorghum) at two pounds per head per day from April 23 to August 13. Steers receiving only pasture gained 1.2 pounds per head per day compared with l .6 pounds by energysupplemented steers. The 0.4 pounds difference resulting from the supplementation is the same as in the previous study. The additional labor of daily feeding must be considered.
Although costs and returns may vary considerable, it is apparent that energy supplementation will be profitable under a wide range of economic conditions.
Comparison of Fescue and Bermudagrass as Summer Graze for Cow-Calf Pairs
Fescue, a cool season grass that thrives in southeastern Kansas, is highly useful for spring and fall grazing. Hot summer months reduce the growth and quality of cool season grasses so cattle on them gain little or nothing. A warm season grass, like Bermudagrass should improve summer gains. This study compared summer grazing on fescue, a cool season grass, with grazing on Bermudagrass.
Thirty-two cow-calf pairs, after two months on a fescue pasture were divided into two groups with 16 placed on fescue pasture and 16 on Bermudagrass.
Average weights for the 56-day period were as follows: The small net advantage of 15 pounds for the Bermudagrass grazed cows and 4 pounds for their calves came on grass that had suffered considerable winter kill and was badly weed infested, while the fescue had been only sparsely grazed and was lush when the trail began.
Lasalocid Compared with Rumensin for Yearling Steers on Pasture
For many years various feed additives have been tested for ability to increase gains or improve efficiency of feed utilization by cattle. One of the most effective has been monensin (Rumens in), a product of Elanco. Lasalocid, a product of Hoffman-LaRoche, Inc., is another one currently under investigation. To compare the two, 48 yearling steers weighing approximately 500 pounds were allotted to six equal groups. Each group was placed on a 10-acre brome pasture and two groups were assigned to one of the three treatments from April 23 to August 13, 112 days: .lJ Least significant difference, p < .05 ::: 0. 2 3.
The additional 2 pounds ene concentrate resulted in the greatest increment of gain, 0.37 pounds r Treatments 3 and 4 were significantly different (P<.05) from 2 ich also di from l. Lasalocid appears to have potential for i ing ins as a a itive to steers on pasture. It, however, does not have a roval r use in any cattle feeding regimen.
Comparison of Source of Nitrogen Fertilizer on Incidence of Grass Tetany
Grass tetany, an intermittent t n costly problem for southeastern Kansas livestock producers, most commonly occurs in cows with calves during a cool, wet spring with rapid, lush g of grass. This study was an attempt to see if different sources rt lizer nitrogen affected mineral levels in blood and plant tissue di tly and if there was any relation to grass tetany.
Interseeding and Fertilizing Lespedeza in Tall Fescue
Lespedeza, a warm-season annual legume, can be interseeded into tall fescue to effectively complement grass production. Managing the mixture for optimum production differs from managing a pure stand of tall fescue with regard to nitrogen fertilization. Two methods of lespedeza interseeding were tested, along with responses of fescue and lespedeza to spring and fall nitrogen applications, and to phosphate-potash fertilization. Interseeding methods tested were broadcast and 1 zip 1 -seeding, with a control. On April 9, 1979, 'Summit' lespedeza seed was broadcast at 20 lb/A or drilled with a Midland 'Zip' seeder at 12 lb/A. Plots receiving spring fertilizer were treated February 27, 1979. Fifty lbfA each of phosphate and potash were applied to half the plots. Nitrogen (N) treatments were control (0 N), 40 N in spring, 40 in fall, or 40 N spring+ 40 N fall. All plots were cut May 15, and lespedezaseeded plots were stand-rated and cut September 4. Fall N was applied September 7, and no further harvests were obtained in 1979 because of the dry fa 11.
First:cut yields contained only fescue because lespedeza seedlings were still small (Table 13). No yield differences were found between interseeding methods, nor between lespedeza-seeded and unseeded fescue. There was a small, nonsignificant (5% level) yield advantage by plots with 50 lb/A of phosphate and potash, and a highly significant (1% level) difference between plots that did and those that did not receive 40 lb/A of spring N.
Lespedeza stands were significantly reduced by spring N. Stands without spring N rated 1.4, while plots receiving 40 lb N/A in spring averaged a 3.2 rating, where 1 represents a perfect lespedeza stand. The two seeding methods produced similar stand ratings.
Second-cutting yields contained mostly lespedeza, so fescue-only plots were not cut. Hence, comparisons for second-cutting and total yields (cut 1 + cut 2) were only among interseeded plots. Broadcast-seeded plots yielded slightly but not significantly more than 'zip'-seeded plots. Adding phosphate and potash significantly increased second-cutting and total yields over control plots. Spring N decreased second-cut yields significantly compared with plots without spring N, but total yields did not differ with N treatment because differences in second-cut yields offset differences in firstcut yields. Jj Rated 1-5, where l is a perfect stand and 5 is no lespedeza. 2/ Anticipated third cutting did not materialize. First cutting was -practically all fescue, and second cutting was mostly lespedeza with some wann-season annual weeds. 3/ Inc 1 udes O N and 40 N fa 11 ( 9-7) treatments. 4/ Includes 40 N spring, and 40 N spring + 40 N fall treatments. 5/ Means of a column within a comparison, followed by the same letter -did not differ at the 0.05 significance level, while means followed by different letters differed at the 0.01 level.
J. L. Moyer, John Meisenheimer, and Tom Glick
The effects of low fertilizer rates on yield, composition, and quality of native hay were measured with or without spring burning on land managed by the Kansas Fish and Game Commission. Treatments, begun in 1976, consisted of burned and unburned blocks containing eight fertility levels -a control and 30 lb N/A with 0, 10, or 30 lb/A of phosphate and/or potash applied annually.
Yields in 1979 averaged 2.48 tons/A at 12% moisture, ranging from 1 .90 tons/A for the control to 3.03 tons/A for the 30-30-30 treatment. Phosphorustreated plots yielded significantly more than plots receiving no phosphate, 2.81 and 2.15 tons/A, respectively. Burning had no significant effect on yields in 1979. Soil phosphorus (P) was higher in burned than unburned plots, and higher in plots that received 30 lb P 7 o 5 ;A than in all other plots.
Soil K in 1979 was higher in plots receiving 30 Tb KO/A than in plots receiving no K. Neither soil pH nor soil organic mafter content were affected by any treatments.
Crude protein content of forage averaged 5.8% in 1979. Fertilizer treatments interacted significantly with burning because burned plots varied less with fertilizers than unburned plots. The latter often seemed higher in crude protein than burned plots. Highest protein contents came when N and K were applied; lowest in the N-P treatments. Forage digestibility was improved by burning the three previous years, but analysis of 1979 forage is yet to be done.
Burning increased the proportion of warm-season perennial grass in the forage in 1979.
Unburned plots averaged 65% warm-season grasses by weight, and burned plots 83%. Fertility had little effect on 1979 composition, since P did not reduce the warm-season grass percentage as it did in previous years. Tall fescue pastures often are fall-fertilized to improve fall and winter pasture. This study, started in August, 1977, was to determine if fall-applied nitrogen benefits spring fescue growth, particularly after a dry fall, and whether method or rate of application affects carryover.
In 1977-78, a wetter-than-normal fall, no spring response to fall N application was found. However, only 7.83 inches of moisture (50% of normal) were received from July through October, 1978, and 15% of the average October amount, so fall forage yields (November 30) averaged only 0.52 ton/A (12% moisture), with no yield differences among treatments.
Spring, 1979 fescue responded to both August and February N applications (Table 14 ). The 50-lb N rate produced the same yield applied spring or fall, but the fall 100-lb rate produced less than 100-lb applied in the spring. The 150-lb fall N application produced the same as the 100-lb spring treatment.
Overall, we found no real yield difference between 11 dribble 11 application of 28% N solution and broadcast dry urea. Optimum rates were usually about 100 lb/A in spring and 50 lb/A in fall. Twenty-four alfalfa varieties were seeded at 12 lb/A in spring, 1978, using 1.5 lb a.i./A of benefin (Balan) preemergence herbicide and 400 lb/A of 6-24-24 fertilizer preplant. Seven of the varieties originated at Federal and State experiment stations; the other 17 were from six seed companies. In 1979, 200 lb/A of 6-24-24 fertilizer were broadcast after the first cutting.
Total yields in 1979 averaged 4.76 tons/A (12% moisture). Yields of the varieties ranged from 4.48 to 5.10 tons/A for four cuttings, none significantly (5% level) different. Cutting dates were May 11, June 15, July 19, and August 21. Second-cutting yields differed significantly, and ranged from 1.02 tons/A or less for 'Cody', Pioneer 531, 1 Kanza 1 , and 'Olympic' to 1.30 tons/A for 'Pacer' and DeKalb 130. Observations for bloom date, lodging, and height were recorded, and plots were harvested September 18. Complete results of the test are in 1979 Kansas Sorghum Performance Tests (Agric. Expt. Sta. Report of Progress 375). Silage yields (70% moisture) averaged 22.5 ton/A, excluding two checks, 1 Atlas 1 and 'Early Sumac', which had poor stands. Silage yields ranged from 15 to 38 tons/A. Four hybrids yielded significantly more silage than the test average, and six others produced the equivalent of 60 bu/A or more of mature grain. Seven entries failed to produce mature grain. All entries bloomed 60 to 85 days after planting, but the test averaged only 29% dry matter, ranging from 24%-37%. Height to flag leaf ranged from 65 to 115 inches and lodging was from 0 to 56%. Studies were initiated in 1979 in Labette and Cherokee counties to study various fertility-tillage management systems for grain sorghum and soybeans. Previous work had indicated that no-till systems performed poorly. Our 1979 results are shown in Table 15.
Tillage systems used included conventional, reduced, and no-till. Several fertility management variables were included.
Results of the 1979 studies show exceptional grain sorghum yields. Reduced tillage gave yields equal to conventional tillage under several fertility management regimes. No-till yields were good, except where UAN was the N source and yields were reduced. Apparently, UAN broadcast on no-till plots was lost to volatilization, as tissue N analysis indicated.
The Labette county site (soybeans), where yields were reduced by a late season dry spell, gave different results. Conventional tillage resulted in highest yields over all fertility management systems, followed by reduced tillage, and no-till gave significantly lower yields than conventional tillage.
These initial results indicate that soybeans are more affected by tillage than grain sorghum. The studies will be continued to further evaluate these fertility-tillage management systems.
Effects of N-P-K Rates and Application Methods on Soybean Yields
Studies were initiated at two locations in southeast Kansas to evaluate rates of N, P, and K applications as well as application methods on yields and tissue composition of soybeans. Soil tests showed both sites low in available phosphorus and potassium. Potassium tripolyphosphate (0-26-25) liquid fertilizer was used to facilitate knifing in of the P and K. The broadcast treatments were applied and incorporated with a springtooth. All fertilizer applications were preplant. Results are shown in Table 16.
Soybean yield response due to fertilizer treatments was not significant in 1979 at either location although adding P and K increased yields somewhat. In Neosho county, knifed applications gave higher yields than broadcast and significantly higher P and K concentrations in soybean plant tissues. Yields at both sites were reduced by a late-season dry period. These studies will be continued in 1980. Studies were initiated in 1979 at two southeastern Kansas locations to evaluate P-K rates, carriers, and application methods on soybean yields. Both sites were low to very low in available phosphorus and potassium according to soil tests. P-K carriers compared were dry (0-46-0, 0-0-60) and liquid (0-26-25). All dry solid fertilizer was broadcast and incorporated. Broadcast and knifed methods were used for the liquid fertilizer.
Soil & Water
Soybean yield responses to P and K rates, carriers, and methods of application were nonsignificant. Results indicate yield benefits when P and K were applied and from knifed applications of P and K. Again, late season drought caused pod abortion and dampened effects of fertilizers. Similar studies will be continued in 1980.
Effects of N, P, and K Rates and Application Methods on Yields and Tissue Composition of Grain Sorghum
Studies were initiated at two locations in southeastern Kansas to evaluate rates of N, P, and K applications and application methods on yields and tissue composition of grain sorghum. N rates used were 0, 50, 100, and 150 pounds of N per acre. All N was applied as NH . P-K rates were 0 and 50 pounds of P 2 o 5 and K?O per acre, as potassium t~ipolyphosphate liquid (0-26-25). ApplTcations were broadcast and knifed for the P and K; where P and K were knifed, they were applied simultaneously with the NH 6 to 8 inches deep. Both sites were low in both available phosphorus and potassium according to soil test. Results are shown in Table 17.
Labette county grain sorghum showed a significant yield response to N and a significant increase in tissue N concentrations with added N. There also was a response to P and K at the Parsons Field and knifed P and K gave the highest yield. P and K rate and method effects on tissue analysis were nonsignificant at Parsons. Yields in Neosho county (Weidert farm) were exceptional. This location had been in fescue for several years and high organic matter probably resulted in no yield response to added N. Adding P and K increased yields, though nonsignificantly, and there was no significant difference in yields from broadcast versus knifed P and K. Similar studies will be continued in 1980.
Effects of Methods of A N and P and N-Form on Yield and Com osition o Winter Wheat
In recent years, the technique of simultaneously injecting anhydrous ammonia with ammonium polyphosphate (10-34-0) 6 to 8 inches deep has proven to be a highly efficient way of fertilizing winter wheat. A study was con-ducte~ in Neosho county in 1979 to determine if the ionic form of fertilizer N, NH 4 or No3, in conjunction with methods of applying N-P affected use of P and winter wheat yields. N application rate totaled 75 pounds per acre and P was applied at 40 pounds of P 0 per acre. N carriers used were urea-a~onium nitrate solution (UAN)~ ~nhydrous ammonia (NH 3 ) (predominantly NH 4 -N) and sodium nitrate (N0 3 -N). Results are shown 1n Table 18. Leaf tissue concentrations of P were generally higher with treatments involving dual knife N-P applications. N tissue generally was highest in treatments receiving knifed N applications.
Grain yields at this location were depressed by hail damage estimated to cause a 40 percent loss. Dual knife N-P using NH resulted in a significantly higher grain yield than any other treatment e~cept the dual knife N-P with UAN. Dual knife N-P with sodium nitrate as the N source gave the lowest of the dual-knife yields.
The data from this study suggest maxium ~fficiency with the dual knife N-P technique is obtained with an ammonium (NH 4 ) form of nitrogen. Because interest continues high throughout southeastern Kansas about different primary tillage operations and no-till, we are evaluating the different tillage operations and no-till at both the Parsons and Columbus fields, using grain sorghum at Parsons and soybeans at Columbus. After the primary tillage operations, which were done on April 16 at both sites, all tilled plots received identical seedbed preparation. Results are summarized in Table 19.
Yield data show that no-till performed poorly in 1979 for both crops. Yields were low despite excellent stands in the no-till plots, which corroborates earlier work with no-till at Parsons. The moldboard plow gave the highest yields at both locations but not significantly higher than other primary tillage operations for grain sorghum. This work will be continued in 1980 with a subsoiler included as a treatment. Table 20.
Rainfall was excellent in May through August with average to above average amounts each month. Corn reached physiological maturity September 2. September and early October were very dry with no rain received in September, which was the critical stage for the Group V Essex beans.
Corn yields were exceptional, thanks to timely rainfall. Even with the timely rains, supplemental irrigation of 0.75 inch at tassel or blister significantly increased yields, but two irrigations were no better than one. One irrigation gave an extra 14 to 18 bushels of corn per acre. On December 1, 1979, corn prices existed that would mean an extra $30 to $40 per acre. Whether or not that would be profitable would depend on type of irrigationJ system pumping costs, and other factors.
Even though the soybeans were irrigated during a time of stress, irrigation did little to increase yields. Two irrigations significantly increased yields but not enough to be economically feasible.
Irrigation studies will be expanded in 1980. A study was initiated in Labette county in 1979 to evaluate the effects of nitrogen, phosphorus, and potassium; and application method on yield and composition of tall fescue. A soil test indicated low P and K (12 lbs/A available P and 85 lbs/A exchangeable K) and a pH of 6.8. Results of the study are shown in Table 21.
Nitrogen was applied at 50, 100, and 150 pounds per acre and phosphorus and potassium at 0 and 40 pounds per acre. All fertilizers were in liquid form, 28-0-0 (UAN) and 0-26-25 (potassium tripolyphosphate). Broadcast treatments were applied through flat spray nozzles. The dribb1e applications involved removing spray nozzles from the boom to apply the fertilizer in a band on the soil surface. Knifed treatments were applied through an injection tube behind a narrow shank 6 to 8 inches deep on 15-inch centers.
Yield data indicate a highly significant response to nitrogen and phosphorus-potassium. Adding 40 pounds of P and K increased forage yields nearly 850 pounds per acre.
Knifed applications gave highest yields -significantly higher than dribble applications and 300 pounds an acre more than broadcast applications. N application rates significantly affected plant N content, which increased as N was increased. Adding 40 pounds of P and K significantly increased N, P, and K in the forage. The knifed application produced significantly higher forage N, P, and K than other methods.
Results of this study indicate that we need to add phosphorus and potassium on low testing soils to obtain maxium production of good quality forage. The 1979 results also indicate that knifed applications, to place fertilizer materials deeper in the root zone, were superior to surface applications. This work will be expanded in 1980. ].! Knifed in on 18-inch centers, 6 to 8 inches deep. Treatments applied March 12; harvest was May 9.
Effects of N, P, and Methods of Application on the Yield of Tall Fescue Forage
R. E. Lamond and J. L. Moyer
A study was continued in Allen county in 1979 to evaluate the effects of nitrogen, phosphorus, and the application method on the yield of tall fescue. This site, on the Stan Dreher farm, had been in fescue several years; soil tests indicated low (11 lb/A) P. Results of the 1979 study and three-year average yields are shown in Table 22.
Nitrogen was applied at 60, 120, and 180 pounds per acre, and phosphorus at 0 and 40 pounds P 2 0s per acre. The fertilizers were broadcast or dribbled. Dribble applications were with spray nozzles removed so fertilizer material was banded on the soil surface.
Yield data indicate significant responses to nitrogen in 1979 and for three-year averages. Adding 40 pounds P?0 5 per acre increased yields significantly in 1979 and produced an additional 400 pounds per acre on the threeyear average yields. Dribble applications produced higher, though not significantly higher, yields than broadcast applications in 1979. Three-year average yields showed no difference between application methods. This is the last year for this study at this location. The three years data tell us that when soil P is limiting, it may be necessary and profitable to include phosphorus as well as nitrogen in our forage fertilization programs. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture.
Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco’s modified eagle’s medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow in DMEM supplemented with colostrum. Sparse cultures of early-passage human and rat fibroblasts that grow readily in DMEM supplemented with serum do not grow in DMEM supplemented with colostrum. Canine kidney epithelial cells (MDCK), when plated sparsely, grow exponentially in DMEM supplemented with day 1 bovine colostrum. The generation time is 26 h, the same growth rate as in DMEM supplemented with calf serum. The MDCK cells can be subcultured and regrown to confluence repeatedly in colostrum-supplemented DMEM. Growth in DMEM supplemented with colostrum does not alter the morphological characteristics of the MDCK cells, which are polygonal, contain microvilli at the apical surface, and are connected by tight junctions and desmosomes. MDCK cells do not proliferate in DMEM supplemented with milk obtained 1 wk after the birth of a calf.
epithelial cell lines and early-passage epitheliallike cells proliferate in colostrum-supplemented medium while early-passage fibroblasts do not .
Source of Milk
Bovine milk was obtained at various times after birth of a calf and provided by Dr. Edward Kingsbury (Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Mass . ) . The milk was obtained from Holstein and Jersey cows and frozen immediately after milking. In this report, day I colostrum is obtained 8 h after the birth of a calf and day 8 milk is obtained 176 h after birth of the same calf.
Preparation of Milkfor Cell Culture
Frozen samples of milk were thawed and then centrifuged in a RC-5 superspeed Sorvall centrifuge (DuPont Instrument Co ., Sorvall Biomedical Div., Wilmington, Del.) at 12,000 g for 30 min. The fat floating at the top of the centrifuge tube was removed and discarded. Cellular debris and other sediment at the bottom of the centrifuge tube were also discarded. The milk was sterilized by filtration through Nalgene filter units (Nalge Co., Nalgene Labware Div., Rochester, N.Y. ) . The presence in milk of casein micelles and other particles makes it difficult to filter milk at a concentration >10% (vol/vol) . Therefore the milk was diluted into Dulbecco's Modified Eagle's Medium (DMEM, Grand Island Biological Co ., Grand Island, N.Y .) at concentrations of <<-10% (vol/vol), prefiltered with an 0.80-micron filter, and subsequently filtered with an 0.45-Am filter . The sterile milk samples were kept frozen at -20°C, with no apparent loss in activity up to at least 3 mo.
Cell Culture
Madin-Darby canine kidney epithelial cells (MDCK), rabbit kidney epithelial cells (RK13), primary calf kidney cells (CK), and primary bovine embryonic kidney cells (BEK) were purchased from Flow Laboratories (Rockville, Md.). Human skin fibroblasts were prepared by explant culture of human foreskin . Human skin Fbroblasts between passages 10 and 18 were used . Rat embryo Fbroblasts of passage 4 were obtained from Dr. K. Steimer (Harvard Medical School, Boston, Mass .) and prepared as described by Steimer and Boettinger (20) . All cells were grown at 37°C in DMEM containing glucose (4 .5 g/liter), penicillin (50 U/ml), and streptomycin (50 lAg/ml), and supplemented with either calf serum (Colorado Serum Co ., Denver, Colo.) or bovine milk prepared as described above. Both the cells grown in DMEM supplemented with serum and those grown in DMEM supplemented with milk were subcultured with 0.1% (vol/vol) trypsin (Grand Island) and 0.02% (wt/vol) EDTA in phosphatebuffered saline lacking calcium and magnesium.
Cell Proliferation
The following protocol was used to measure cell proliferation. Cells were detached by incubation with 0.1% (vol/vol) trypsin and 0.02% (wt/vol) EDTA made up in phosphate-buffered saline lacking calcium and magnesium. The cells were resuspended in unsupplemented DMEM at a concentration of -l0" cells/ml, and 1 ml of cells was plated into each well of a 24-well microtiter plate (16-mm diameter, Costar, Data Packaging, Cambridge, Mass.). Between 2 and 6 h after plating, the DMEM containing unattached cells was removed and the attached cells were detached with trypsin and counted in aCoulter modelZF electronic particle counter (Coulter Electronics Inc., Hialea, Fla.) . The plating efficiency under these conditions was -25-50%. The attached cells in replicate wells were then fed either with unsupplemented DMEM or DMEM supplemented with milk or serum. On every 3rd or 4th d, duplicate wells were counted in the Coulter counter and the rest of the cells were refed with the appropriate fresh medium .
Electron Microscopy
Cells were plated onto sterile Millipore filters (25 mm, type HA, 0.45 micron, Millipore Corp., Bedford, Mass .), as described by Misfeldt et al. (13). The cells were grown either in DMEM supplemented with milk or in DMEM supplemented with serum. After reaching confluence, the cells on the filter were fixed with 2.5% glutaraldehyde. The fixed cells were cut into thin sections and analyzed by electron microscopy, as described by
RESULTS
Cultures of primary bovine embryonic kidney cells (BEK) and primary calfkidney cells (CK) contain both epithelial cells and fibroblasts when grown in DMEM supplemented with serum . The epithelial cells are eventually overgrown by the more rapidly dividing fibroblasts ( Fig . la and lb) . A colony of CK epithelial cells surrounded by CK fibroblasts is clearly seen in Fig. l b . The pattern ofgrowth of mixed kidney cell cultures is different when the growth medium is DMEM supplemented with day 1 colostrum. First, the cell density of BEK and CK cells grown in DMEM supplemented with day 1 colostrum is -10-15% of those grown in DMEM supplemented with serum. Second, the cells that grow in colostrum are cuboidal and resemble epithelial cells; there is apparently no growth of fibroblasts ( Fig. lc and ld) . The shape of the epithelial-like cells changes little when colostrum is replaced by serum . The apparent lack of fibroblast growth in DMEM supplemented with colostrum was verified in experiments with earlypassage human and rat fibroblasts . Sparse cultures of human fibroblasts ( Fig. 2) and rat fibroblasts (Table I) were unable to grow in DMEM supplemented with colostrum . However, the sparse cultures did grow readily in DMEM supplemented with serum. The colostrum is not toxic to the fibroblasts ; the growth of these cells resumes when day 1 colostrum is replaced with serum (Fig . 3) .
The ability of epithelial cells to grow in medium supplemented with colostrum was investigated using MDCK epithelial cells. MDCK is an established canine kidney epithelial cell line that preserves the structure and function of kidney epithelium (13,18) . The growth of MDCK cells in DMEM supplemented with various concentrations of milk and serum is shown in Fig . 4 . The milk used was either day 1 colostrum or milk obtained 1 wk later (day 8 milk) . The optimal concentration of day 1 colostrum for the growth of MDCK is 2.5% (vol/vol), whereas the optimal concentration of calf serum is 5% (vol/vol) . At (Table II) . Observations with both light and electron microscopes indicate that there are no morphological differences between MDCK cells grown in serum and MDCK cells grown in colostrum. In either medium, MDCK cells grow and form a confluent monolayer of polygonal cells. The morphology is characteristic of epithelial cells. Fig. 6 a shows an electron micrograph of a thin section cut perpendicular to the plane of MDCK cells grown in DMEM supplemented with 2.5% (vol/vol) day 1 colostrum. The apical surfaces of the MDCK cells contain microvilli projecting into the medium above. The cells are connected to each other on the lateral side by an abundance of interdigitating processes. At a higher magnification, tight junctions are seen at the apical surface and several well-developed desmosomes are seen connecting the two MDCK cells (Fig . 6b) . There is no evidence that growth of MDCK cells in colostrum leads to any adverse effects on epithelial morphology.
DISCUSSION
Bovine milk obtained within 8 h after birth of a calf, i.e., colostrum, can replace serum for the growth of sparse cells in culture. However, colostrum acts selectively in supporting the growth of cells in culture. For example, although epithelial cells proliferate, human, rat, and bovine earlypassage fibroblasts do not. The lack of fibroblast growth is apparently not due to the presence in colostrum of toxic or inhibitory factors. Possibly colostrum contains specific factors necessary for epithelial growth but lacks those needed for fibroblast growth .
Fibroblast overgrowth is still a major problem in the culture of uncloned epithelial cells. There have been numerous but inconclusive attempts to solve this problem by the use of selective media that inhibit fibroblast growth . These include media in which L-valine is replaced by D-valine (4), media in which arginine is replaced by citrulline (21), and media lacking tyrosine (2) proach to overcoming the problem of fibroblast overgrowth .
Although colostrum, in the absence of serum, supports the growth of such epithelial cells as the MDCK cell line, milk obtained as early as a week after the birth of a calf is completely inactive. The failure of MDCK cells to proliferate in milk obtained a week after birth or later does not appear to be the result of inhibitors in milk. Recently, we found that MDCK will grow in milk obtained 1 wk after birth, if the milk is supplemented with human transferrin .l In fact, milk supplemented with transferrin is as active as colostrum in stimulating MDCK growth . Sato and his colleagues (6,8,12,19,22) have demonstrated that the serum requirement for the growth of GH 3 cloned ratpituitary cells, HeLa cells, melanoma cells, MDCK cells, and embryonic carcinoma cells can be replaced by mixtures of hormones, mitogens, and other factors. Transferrin is the only component whose presence is required in each mixture and therefore may be essential to the growth of cells in culture. Transferrin is also found in bovine milk (5); perhaps the levels of transferrin decrease as the lactation period proceeds. It is known that the number of cells (15) and the levels of immunoglobulins (14) and lactoferrin (3,17) decline rapidly in the postpartum period . Colostrum may be ' M. Klagsbrun and R. Packard. Manuscript in preparation .
an exceptionally rich nutrient medium that is fortified with growth factors, hormones, and immunoglobulins. These factors may be highly important for the growth and development of cells and tissue in the newborn during the first few days of life . As the newborn matures, the need for such factors declines. The decline in these factors may be the reason that effective use of milk in cell culture is limited to milk obtained immediately after birth, the colostrum. | v3-fos |
2019-03-19T13:05:16.532Z | {
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} | s2 | Seeding year alfalfa population development as influenced by weed competition and density of establishment
The research in intraspecific competition within an alfalfa stand and interspecific competition between alfalfa and weeds was begun on the Michigan State University farm in East Lansing in 1972. Alfalfa seeding densities of 50, 400 and 800 seeds/m2 were used to determine intraspecific competition. Interspecific competition between alfalfa and weeds occurred mainly in noncontrolled alfalfa stands at various levels of seeding densities. Plant competition was evaluated with importance values and with relative crowding coefficients. Both measurements were found to be suitable for this kind of study. Importance values primarily indicated the quantity relationships of different species. Relative crowding coefficients mainly characterized the competitive ability of a species in a mixed stand. The importance of alfalfa exceeded the importance of weeds in noncontrolled and herbicide controlled stands at the seeding rates of 14 and 7 kg/ha respectively. Relative crowding coefficients show the competitive ability of alfalfa with regard to weeds. In the case of intraspecific competition there was no change in the crowding coefficient of alfalfa in respect of weeds when the seeding rate of alfalfa was increased beyond 9 kg/ha. The crowding coefficient of weeds presented equal values under all treatments thereby indicating the variability and plasticity of weeds. The maximum yield level was obtained in all treatments with the alfalfa seeding rate of 9 kg/ha. The total biomass production per unit area was equal in the noncontrolled system at all seeding densities and in the herbicide controlled system at seeding rates of 9 kg/ha or more.
Introduction
Plants compete for light, moisture and nutrients. Competition occurs between different species and among pure stands. Competition has been studied by way of mathematical models, laboratory and field experiments with single populations or limited mixtures and with influences from natural and seminatural populations growing in mixed communities with or without experimental manipulation. Me Intosh (1965) noted that each species has a genetic potential of response to the environment which determines if and how well it can grow on a given site. Its success is not solely dependent on site conditions, however, but is to a large degree controlled by interaction with other plants. The familiar measures of the competitive effect are reduction in size or reduction in reproductive capacity (plasticity) and reduction in number of plants (mortality). Cook (1965) has stated that intraspecific competition is mainly characterized by plastic response, while interspecific competition mainly results in mortality. Sagar and Harper (I960), based on their experiments and observations, suggest that cohabitation of mixed populations may, in some cases, be maintained by each species suffering more from intraspecific competition than from interspecific competition.
An important point concerning the plant community is the effect of the mixture of species on the utilization of site resources. The conventional idea held by ecologists is that diversified utilization of the site resources should result in greater productivity and efficiency. Donald (1963) has noted, however, that the yield of a mixture of herbaceous plants is usually less than that of a pure stand of the higher yielding component.
Perennial forage crops represent the category in which the economic yield is the biological yield, consequently the yield response to an increasing plant population is asymptotic. In this case, reports Holliday (1960), it is important to get a stand dense enough to obtain maximum yields, but if the stand is too dense, the only loss is from greater seeding expence. From the ecological standpoint the main aim of this field study is to examine the interspecific competition of an alfalfa stand. From the agricultural standpoint the main idea is to find the optimum seeding rate and the proper management technique. In the case of a low seeding rate, weed competition can be harmful, and if the stand is too dense, the seeding expences are unnecessarily high. With clear seeding and harvesting in the year of seeding, stand density plays an important role in obtaining the maximum biological and economical yields.
1. Establishment
The experiment was established on the Michigan State University farm in East Lansing in 1972. The soil was a productive, well drained Hillsdale loam soil with pH 6.8. Saranac, a Flemish type, vigorous, early developing variety was selected. The seed was well prepared prior to seeding. Commercial fertilizer totalling, NPK 300 kg/ha (0-14-42) was broadcast and incorporated in the soil by discing. Germination was 93 % for the alfalfa. Seeding rates were adjusted to 100 % germination according to the actual germination percentage and seed weight. Saranac alfalfa was clear-seeded May 1, 1972. Establishment was done by using a Nursery planter. The plot size was 0.7 x 7.8 m consisting of 5 rows, 15 cm apart. A compeletely randomized split-plot design was used. Herbicide applications were 750 g/ha 2,4-DB and 1.0 kg/ha of dalapon.
Weed control treatment was timed when plants were in the 2-leaf stage. Seeding rates were 1 kg/ha, 9 kg/ha and 18 kg/ha representing densities of 50, 400 and 800 seeds/m 2 .
Cuttings
The alfalfa was harvested two times in the seeding year. Due to a slightly late seeding date, the first harvest was delayed until August 8, a period of 100 days, which is satisfactory for plant development. The second harvest was postponed until October 26, a relatively late cutting date, but a date that is possible under Michigan growing conditions. A period of 79 days elapsed between the two cuttings.
The plots were divided into 3 equal parts, and within each part a 0.21 m 2 area of plants was removed by using a shovel. Both the tops and roots of the alfalfa and weeds were stored in plastic bags for further anlysis. By using sampling techniques equivalent to gradient analysis in ecological studies, the treatments were replicated six times and covered the entire weed population of the area.
3. Laboratory studies
In the laboratory studies each sample was separated into categories of a) alfalfa, b) weeds and c) inert material. Weeds were indentified and the tops and the roots were separated. Alfalfa and the different weeds were counted. After root washing, the tops and roots of the alfalfa and the weeds were allowed to dry for 3 days at 75°C after which the dry matter production was determined for each sample. In the second cutting, the root dry matter production which was determined for each sample, was sampled from the top 6 inches of the root.
4. 1. Importance values, determinants of competition
In this study the main phytosociological characters of relative frequency, relative density and relative dominance of different species were determined.
The synthetic importance value represents these three values together. The characters are the measurements used in gradient analysis in ecolocical measurements.
The following formulas were used (Curtis andMe Intosh 1950, Whittaker 1967 where constants p and a can be calculated. According to the equation, by raising the plant density one approaches the theoretical maximum yield level (Fig. 1 A).
If both sides of the equation obtain inverse values the equation follows the formula: (2) a =-= = = Z p + Z-1 p Z + Z • Z-1 p z + 1 z According to the equation, the yield of an invidual plant approaches the upper limit of the theoretical maximum yield as the number of plants decreases and the growing space of an invidual plant increases ( Fig. 1 B).
If both sides of the equation (2) obtain inverse values the equation follows the formula: If the inverse values of the yield of the invidual plants and the corresponding densities are drawn between the co-ordinating axes, the relationship is linear and the empirical constants Q and /? Q can be determined by linear regression.
The relative crowding coefficient characterizes not the process of crowding itself but only the result of this crowding (de Wit 1960).
Results and discussion
3. 1. Cultivated plant community and plant competition described by importance values Gradient analysis is a research approach for studying the spatial pattern of vegetation. It seeks to understand the structure and variation of the vegetation of a certain surface area in terms of gradients in space of variables on three levels environmental factors, species populations and characteristics of communities. Gradient analysis and classification are alternative approaches to the vegetation of an area (Whittaker 1967). Gradient analysis of vegetation can be a direct gradient analysis with the procedures of a ransect along a single gradient, sometimes ordination measurements of hyperspaces and evolution. Gradient analysis can also contain similarity measurements with quantitative classification, factor analysis, early Wisconsin gradient analysis or Wisconsin comparative ordination analysis (Whittaker 1967). Each method necessitates a number of terms to be defined in advance. These terms are listed ans discussed by Curtis and Me Intosh (1950) and Whittaker (1967). The most important terms are frequency, density, abundance, constancy, presence and dominance consisting of species biomass or production as an expression of the amount of organic matter produced, or energy bound, per unit of ground surface per unit of time.
Results:
Importance values of different crop species represent added values of relative frequency, relative density and relative dominance. In this particular study the importance values were used to show plant population changes with herbicide controlled and noncontrolled field conditions at several alfalfa seeding rates. Relative frequency, density, dominance and importance values in the first harvest are shown in Table 1 and from the second harvest in Table 2.
A. First harvest
Relative frequency values: In uncontrolled field conditions at a low seed rate of alfalfa, witchgrass (Panicum capillare) and prostrate pigweed of the species group Amaranthus spp had the sama frequency, followed by red root pigweed (Amaranthus spp) and purslane (Portulaca spp) (Table 1). When the herbicide was applied, alfalfa, purslane and witchgrass had the highest frequencies and both pigweeds were usually eliminated by the herbicide treatment.
At 9 kg/ha seeding density alfalfa had the same RF-value as red root pigweed (Amaranthus spp), purslane (Portulaca spp) witchgrass (Panicum capillare) and prostrate pigweed (Amaranthus spp) in the plots receiving no weed control ( Table 1). The frequency of most weeds was lowered under field conditions with weed control. However, purslane and witchgrass retained the same RF-values as alfalfa.
At the alfalfa seeding rate of 18 kg/ha, RF-values without weed control were the same for alfalfa, purslane and withchgrass. When the herbicide was applied the trend for the weed species, previously mentioned, was the same as for the 9 kg seeding rate. The RF-values of alfalfa and weeds were equal at all seeding rates in controlled and noncontrolled conditions (Fig. 2). Rel .
Re l .
I m p o r t .
I m p o r t . Rel .
Re l .
I m p o r t .
I m p o r t . Relative density values: At the lowest seeding rate and without weed control purslane had approximately three times the density value of alfalfa (Table 1). Red root pigweed and witchgrass ranked intermediate in their values. Under controlled conditions, purslane was the only important weed.
At the 9 kg/ha alfalfa seeding rate purslane was again the commonest weed component for both controlled and noncontrolled conditions. The trend at the 18 kg/ha seeding rate was similar to that found for the 9 kg/ha seeding rate. Only purslane (Porlulaca spp) had the same importance in both systems.
The relative density of alfalfa exceeded the relative density of all weeds at the alfalfa seeding rate of 9 kg/ha in noncontrolled field conditions with a slightly lower rate in controlled conditions (Fig. 2).
Relative dominance values:
At the low seeding rate for alfalfa in noncontrolled field conditions red root pigweed (Amaranthus spp) had a very strong relative dominance value. It was about three times that of alfalfa. When weed control was used, the pigweeds were mostly eradicated and purslane exceeded the level of alfalfa (Table 1).
At the seeding rate of 9 kg/ha in noncontrolled field conditions red root pigweed was of major importance. However, its relative dominance value was lower than that of alfalfa. In the herbicide controlled system the dominance values of weeds were very low (Table 1).
At the high rate of seeding purslane was of importance in both the herbicide controlled and noncontrolled conditions as was pigweed in the noncontrolled conditions.
The relative dominance of alfalfa in respect of the relative dominance of weeds under noncontrolled field conditions showed the same trend as with the values of relative density (Fig. 1). In the case of the herbicide controlled stand the dominance of alfalfa exceeded the dominance of weeds at a very low rate of seeding.
Important values:
At the low rate of seeding, weeds competed very strongly with alfalfa when no herbicide was used. The Amaranthus species in Michigan conditions seemed to be the greatest problem for alfalfa. Purslane was another big problem.
Other strong competitors were witchgrass and lambsquarter (Chenopodium album). In the herbicide treated stand purslane played an important role as a main competitor with alfalfa (Table 1).
At the seeding rate of 9 kg/ha of alfalfa and when no herbicide was used, red root pigweed, purslane and witchgrass had a combined importance value similar to that of alfalfa. In the herbicide controlled stand purslane and witchgrass were the two most important weeds (Table 1).
The importance values (Fig. 2) show that in the noncontrolled field conditions the importance of alfalfa in respect of weeds increases linearly from the seeding rate of 1 kg/ha up to the studied level of 18 kg/ha. At the same time the importance of weeds decreases, also linearly. In the case of the weed controlled stand, and with the seeding rate greater than 9 kg/ha, the changes in importance values of alfalfa and weeds are of minor importance. The values for alfalfa and weeds are equal at the seeding rates of 14 kg/ha and 7 kg/ha in the noncontrolled and controlled stands respectively.
B. Second harvest
Weeds did not play an important role in the second cutting. The first harvest eliminated most of the annual broad-leaved weeds although some red clovers, ladino clovers and annual grasses were found in the second cutting.
The importance value of grasses seems to play a certain role in a 1 kg/ha alfalfa stand, no importance was however, found in the other treatments (Table 2). Red clover seemed to invade the areas where some of the weed species had been eliminated, but this was of little consequence.
3. 2. Cultivated plant community and plant competition described by crowding coefficients Competition among plants can be either intraspecific or interspecific. Intraspecific plant competition takes place within a pure stand. In the mixed stand the competition is mainly interspecific but also intraspecific. The competitive ability of a species is not an absolute factor but is influenced by the composition of the stand and environment (Baeumer 1964). Crowding coefficients describing competition among species have been introduced by several plant scientists.
A parameter which describes the development of the mixed stand adequately is the relative reproductive rate (RRR) introduced by de Wit and van den Bergh (1965). Another method widely used is the one introduced by Lampeter (1960) and applied by Hofer (1970) and Erviö (1972). The calculation method used in this study is a modification of the method introduced by de Wit (1960).
Results:
At a low seeding rate of alfalfa the interspecific competition of alfalfa in respect to weeds takes place especially when there is no weed control.
However, alfalfa competes well with weeds at low population densities of alfalfa (Table 3). The relative crowding coefficient of alfalfa at one kg/ha seed rate in a stand without weed control was three times that found in the stand where weed control was used. The same trend can be seen among yields (Table 5). The increase in the yields of alfalfa was relatively low in respect to the decrease in the yields of weeds when no control vs. weed control was used.
When the seed rate of alfalfa was increased beyond the population densities of 9 kg/ha, weeds no longer played an important role and the crowding coefficients (K aw ) evened out when both no control and weed control were used.
In the case of population density developing from the 9 kg/ha seed rate or more, there was vigorous intraspecific competition of alfalfa and no changes in K aw could be found (Table 3).
It is important to note that in the competition of weeds in respect to alfalfa (K wa ) no changes could be found (Table 3). The same trend was observed when no control was used or the stand was controlled by herbicides. Table 5. Seeding year top, root and total yields tons/ha of alfalfa and weeds, controlled and without weed control seeded at tree densities and cut two times.
Discussion
Among pure stands only intraspecific competition exists. In mixed stands, or in the stands where weed control has not been successful, both interspecific and intraspecific competition can be found. The growing potential of an individual plant is determined by the relative dominance of a plant or the population density of a species (Baeumer, 1964). In this particular study, both the applied ecological approach and the mathematical model suitable for quantitative measurements were used in evaluating the spring growth of alfalfa and weeds. When studying the importance values of the stand it was found that the same weeds which were not affected by the herbicide treatment were not controlled by the biological weed control at the high alfalfa seeding rates. Importance values show that a crop competes best with weeds at high population densities as shown by Mann and Barnes (1945, 1947, 1949, 1950, Granström (1962), Horowitz (1966), Erviö (1972). Importance values also describe adequately the requirements of seeding rates for maximum yield in herbicide controlled and noncontrolled systems as observed in this study and in alfalfa studies by Pulli (1973).
The crowding coefficients primarily show the competitive ability of alfalfa and weeds, not the dominance situation or importance of the species at the moment. As a result of competition within the stand the equilibrium stage was reached at the population density of 9 kg/ha ( Table 3).
The equilibrium stage is reached by means of regulating both the vegetative growth of the stand and the number of inviduals as shown by Harper (1960) and Harper and Gaijic (1961). It is interesting to note that the weeds generally have a very stable competitive ability at any alfalfa density in controlled or noncontrolled systems. This study clearly showed that weeds are very adaptable to various growing conditions due to the versatility between species. The excellent competitive ability of alfalfa could be seen at low seeding rates when no herbicide was used ( Table 3). The damaging effect of herbicides can be seen especially at low seeding rates of alfalfa. Table 4. The results show that the aboveground yield of alfalfa in a weed controlled system is more than two times greater than that in a noncontrolled system. The competition is more equal among shoots than among roots. With herbicide treatment the weeds are about five times smaller in controlled than in noncontrolled systems. The change in plant size is more stable and more gradual without weed control than in a controlled system when the population density of alfalfa is increased (Fig. 3). The decrease in the number of weeds in a controlled system in not drastic compared to the noncontrolled system, but the size of the weed plant is smaller. Fig. 3. Effect of the number of plants or the seeding rate of alfalfa on the dry matter weight of the alfalfa and weed shoot and root production g/plant in controlled and noncontrolled growing conditions.
4. 2. Yields of alfalfa and weeds
Theoretical maximum yields; The calculated theoretical maximum yields of a unit area of alfalfa and weeds were obtained according to the way described by the Wit (1960). The results are shown in Table 4. Without weed control the calculated theoretical maximum yield of weeds in the noncontrolled system was more than twice that of alfalfa. In the controlled system the relationships were reversed. It is obvious that the competition took place mostly between the underground parts of plants as indicated by the root yields of 3.3 tons/ha of alfalfa and 3.6 tons/ha of weeds in the noncontrolled system. In the controlled system there was no room for weed roots as represented by the root yields of 4.6 tons/ha for alfalfa and 0.4 tons/ha for weeds.
Yields in cuts:
The maximum yield levels of shoots and roots in each cut and in total yields were obtained at the seeding rate of 9 kg/ha (Table 5). Due to the experimental design, where herbicide treatments were in the main plot with only two replications, a statistical difference between treatments could not be found, although the differences were in favour of the controlled system. The negative correlation between the alfalfa and weed yields can be observed when the population density of alfalfa is increased. The weeds played an important role in the first cut at all seeding densities in the noncontrolled system and only at the lowest seed rate in the controlled system. In the second cut the weed problem was quite unimportant. The calculated yield curves of the stand spring growth obtained by the means of de Wit (1960) show the critical growth curves of alfalfa and weeds at different population densitied (Fig. 4). Fig. 4. Effect of the seeding rate of alfalfa on the shoot and root production of alfalfa and weeds (DM tons/ha) in weed controlled and noncontrolled growing conditions. Total biomass: The total biomass yields of alfalfa and weeds in two cuts are presented in Fig. 5. The most important observation here is that the combined root, top and total biomass yields of alfalfa and weeds are equal at any seeding rate of alfalfa when no herbicide has been used in the management technique. The same yield levels were obtained also in the weed controlled system with the seeding rate of 9 kg or more of alfalfa seed per hectare. At the 1 kg/ha seeding rate in the noncontrolled system the total biomass of weeds was 58.3 % of the total biomass per unit area (Table 6). At the same population density in the controlled system the corresponding value was 32.4 %, indicating that there were too few plants per unit area to obtain the maximum biomass yields.
The relative proportions of alfalfa and weed root and top yields from the total biomass per unit area are presented in Table 6. Alfalfa roots have a high competitive ability compared to weeds and the root yield was very stable in both controlled and noncontrolled systems beyond the seeding rate of 9 kg/ha.
Most of the changes occurred in aboveground competition giving a clear indication of the intraspecific and interspecific competition in the noncontrolled system and the predominantly intraspecific competition in the weed controlled system. Fig. 5. Root, shoot and total biomass production of alfalfa and weeds at different seeding rates of alfalfa in weed controlled and noncontrolled growing conditions.
4. Discussion
Plants under natural conditions grow as heterogenic societies and in cultivated areas as limited mixed populations or as monocultures. However, also a monoculture is affected by the number of weeds even in controlled conditions. Competition between plants either in a monoculture or in a mixed stand develops if any one of the growth factors is limited. Baeumer (1964) has stated that the greater the seeding rate the earlier the plants fill their space requirements. During the period without space limitation the plants grow at the same rate at all seeding densities. When the space requirements have been met, competition between the plants slows down the growth.
The decrease in growth is greater the denser the populationis. In dense populations the competition for light is one of the most dominant factors causing morphological changes among plants (Stern 1965). Baeumer's and Stern's observations can be found also in this study. In noncontrolled growing conditions (Fig. 3) the changes in plant size are gradual and less radical due to the heavy competition between alfalfa and weeds already at the 1 kg/ha seeding rate of alfalfa. In controlled growing conditions space requirements are met mainly around the seeding rate of 5 kg/ha (Fig. 3).
The calculated theoretical maximum aboveground yields of alfalfa (Table 4) and the yield curves of alfalfa and weeds (Fig. 4), obtained according to the way introduced by de Wit (1960), show that the theoretical maximum yield of alfalfa in controlled growing conditions in the first cut of the seeding year was about 22 % less than in noncontrolled growing conditions. The development of the alfalfa DM yield was less steep in the case of noncontrolled growing conditions compared to the curves for the controlled system (Fig. 4). The optimum seeding rate of alfalfa, 9 kg/ha, was equal to that obtained by Pulli (1973). In less favourable growing conditions and with the use of biological weed control the seeding rates of 16-33 kg/ha (Tesar & Jackobs 1972) and 20-30 kg/ha, as recommended by Oswald (1959), Kemenesy and Manninger (1968) and Multamäki (1965), are needed for favourable growth and development in the year of seeding when no companion crop is used.
As regards biomass production, comparing weed controlled and noncontrolled systems, the results agree with the summary of Odum (1971), that it may be stated that nature maximizes for gross production whereas man maximizes for net production. In a monoculture a certain population density is required in order to obtain the maximum yield. Beyond that environment dependent population density the only losses are from higher seeding expences.
Summary and conclusions
The study dealing with the intraspecific competition among alfalfa stands with different population densities and with the interspecific competition between alfalfa and weeds was conducted on the Michigan State University farm in East Lansing in 1972. An ecological approach to studying plant competition was made by testing an applied form of gradient analysis widely used in ecological mapping. The research involved a complementary study to | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | A semi–automatic system for subsampling heterogeneous foods
Introduction Chemical analysis of foods is preceded by subsampling the original sample. Reasonably homogeneous foods may present few problems, but the subsampling of heterogeneous materials requires care to assure meaningful analytical results. One effective solution to the problem, where sample size permits, is to homogenize the entire food item and subsample the homogenate. The success of this homogenization, the maintenance of homogeneity during subsampling and the protection of subsamples from degradation are crucial to the validity of subsequent nutrient determinations. For some time in the authors’ laboratory, heterogeneous consumer food items, such as franchise hamburgers, french-fried potatoes and pastries, have been sampled after making homogeneous slurries in a Sorval* Model 17105 Omni-Mixer (Du Pont Company, Wilmington, DE 19898). Briefly, the manual procedure for slurry preparation and subsampling has been to weigh the sample in an OmniChamber, to add a predetermined quantity of water containing antioxidants, to displace chamber air with nitrogen and to homogenize. Often it was necessary to add additional water to thin the slurry, cover again with nitrogen and continue homogenization. Following homogenization, the chamber and cover assembly were dismounted from the motor, the cover assembly was removed and subsamples were scooped out into small containers. Taking replicate subsamples manually is a slow and tedious process during which significant moisture loss and oxidation may occur. It is often further complicated by the need to rehomogenize samples which tend to separate. During each homogenization, sample is retained inside the shaft assembly; it is therefore necessary to completely disassemble it for proper cleaning and to prevent any cross contamination between samples. Many homogenizers are commercially available, but none can be readily automated, or has the combined capabilities for large particle size reduction, simultaneous homogenization and subsampling, inert atmosphere sample protection and cleaning without disassembly. Consequently, a system has been developed in the authors’ laboratory for preparing and subsampling slurries of food items based on a modified Omni-Mixer. The system is easily automated and operates faster providing more uniform results than manual methods. Although the system was devised to explore the feasibility of fully automatic sample preparation, it is valuable as a stand-alone unit where multiple representative aliquots of heterogeneous materials are needed. Details of the system’s construction and operation are presented in this paper with some experimental results illustrating its performance. Its limitations and potential are also discussed.
Introduction
Chemical analysis of foods is preceded by subsampling the original sample. Reasonably homogeneous foods may present few problems, but the subsampling of heterogeneous materials requires care to assure meaningful analytical results. One effective solution to the problem, where sample size permits, is to homogenize the entire food item and subsample the homogenate. The success of this homogenization, the maintenance of homogeneity during subsampling and the protection of subsamples from degradation are crucial to the validity of subsequent nutrient determinations. For some time in the authors' laboratory, heterogeneous consumer food items, such as franchise hamburgers, french-fried potatoes and pastries, have been sampled after making homogeneous slurries in a Sorval* Model 17105 Omni-Mixer (Du Pont Company, Wilmington, DE 19898).
Briefly, the manual procedure for slurry preparation and subsampling has been to weigh the sample in an Omni-Chamber, to add a predetermined quantity of water containing antioxidants, to displace chamber air with nitrogen and to homogenize. Often it was necessary to add additional water to thin the slurry, cover again with nitrogen and continue homogenization. Following homogenization, the chamber and cover assembly were dismounted from the motor, the cover assembly was removed and subsamples were scooped out into small containers. Taking replicate subsamples manually is a slow and tedious process during which significant moisture loss and oxidation may occur. It is often further complicated by the need to rehomogenize samples which tend to separate. During each homogenization, sample is retained inside the shaft assembly; it is therefore necessary to completely disassemble it for proper cleaning and to prevent any cross contamination between samples.
Many homogenizers are commercially available, but none can be readily automated, or has the combined capabilities for large particle size reduction, simultaneous homogenization and subsampling, inert atmosphere sample protection and cleaning without disassembly. Consequently, a system has been developed in the authors' laboratory for preparing and subsampling slurries of food items based on a modified Omni-Mixer. The system is easily automated and operates faster providing more uniform results than manual methods. Although the system was devised to explore the feasibility of fully automatic sample preparation, it is valuable as a stand-alone unit where multiple representative aliquots of heterogeneous materials are needed. Details of the system's construction and operation are presented in this paper with some experimental results illustrating its performance. Its limitations and potential are also discussed.
Principles of operation
Preliminary experiments in the authors' laboratory with complex foods showed that the sample size reduction and mixing, such as provided by the Omni-Mixer at viscosities near 3000 centipoise, yield analytically homogeneous slurries. Samples containing sensitive nutrients may be processed without damage when degassed solvents, antioxidants and inert atmospheres are used. For slurries that tend to separate, mixing must be continued during subsampling, and the flow of slurry in the sample lines must be maintained to provide representative subsamples. Disassembly of the unit for cleaning was unnecessary when the slurry was prevented from entering the drive shaft assembly and when all working surfaces could be cleaned by turbulent wash solution.
The six operating states of the slurry subsampling system are illustrated in Figure 1 In practice, a 450 ml Omni-Chamber is weighed, sample is added, and the combined weight is determined. The system is then switched to 'Load/Unload', which corresponds to amount of solvent has been added to the chamber, the 'Homogenize' state is activated, and the mixer is turned to full power. This state is illustrated in Figure (c). During homogenization, nitrogen flows through the shaft assembly, into the chamber and out through the adaptor recess and vacuum solvent port. In this way contamination of the shaft assembly is prevented and the sample is protected. If after several minutes the motor fails to reach a specified RPM, additional solvent can be added by returning the system to 'Dilute' and adding solvent from tte syringe pump until the desired RPM is indicated.
After the homogenization is complete and the slurry is of the proper viscosity, the motor power is reduced to 25%. A flow of slurry is established between the homogenization chamber and dispenser head by nitrogen pressure and from the dispenser head to the bypass container, by a vacuum. When the slurry lines are wetted and a uniform flow obtained, subsamples may be taken by switching from 'Bypass' to 'Dispense' until the desired amount of subsample has been dispensed. This is shown in Figures d and e. Returning the system to 'Bypass' stops the subsampling process and diverts the flow of slurry back to the bypass container. Multiple subsamples may be taken by alternating between the 'Bypass' and 'Dispense' modes. In the 'Wash' state, cleaning solution is drawn through the shaft assembly by a vacuum * Mention of a trade-mark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suited. Volume 2 No. January 1980 applied at the adaptor recess. This is shown in Figure f. This filling operation is carried out with the mixer at 25% power.
When the chamber is full of wash solution, the system is switched to 'Bypass', the mixer turned to full power, and the syringe pump triggered. In this way, all interior surfaces are washed and waste is purged into the bypass container. After several 'Wash-Bypass' cycles, the system is switched to Load/ Unload', the empty Omni-Chamber is removed, and a new sample can be introduced.
Instrumentation
The Omni-Mixer was modified to provide four access ports and to withstand moderate operating pressures. For details of the modified Omni-Mixer refer to Figure 2. The coupling adaptor was beveled, so that O-rings could be fitted against both the motor casing and the chamber lid. A teflon flap seal was pressed between the motor and rubber coupling. The upper set screw in the rubber coupling was magnetized, and the coupling adaptor was-drilled and tapped adjacent to the set screw to accept an inductance sensor.
Three holes were drilled through the chamber lid to match the slurry, lower recess and upper well ports in the teflon adaptor. The adaptor was sealed against the chamber and shaft assembly by O-rings and was configured to permit nitrogen, solvent or wash solution to enter a well in the top of the adaptor. A recess in the bottom of the adaptor allows a vacuum to be applied to the homogenization chamber and also the solvent to be pumped through the recess for cleaning purposes. A teflon tube was fitted on the drive shaft between the rulon bearings to reduce the dead volume in the shaft assembly. The flow of nitrogen, dilution solvent or wash solution through the shaft assembly was facilitated by drilling a hole above the lower rulon bearing and enlarging the upper hole which is at the bottom of the teflon adaptor well. The Omni-Mixer knives are, as supplied, blunt on the leading edges, and collagen fibers wound around the knives and shaft assembly when raw meats were homogenized. Sharpening the leading edges enabled raw meats to be processed without difficulty.
During the 'Bypass' and 'Dispense' modes of operation, slurry flow is controlled by the chamber valve and bypass valve. These were machined from teflon rod and moulded teflon tube and are pneumatically actuated. O-rings at both ends of the slider prevent control gas from entering the slurry lines; however, only press fitted teflon surfaces separate the input and output lines when the valves are closed. Both valves are identical except for a cross drilling in the bypass valve that permits the tube to the bypass chamber to be emptied while a subsample is being dispensed.
The dispenser head was designed in an open, three port venturi configuration and machined from teflon rod. Figure 4 illustrates its construction. As slurry flows through the dispenser head in the 'Bypass' mode, a partial vacuum in the bypass line and the slurry's own momentum prevent unintentional dispensing of sample. Any material retained in the subsample orifice is drawn into the bypassed slurry or wash flow. Nitrogen flows through a hole next to the subsample orifice to protect the dispensed subsample and to fill the homogenization chamber as it is being loaded with sample. The angle between dispenser head ports was chosen to minimize turbulence in the bypassed flow yet optimize venturi suction at the subsample orifice.
Proper operation of the system depends on the final slurry viscosity, which is measured as a function of mixer RPM. Its Brown & Slover Semi-automatic system for subsampling heterogeneous foods sensor in the coupling adaptor adjacent to the magnetic set screw. Figure 5a is a schematic of the tachometer. The potentiometer can be adjusted to give full meter deflection for any RPM. Nitrogen and solutions are controlled by Angar scientific three-way model 250 TFE solenoid valves and the vacuum is controlled by an Asco two-way model 104R solenoid valve. All valves are multiplexed from six switches to facilitate changing machine states, as shown in Figure 5b.
Each switch fully enables a single National 7447 decoder/ driver. Jumper wires connect to select Teledyne P/N 601-1401P relays, which activate the Angar valves. The definition of any state can be altered by simply changing jumper wires between the relays and the individual decoder/drivers.
Detailed schematics and operating instructions are available upon request.
System evaluation
Slurries of raw and franchise hamburger, salad dressing, french fries and pastries were analyzed from samples prepared and subsampled by both manual methods and the dispenser system. Samples requiring dilution to obtain proper viscosity were diluted with a solution of 4g pyrogallol per litre of distilled water. The apparatus was cleaned with a 20% solution (v/v) of ethanol and distilled water.
The dispenser system was evaluated and compared with manual subsampling on the basis of precision in determination of moisture (1) on replicate 2g subsamples, and of total lipid (2) and fatty acids (3)on chloroform-menthol extracts of replicate 10g subsamples. The result of these comparisons are shown in Table 1. The data show that precision was improved by use of the dispenser system. No significant differences were found in moisture, total lipid or the absolute fatty acid compositions between subsamples automatically dispensed and taken manually. The thoroughness of the cleaning cycle was determined by rinsing the disassembled unit with a mixture of chloroform and methanol, partitioning with water and measuring the total lipid in the resulting lower phase. There was no significant cross contamination between samples after four 'Wash-Bypass' cleaning cycles ( Figure 6). With the present system the feasibility of automatic slurry handling has been demonstrated; this improves subsample uniformity and reduces the operation time for staff. With the system described here, sample charges ranging from a maximum of 400 ml to a minimum of 50 ml can be processed. At about 20 psig chamber pressure, subsamples may be taken at the rate of ml per second in aliquots as small as 0.2 ml. Most samples can be homogenized in 5 minutes, and the system can be washed in 3 minutes.
The system is limited primarily because a gas pressure must be used to pump the homogenate. Adjustments,in nitrogen pressure are sometimes necessary to offset differences in the flow shear rate between slurries of similar viscosity, and time was not a reliable measure of subsample size. Where many small subsamples are required, typically 30% of the slurry is lost to the bypass chamber. In practice samples are collected and stored in inexpensive glass containers and transferred to stainless steel Omni-Chambers for homogenization and subsampling. It would be more efficient to store and homogenize samples in the same vessel, but glass would probably be incompatible with even the moderate operating pressures described. In order to permit the use of glass jars for sample collection, storage and homogenization, a peristaltic pump will be used to replace the gas pressure for fluid transport.
Further plans for this device include the addition of microprocessor control for the operation' of valves and pumps. | v3-fos |
2019-04-01T13:15:59.253Z | {
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} | s2 | Normal and light weight oats as feed for growing pigs
The feed value for pigs of five lots of oats was studied by digestibility and feeding trials. Two of the oats were very light (36—39 kg/hl), three normal weight (51—56 kg/hl). The most apparent difference between the light and normal oats was the contents of starch and crude fibre and the digestibility of NFE. Clear differences were also found in the colours of meals, although all oats were light-coloured varieties. The difference in the energy value between the best and the poorest was 1 3 % by the digestibility and 1 5 % by the feeding trial. The difference in the economic return on the oats was 23%. The energy values of the three normal oats were very much alike. One of the two light oats was darker in colour and poorly palatable due to damage during harvest. Its energy value was found lower in the feeding trial than in the digestibility trial. Our results show that light weight oats, if not damaged, are useful feed for pigs, even though their nutritive values arc lower. The feed value of damaged oats, on the other hand, could not be predicted from the chemical composition, nor sufficiently even from the results of the digestibility trial. The results were tested by one-way variance analysis and the differences between means by the Tukey-tcst.
Introduction
Although oats are not suitable as the sole cereal feed for growing pigs, a certain amount is generally incorporated into the pig rations in Finland.
The nutritive value of oats is more variable than that of barley due to the greater variation in the contents of hulls and starch (SALO 1978 a). The different growth conditions in various parts of Finland accentuate the quality variation (VARIS et ai. 1979).
The feed value of oats is principally determined by the proportion of hulls (THOMKE 1960, SIBBALD andPRICE 1977), which in composition are equal to rough straw and thus not utilizable by pigs. Oats rich in hulls have a lower digestibility of NFE, because the NFE of such oats contains less starch and more hemicellulose and lignin. The pigs digest the starch of oats totally, but scarcely one third of the hemiccllulosc (SALO 1971).
The purpose of this experiment was to study the significance of volume weight as a parameter for the nutritive value of oats for growing pigs. The intention was that the oat lots differ evenly in volume weight, but re-testing revealed that two of the five lots were very light, the other three normal. The experiments were performed as a digestibility trial in the Department of Animal Husbandry, and as a feeding trial at the Swine Research Station.
Materials and methods
The same five oat lots (Table 1) were used in the digestibility and feeding trials. The oats were milled using a 3 mm sieve.
The digestibility trials were carried out with six castrated Yorkshire pigs, weighing 5 s-Bo kg. A vermicide was administered before the trial. The experiment was performed using a total collection method, as two 3x3 Latin square (one barley sample as the sixth feed). The preliminary period was 12 days for the first trial and 7 days thereafter. The collection period was 5 days.
A spray-dried skim milk powder was used as protein supplement for 16 %ofthe diet (120 g DCP/F.U.). Minerals and vitamins were given according to standards.
The daily rations of the three periods were 2.4; 2.6 and 2.6 F.U./d (F.U. = 0.7 kg starch). The animals were fed twice daily. Oats, skim milk powder and mineral and vitamin mixtures were weighed separately and given mixed with two volumes of water. The pigs ate the ration immediately and were then offered water.
The faeces and urine were collected in the morning and samples of fixed amount were taken for analysis. The faeces samples were preserved frozen during the period. At the end of the period, spatterings of feed were collected from a plastic sheet placed in front of the trough, and subtracted from the total amount of feed given.
The dry matter determinations were made at 103°C, and the samples for analysis dried in vacuum at 40-50°C. Feeds and faeces were milled using a sieve of 0.5 mm.
The common feed analyses were made according to standard procedures. The starch was determined by amyloglucosidase method (SALO and SALMI 1968).
The digestibility coefficients for skim milk powder were taken from Feed Tables (ERIKSSON et al . 1972). The digestibility of oats was then calculated by subtraction. The metabolizable and net energy values were calculated using factors and models of NJF's Feed Tables (ANON. 1969). The results were tested by one-way variance analysis and the differences between means by the Tukey-tcst.
Results and discussion
The oats formed two groups: the light (under 40 kg/hl) and medium weight (51 -56 kg/hl). The clearest difference between the groups was the starch content (Table 1). This was reflected in the digestibility of NFE (Table 2) and proved to be a better parameter for the digestibility of NFE than the crude fibre. The digestibility coefficients of NFE found here agree well with those of oats quality classes 1 and 3 in the Feed Tables (ANON. 1969, ERIKSSON et al. 1972. No significant difference between light and good oats appeared in the crude protein digestibility, or in the nitrogen balances ( Table 2). The digestibility of protein tended to increase, however, with both high protein content and high volume weight. The digestibility coefficients for protein were equal to those recorded in some experiments (ANON. 1971, KELLNER and BECKER 1971, SALO 1971, ANDERSON et al. 1978), but a little higher than the figures presented in most Feed Tables. The same could be noted also for the fat digestibility. The pigs digested fat and protein of oats better than those of barley (SALO 1978 b).
The digestibility of crude fibre was small and dependent on the individual pig. The level was the same as earlier found for the cellulose of oats (SALO 1971).
The differences between energy values were principally dependent on the digestibility. Between the two light oats, however, the chemical composition was more determinative. The difference between the best and the poorest oats was 13%.
The energy values found here agree well with values calculated from the digestibility coefficients of oats of like crude fibre contents given in Feed Tables (ANON. 1969, ERIKSSON et al. 1972). The only apparent exception is the oats No 1. for which the fibre content predicts too high an energy value. Starch seems again to be a better parameter than fibre.
Material and methods
The experiment was performed with the same five oat lots as the digestibility trial (Table 1). Sixteen pigs for each oats lot were fed individually from 24.5 kg to 90 kg liveweight gain. (Oats No 1 was sufficient only for eight pigs up to a ten weeks period).
Each oat lot was fed as equal daily dry matter rations. As a protein supplement each pig received 100 g soya bean meal plus 100 g fish meal daily. The digestible crude protein content of the diet was thus at least 15.5 % at the beginning and 9.3% at the end of the trial (169-107 g DCP/F.U.). The purpose was to give so high a protein supplement that the different protein contents of individual oat samples could not influence the growth rate of animals. The minerals and vitamins were supplied according to standards.
Results and discussion
The growth results (Table 4) agreed well with those of the digestibility trial, that is, the feed efficiency (kg feed/kg again) of oats No 2 was 15 % and the growth rate 11 % lower compared with the best oats. The difference in the economic returns on the oats was still higher (23 %), however, because the light oats required a longer feeding period. Nevertheless it should be emphasized, that even with oats as light as 39 kg/hl the growth rate of pigs was nearly 700 g per day. The results found here agree well with an earlier trial (ALAVIUHKOLA 1978) where two lots of oats were compared, one weighing 45 kg/hl, the other 53 kg/hl. In that trial the difference between the feed efficiency was 11 % and the daily gain 8%.
The oats of lot 1 were sufficient only for a ten-week trial. The growth results were clearly worse than the digestibility results, due to the damage to the crops at harvest time. The dark colour and poor palatability are indicative of such damage. All the oats were light-coloured varieties, but even so there were clear colour differences, as the reflectometer values of meals below indicate. (A reflectometer measures the intensity of light reflected.) The colour seems to correspond rather well to the feeding values. The daily intake of the No 1 oats was lower than the others, which explains the poorer growth rate. In the digestibility trial this factor had no effect, because the pigs were larger and the amount of feed was adjusted according to he palatability of the poorest oats.
We would conclude that although volume weight is not an accurate criterion for the energy value, it does offer a practical tool for the rough division of oats into different classes. The starch and crude fibre determinations provide a more reliable basis for this classification. On the other hand the chemical composition does not reveal the possible harvesting damage to crops, and just that may quite decisively affect the nutritive value. To some extent such damage can be judged from the appearance and the palatability of the oats.
The protein value of the oats, for its part, generally correlates positively with the energy value. This rule has exceptions, however, one example being the oats No 1 of the present study. | v3-fos |
2018-12-21T20:47:54.306Z | {
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} | s2 | Improved techniques for assayinq protein concentration in geminating Neurospora conidia
Improved techniques for assayinq protein concentration in geminating Neurospora conidia. This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol27/iss1/15 Christensen, R. L., D. Wolens, and J. C. Schmit. Improved techniques for assaying protein concentration in germinating Neurospora conidia. The measurements of the specific activities of enzymes during developmental sequences, such as conidial getmination, are directly related to the accuracy of the protein determinations (Schmit and Brady 1976 Bacterial. Rev. 4O:l). A permeabalization procedure (Basabe Sal. 1979 Xiial. Biochem. 92:356; Christensen and Schmit 1979 Neurospora Newsletter 2&:13) can be used to assay various enzyme activities during conidial germination. We have developed a protein assay which uses the same samples that are peneabilized for enzyme assays. This procedure eliminates many of the problems that are associated with accurately measuring the specific activity of enzymes. Protein concentration of permeabalized conidia was measured using an adaptation of the "Ho-Rad Protein Assay" (Bulletin 1069, February 1979, Bio-Rad Labs.). The concentrated dye was diluted 1:4 with double distilled water and filtered. The protein standard (bovine gamma globulin) was diluted analytically to 1.4 mg/ml, and a standard curve was prepared using protein concentrations from 0.2 to 1.4 mgfml. Permeabalized conidia were prepared for the protein assay by vortexing 300 ~1 of the sample, containing 5 to 10 mg of conidia, with 300 mg of acid washed sand for two minutes. As soon as the sand settled, the conidial suspension was removed and stored at 4qC. The protein content of the sample was determined by mixing 20 ~1 of the ground cells with 1.0 ml of diluted dye. After ten minutes, the tubes were gently mixed, the absorbance was measured at 595 nm, and the protein content calculated by comparison to a standard curve prepared at the same time. The assay is very sensitive; therefore, care must be taken to insure that all glassware is thoroughly clean. Figure 1. -Protein and dry weight levels of germinating conidia. Conidia of nada strain (FGSC 2688) were dry harvested and then innoculated at 1.5 mg/ml in either Vogel's minimal medium (Vogel 1964 Am. Nat. 98:435) with 2% glucose, or in distilled water. The cultures were shaken at 150 t-pm at 24oC. The dry weight was measured in 1.0 ml samples that were harvested on a preweighed filter and dried at 90°C for 24 hours. The percent germination was determined (Schmit and Brady 1975 J. Bacterial. 124:232). The protein levels were assayed as described in the text. Symbols: (0 ), dry weight; (I ), percent germination; (0 ), protein concentration in minimal clucose medium; (A ), protein concentration in distilled water; (A ), protein concentration in minimal glucose medium with 36 UM cycloheximide. Protein content increases during conidial germination with the same doubling time as the dry weight (Figure 1). When cycloheximide is added to the germination medium OP when conidia are incubated in distilled water, there is no increase in protein content. The protein assays were found to be very reproducible; duplicate samples of the same preparation of permeabalized conidia that were ground with sand and then assayed for protein varied less than i5%; duplicate assays of the same preparation of ground cells varied less than i3%. The protein assay was linear up to about 0.70 optical density units. This corresponded to a maximum of 28 ug of protein per assay. The major cause of scatter in the data in Figure 1 is due to errOP in the initial sampling of germinating conidia. Conidia have a tendency to clump during germination, and it is difficult to remove uniform samples. The error due to clumping does not affect the specific activity calculations because both protein content and enzyme activity are assayed in the same sample of permeabalized cells. Minor errors in the protein assays of conidia incubated in distilled water or with cycloheximide are exaggerated in Figure 1 because the data are graphed on a logarithmic scale. School of Medicine and Oepartment of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901. __ ____ ~ ~ -
Improved techniques for assayinq protein concentration in geminating Improved techniques for assayinq protein concentration in geminating Neurospora conidia. Neurospora conidia.
Abstract Abstract
Improved techniques for assayinq protein concentration in geminating Neurospora conidia.
The measurements of the specific activities of enzymes during developmental sequences, such as conidial getmination, are directly related to the accuracy of the protein determinations (Schmit and Brady 1976 Bacterial. Rev. 4O:l). A permeabalization procedure (Basabe Sal. 1979 Xiial. Biochem. 92:356; Christensen and Schmit 1979 Neurospora Newsletter 2&:13) can be used to assay various enzyme activities during conidial germination. We have developed a protein assay which uses the same samples that are peneabilized for enzyme assays. This procedure eliminates many of the problems that are associated with accurately measuring the specific activity of enzymes.
Protein concentration of permeabalized conidia was measured using an adaptation of the "Ho-Rad Protein Assay" (Bulletin 1069, February 1979, Bio-Rad Labs.). The concentrated dye was diluted 1:4 with double distilled water and filtered. The protein standard (bovine gamma globulin) was diluted analytically to 1.4 mg/ml, and a standard curve was prepared using protein concentrations from 0.2 to 1.4 mgfml.
Permeabalized conidia were prepared for the protein assay by vortexing 300 ~1 of the sample, containing 5 to 10 mg of conidia, with 300 mg of acid washed sand for two minutes.
As soon as the sand settled, the conidial suspension was removed and stored at 4qC. The protein content of the sample was determined by mixing 20 ~1 of the ground cells with 1.0 ml of diluted dye.
After ten minutes, the tubes were gently mixed, the absorbance was measured at 595 nm, and the protein content calculated by comparison to a standard curve prepared at the same time. The assay is very sensitive; therefore, care must be taken to insure that all glassware is thoroughly clean. Protein content increases during conidial germination with the same doubling time as the dry weight (Figure 1). When cycloheximide is added to the germination medium OP when conidia are incubated in distilled water, there is no increase in protein content.
The protein assays were found to be very reproducible; duplicate samples of the same preparation of permeabalized conidia that were ground with sand and then assayed for protein varied less than i5%; duplicate assays of the same preparation of ground cells varied less than i3%. The protein assay was linear up to about 0.70 optical density units. This corresponded to a maximum of 28 ug of protein per assay.
The major cause of scatter in the data in Figure 1 is due to errOP in the initial sampling of germinating conidia. Conidia have a tendency to clump during germination, and it is difficult to remove uniform samples. The error due to clumping does not affect the specific activity calculations because both protein content and enzyme activity are assayed in the same sample of permeabalized cells.
Minor errors in the protein assays of conidia incubated in distilled water or with cycloheximide are exaggerated in Figure 1 | v3-fos |
2017-07-29T04:25:17.705Z | {
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} | s2 | Genetic differences in feed utilization by dairy cattle
are about 53 Heat is, however, related much more closely to total protein synthesis, most of which takes place in tissues other than "meat". Ways and means of mani-pulating protein synthesis and the energy cost of growth by nutrition, anabolic agents and anti- microbial growth promoters are considered. Livestock production under grazing is characterised by inability to directly measure feed intake, an interactive pasture-animal complex, marked seasonal fluctuation in feed quantity and quality, high animal maintenance costs and relatively low production levels. Harvesting ability of the animal, resilience to feed fluctuations, resistance to disease or stress and voluntary feed intake contribute importantly to total feed efficiency. Greatest scope for genetic impro- vement however is through increasing feed conversion efflciency. The high proportion of maintenance to total feed requirements in a free-grazing system calls into question the effectiveness in improving total productivity of traditional evaluation and selection on yields per animal. An "efficiency index", equivalent to the yield of an animal of average liveweight having similar predicted efficiency, is proposed for adjusting observed yields for the effect of liveweight on feed conversion efficiency. In many situations the efficiency index can be approximated by a linear function of yield and liveweight, to which standard selection index methods can be applied to optimise genetic progress, given appropriate genetic parameters for liveweight and production traits. The procedure is illustrated for single-purpose dairy production using New Zealand data. Feed efficiency analysis of dairy cows feed according to their peoduction potential and belonging to extremely different genotypes (from the Holstein to the Charolais breed) show that both between and within genotypes, the very first factor of variation is the milk production yield. The second factor observed is the magnitude of weight loss after calving which is related to a mobilization of energy stores. Using the energy system of I,EROY, it was not possible according to the analysis of between and within genotype variations to determine the exact contribution of body store mobilization to milk production. The energy equivalence of the weight change seems to vary from one genotype to another; it should be more accurately defined by taking into account the variations in the feeding level and in the proportion of concentrates in the diet which affects the utilization rate of the rations. Selection for production of milk or fat corrected milk can be expected to result in an au- tomatic increase in gross feed efficiency. The effectiveness of indirect selection is expected to be 70 to g5 per cent as great as direct selection. Correlated responses of body weight change are less well known. Magnitude of genotype by environmental interactions, de finedby energy in the diet, from most work seems negligible or small, but may be real for some comparisons. Interactions of such small magnitude between genotype and nutritional regimes could not have real for practical breeding decisions. Because genetic differences in milk are more easily measured on diets higher in energy concentration than all forage diets, the potential reduction in genetic gain for milk production when selecting on all forage diets could be greater than errors in selection caused by interactions, particularly if the interactions could be minimized by rescaling the data. In 23 pairs of monozygous cattle twins the relations between feed intake, milk yield and milk constituents were examined. Between milk protein content during 7th to 12th week of lactation and energy intake per kg F.C.M. there was a correlation of r = o.4i. Presumably the protein content can be used as indicator for feed intake for selection purpose. The feed intake has been recorded between 215 and 370 days of age on 118 bulls of the Belgian Blue and White breed, 64 of the conventional or dual-purpose type and 54 of the double-muscled type, reared and fattened on a high energy diet (z.8 M cal ME per kg DM of a concentrate fed ad libitum). The average feed consumptions, expressed in kg DM, adjusted for the metabolic weight were : 6 y2 (conventional) and 6 442 (double-muscled). They are significantly different (P < 2 per cent). The average feed efficiencies, expressed in kg DM per kg weight gain and adjusted for the metabolic weight, were : 5 5y (conventional) and 5 143 (double muscled) and are significantly different (P < 2 per cent). The difference between the conventional and the double-muscled regarding their AP /AL ratio (daily protein deposition on daily lipid deposition) seems to be of the order of ¢o per cent. A difference of this magnitude accounts for a difference in feed efficiency similar to that observed, that is, of about 0.4 kg per DM per kg weight gain. 42 AI-bulls of the Swedish Red and White breed have been slaughtered at an average age of 28.6 months when they had produced 30 ooo doses of semen. The body composition varied to a large extent. If the values were corrected to the same carcass weight 444 kg, the leanest bull had in total 122 kg less fatty tissue deposited than the fattest one. Expressed in energy units the leanest had only 54 per cent as much energy as the fattest. The difference in total value of retail cuts amounted to more than i ooo Sw Cr. or about 20 per cent in favour of the leanest one. The correlation between estimated breeding value for growth rate and body com- position turned out to be close to zero. Further studies will reveal if the body composition of the AI-bulls is worth while to consider in the selection of dual purpose bulls.
Livestock production under grazing is characterised by inability to directly measure feed intake, an interactive pasture-animal complex, marked seasonal fluctuation in feed quantity and quality, high animal maintenance costs and relatively low production levels. Harvesting ability of the animal, resilience to feed fluctuations, resistance to disease or stress and voluntary feed intake contribute importantly to total feed efficiency. Greatest scope for genetic improvement however is through increasing feed conversion efflciency.
The high proportion of maintenance to total feed requirements in a free-grazing system calls into question the effectiveness in improving total productivity of traditional evaluation and selection on yields per animal. An "efficiency index", equivalent to the yield of an animal of average liveweight having similar predicted efficiency, is proposed for adjusting observed yields for the effect of liveweight on feed conversion efficiency. In many situations the efficiency index can be approximated by a linear function of yield and liveweight, to which standard selection index methods can be applied to optimise genetic progress, given appropriate genetic parameters for liveweight and production traits. The procedure is illustrated for single-purpose dairy production using New Zealand data. Feed efficiency analysis of dairy cows feed according to their peoduction potential and belonging to extremely different genotypes (from the Holstein to the Charolais breed) show that both between and within genotypes, the very first factor of variation is the milk production yield. The second factor observed is the magnitude of weight loss after calving which is related to a mobilization of energy stores. Using the energy system of I, EROY , it was not possible according to the analysis of between and within genotype variations to determine the exact contribution of body store mobilization to milk production. The energy equivalence of the weight change seems to vary from one genotype to another; it should be more accurately defined by taking into account the variations in the feeding level and in the proportion of concentrates in the diet which affects the utilization rate of the rations. GENETIC Selection for production of milk or fat corrected milk can be expected to result in an automatic increase in gross feed efficiency. The effectiveness of indirect selection is expected to be 70 to g 5 per cent as great as direct selection. Correlated responses of body weight change are less well known. Magnitude of genotype by environmental interactions, de finedby energy in the diet, from most work seems negligible or small, but may be real for some comparisons.
Interactions of such small magnitude between genotype and nutritional regimes could not have real consequences for practical breeding decisions. Because genetic differences in milk production are more easily measured on diets higher in energy concentration than all forage diets, the potential reduction in genetic gain for milk production when selecting on all forage diets could be greater than errors in selection caused by interactions, particularly if the interactions could be minimized by rescaling the data. In 23 pairs of monozygous cattle twins the relations between feed intake, milk yield and milk constituents were examined. Between milk protein content during 7 th to 12 th week of lactation and energy intake per kg F.C.M. there was a correlation of r = o. 4 i. Presumably the protein content can be used as indicator for feed intake for selection purpose. The feed intake has been recorded between 2 1 5 and 370 days of age on 1 1 8 bulls of the Belgian Blue and White breed, 6 4 of the conventional or dual-purpose type and 54 of the doublemuscled type, reared and fattened on a high energy diet (z.8 M cal ME per kg DM of a concentrate fed ad libitum).
The average feed consumptions, expressed in kg DM, adjusted for the metabolic weight were : 6 y 2 (conventional) and 6 442 (double-muscled). They are significantly different (P < 2 per cent). The average feed efficiencies, expressed in kg DM per kg weight gain and adjusted for the metabolic weight, were : 5 5 y (conventional) and 5 143 (double muscled) and are significantly different (P < 2 per cent).
The difference between the conventional and the double-muscled regarding their AP /AL ratio (daily protein deposition on daily lipid deposition) seems to be of the order of ¢o per cent. A difference of this magnitude accounts for a difference in feed efficiency similar to that observed, that is, of about 0 . 4 kg per DM per kg weight gain. AI-bulls of the Swedish Red and White breed have been slaughtered at an average age of 2 8.6 months when they had produced 30 ooo doses of semen. The body composition varied to a large extent. If the values were corrected to the same carcass weight 444 kg, the leanest bull had in total 122 kg less fatty tissue deposited than the fattest one. Expressed in energy units the leanest had only 54 per cent as much energy as the fattest. The difference in total value of retail cuts amounted to more than i ooo Sw Cr. or about 20 per cent in favour of the leanest one. The correlation between estimated breeding value for growth rate and body composition turned out to be close to zero. Further studies will reveal if the body composition of the AI-bulls is worth while to consider in the selection of dual purpose bulls. | v3-fos |
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} | s2 | Postpollination Phenomena in Orchid Flowers. IX. Induction and Inhibition of Ethylene Evolution, Anthocyanin Synthesis, and Perianth Senescence
Auxin (naphthaleneacetic acid [NAA]) application to Cymbidium stigmas (25 μg/flower) induced high rates of ethylene evolution by the flowers within 18 h of the treatment Actinomycin D reduced the rate of ethylene evolution if applied with the auxin or 2 h after it Cycloheximide reduced ethylene production regardless of time of application Puromycin had an inhibitory effect if applied 1 or 2 h after the auxin Ethionine, if applied with the auxin or 1 or 3 h after it, induced more rapid ethylene production and higher evolution rates than NAA alone Applications of ethionine 2 h after the auxin reduced the levels of ethylene evolution but not the rates or induction time Anthocyanin levels decreased when each inhibitor was applied together with the auxin Actinomycin D, cycloheximide, or puromycin, given 1 h after auxin application, did not reduce anthocyanin levels, whereas ethionine did These results indicate that (1) initial synthesis of anthocyanins may depend primarily on preexisting RNA and proteins, (2) subsequent production requires de novo RNA and protein synthesis, and (3) ethionine may be a specific inhibitor of anthocyanin production Of the additional postpollination phenomena exhibited by Cymbidium flowers, some are sensitive to RNA and protein synthesis inhibitors, and others are not
Introduction
Orchid flowers, if unpollinated, remain alive for long periods of time. Some flowers, which stay fresh for up to 3-4 mo in the absence of pollination, start to show signs of senescence or exhibit typical postpollination phenomena shortly (in some orchids, within 15 min) after being pollinated (DECKER 1941; PODDUBNAYA-ARNOLDI and SELEZNEVA 1957; VAN DER PIJL and DODSON 1966;ARDITTI 1979).
Depending on species or genus, pollination can cause a variety of developmental, physiological, and biochemical events, including wilting, senescence, greening, fading, or anthocyanin formation in some or all segments of the perianth (ARDITTI 1976(ARDITTI , 1979. Auxins can mimic certain pollen effects and initiate a number of postpollination phenomena in many orchids, including Cymbidium (ARDITTI 1979). One of these is ethylene evolution; this hormone has been implicated in control of several postpollination processes (BURG and DIJKMAN 1967;ARDITTI, HOGAN, and CHADWICK 1973;ARDITTI 1979). However, the time course of ethylene production and factors which regulate it and its effects remain unresolved. Co.) were selected, surface-sterilized, and maintained as described by ARDITTI et al. (1973). 1 Dedicated to the memory of ROBERT I. NORTON, formerly of Dos Pueblos Orchid Co., a longtime friend and a strong supporter of orchid research.
2 Address for reprint requests. TREATMENTS.-Naphthaleneacetic acid (NAA) (25 ,ug/flower), ethionine (10 ,ug/flower), and puromycin (1 ,umol/flower) were applied in liquified lanolin pastes. Actinomycin D (10 ,ug/flower) and cycloheximide (10 ,ug/flower) were administered in 0.2% aqueous agar. All substances were applied to stigmas in 5-10 ml drops. Previous work indicated that these substances were effective when administered in this fashion (ARDITTI and KNAUFT 1969). Treated flowers were placed singly in 1-liter jars covered by lids fitted with ampule caps (ARDITTI et al. 1973) and maintained under 16-h photoperiods and 22 C. Untreated, pollinated, and emasculated flowers, as well as blossoms treated with agar, lanolin, and agar plus lanolin, both in and out of jars, served as controls. All treatments were replicated three times. ETHYLENE DETERMINATIONS.-Evolution of ethylene was determined by gas chromatography (STEEN and CHADWICK 1973) at 2, 5, 9, 18, 20, 24, 40, 42, 66, and 70 h after treatments. A 5-ml sample of gas was removed from the flask for chromatography and replaced with room air at each time interval. The rate is expressed as nanomoles ethylene per flower per hour.
EXTRACTION AND MEASUREMENT OF ANTHOCYAN-
INS.-Anthocyanins were extracted from preweighed labella (lips, median petals) and gynostemia (columns) by maceration with a glass rod and steeping in 1% HCI in methanol (ARDITTI and KNAUFT 1969).
Following centrifugation to remove debris, the extracts were adjusted to a constant volume (25 ml) and their absorbance at 525 nm was determined with a Beckman DBG spectrophotometer. Anthocyanin concentration is expressed as A525/g fresh weight (FW) tissue (FURUYA and THIMANN 1964). Since swelling may dilute anthocyanin content, data from swollen gynostemia were corrected using average weights of untreated ones from freshly cut flowers as a correction factor. FLORAL SEGMENTS.-Wilting, aging, loss of curvature, and stigmatic closure are described in subjective terms. Swelling was measured as increase in width along the lower edge of the stigma.
ETHYLENE
EVOLUTION.-Application of NAA and pollination resulted in marked increases of ethylene production ( fig. 1). Ethylene evolution was not affected by agar or lanolin alone or in combination ( fig. 2).
Ethylene evolution was reduced by actinomycin D to levels lower than those of untreated flowers ( fig. 3). When the inhibitor was applied concurrently with NAA or 2 h after it, ethylene production was reduced noticeably. If applied 1 or 3 h after the auxin, actinomycin D redu ced only slightly the NAA-induced ethylene evolution ( fig. 3).
The rate of ethylene evolution by cycloheximidetreated flowers was slightly higher than that of unpollinated ones ( fig. 4). Cycloheximide applied together with NAA or 1, 2, or 3 h after it considerably reduced the auxin-induced ethylene production.
Application of ethionine resulted in a rapid stimulation of ethylene evolution ( fig. 5). In the presence of NAA, ethionine applications at 0, 1, or 3 h brought about an immediate increase which was initially much higher than that resulting from auxin treatments ( fig. 5). During the first 18 h, rates of ethylene evolution by ethionine-treated flowers were always higher than those by blossoms supplied with auxin alone.
Evolution of ethylene was not appreciably affected by puromycin alone during the initial 40 h, but subsequently it was lower than in the controls ( fig. 6). Application of puromycin at 0 h slightly enhanced the rate of ethylene production; if applied later it was inhibitory. Auxin-initiated anthocyanin production in both labella and gynostemia was reduced when actinomycin D was applied together with NAA. This held true for both corrected and uncorrected values (Act D application curves vs. NAA bars in the insert of fig. 3). Anthocyanin levels, with the exception of corrected columns at 3 h, were raised to above those of the control when actinomycin D was applied 2 or 3 h after NAA treatments ( fig. 3). Actinomycin D alone raised anthocyanin levels in comparison with those in unpollinated flowers (figs. 2, 3).
The NAA-enhanced anthocyanin production in labella was reduced when cycloheximide was applied at time 0, 1 h later, or 3 h after the auxin ( fig. 4). Anthocyanin levels in both gynostemia and labella increased when cycloheximide was applied 2 h after the auxin. Pigment concentrations in gynostemia and labella were raised by treatments with cycloheximide only ( fig. 2, insert of fig. 4).
No increases in anthocyanin content resulted from ethionine treatments. The NAA-induced anthocyanin synthesis in both gynostemia and labella was inhibited by this analog of methionine, even when applied 3 h after the auxin (figs. 2, 5). Applications of NAA together with ethionine (time zero) reduced anthocyanin levels to below those brought about by either substance alone (figs. 2, 5).
Anthocyanin content was raised by puromycin in both gynostemia and labella ( fig. 6). The anthocyanin-enhancing effect of NAA was reduced when puromycin was applied at time zero, but the inhibitor had no effect if applied 1 h after the auxin ( fig. 6). Anthocyanin concentration was enhanced slightly in gynostemia and considerably in labella when puromycin was applied 2 h after NAA (figs. 2, 6). WILTING OF SEPALS AND PETALS.-Emasculation, pollination, and NAA treatments caused some wilting of sepals and petals (table 1). Lanolin and agar, applied singly or in combination, did not. The NAAinduced wilting was inhibited by actinomycin D, cycloheximide, ethionine, or puromycin regardless of application time (table 1).
GYNOSTEMIA AND STIGMAS.-Pollination and NAA treatments induced swelling and straightening of gynostemia as well as stigmatic closure (table 1). The effects of NAA were inhibited by ethionine and cycloheximide but not by actinomycin D or puromycin (table 1).
Discussion
ETHYLENE.-Production of ethylene can be blocked by RNA synthesis inhibitors (ABELES 1973). In Cymbidium, evolution of the gas during the first 18 h was reduced only slightly by actinomycin D (fig. 3). One reason for this may be that de novo RNA synthesis is involved minimally or not at all in the initial response. A second possibility is that actinomycin D may not be reaching the site(s) of ethylene evolution in sufficient amounts. Auxin applied to orchid stigmas is translocated to parts of the gynostemium (BURG and DIJKMAN 1967) and the rostellum, the major site of ethylene production in orchid flowers (ARDITTI 1979), where it induces evolution of the gas. Uptake and translocation of actinomycin D may be limited (TAO and KHAN 1976) and slower than auxin movement (ABELES and HOLM 1967). High rates of ethylene evolution require protein synthesis (STEEN and CHADWICK 1973), and some of our findings indicate the same. Production of the gas is reduced by administration of cycloheximide ( fig. 4), ethionine 2 h after the auxin ( fig. 5), and puromycin 1 or 2 h following NAA ( fig. 6). Exceptions are the lack of inhibition by puromycin and when applied at time zero ( fig. 6) and ethionine treatments at 0, 1, and 3 h ( fig. 5).
Increases in ethylene evolution which follow application of ethionine ( fig. 5) suggest that this methionine analog serves as a precursor for the extant system (STEEN and CHADWICK 1973) in Cymbidium as it does in Ipomea tricolor flowers (KONZE, SCEIL-LING, and KENDE 1978), but not in apple plugs (VANG 1974). In another model system, ethvlene was formed from the ethyl moiety of ethionine (SHIMOKAWA and KASAI 1967).
The failure of ethionine applied at 2 h ( fig. 5) to cause a marked rise in ethylene evolution may suggest that (1) during this time neither the inducible nor the extant system is fully operative and so cannot utilize it as a substrate, and/or (2) ethionine acts as an inhibitor of protein synthesis required for activation of the inducible system. Cycloheximide increases movement of auxin into perianth segments of Angraecum and reduces its levels in gynostemia (STRAUSS 1976). In pea roots, cycloheximide blocks the protein synthesis-dependent phase of ethylene production (STEEN and CHAD-WICK 1973). If it has the same effects in Cymbidium, cycloheximide would inhibit the early burst of ethylene synthesis by (1) reducing auxin levels and/or (2) limiting the inducible system (STEEN and CHAD-WICK 1973) through the blocking of de novo protein synthesis. Some of the effects of puromycin can also be explained in terms of its effects on the proteinsynthesis-dependent pathway of ethylene evolution.
ANTHOCYANINS.-Our findings that auxin and ethylene increase anthocyanin levels in Cymbidium flowers confirm previous reports (ARDITTI and FiscH 1977). One possibility is that NAA (either directly or via enhanced ethylene evolution) stimulates production of an unstable RNA required for anthocyanin synthesis or at least stabilizes it (RAD-NER and THIMANN 1963;MOHR 1969). Increased RNA synthesis in Phalaenopsis following pollination (ARDITTI and FLICK 1976, p. 37) lends support to this view. Under such circumstances it is reasonable to assume that a certain amount of de novo protein
BOTANICAL GAZETTE
[DECEMBER synthesis may also occur, and our results suggest that this is indeed so.
Anthocyanin content is lowered when each inhibitor is applied at time zero (figs. 3-6). One hour after the application of NAA, anthocyanin synthesis is inhibited only by ethionine (figs. 3-6). This agrees well with the time required for disturbance effects to spread from the column (GESSNER 1948) and the results of surgical experiments (ARDITTI and FLICK 1974).
Pigment levels are actually increased by actinomycin D, cycloheximide, and puromycin when applied 2 h after the auxin (figs. 3, 4, 6), whereas NAA-induced anthocyanin synthesis is blocked by cycloheximide applications 3 h after the auxin (fig. 4). These results suggest that (1) initial production of anthocyanins may require de novo synthesis of RNA and protein but is primarily dependent on preexisting substances; (2) subsequent biosynthesis of anthocyanins in Cymbidium flowers depends almost entirely on newly produced RNA and/or proteins as it does in Sorghum vulgare (CRAKER 1971); such two-stage hormonal effects are not uncommon and have been reported in barley seeds (BEN TAL and VARNER 1974) and pea root-tips (STEEN and CHADWICK 1973); or (3) due to faster translocation from the stigma, auxin reaches the site of action and acts prior to the arrival of inhibitors.
Ethionine inhibits protein synthesis and appears to be a specific inhibitor of anthocyanin production (THIMANN and RADNER 1955;SCHRANK 1956;FAUST 1965;ARDITTI and KNAUFT 1969). This explains its effects on anthocyanin production in Cymbidium flowers ( fig. 5). ETHYLENE AND ANTHOCYANIN PRODUCTION.-Production or destruction of anthocyanins in orchids can be initiated by either auxin or ethylene (BURG and DIJKMAN 1967;ARDITTI et al. 1973), and since NAA applications bring about ethylene evolution, it may be that auxin effects are ethylene mediated.
Ethylene-and auxin-regulated phenomena can be blocked by inhibitors of DNA-dependent RNA synthesis such as actinomycin D (ARDITTI and KNAUFT 1969;ABELES 1973). There are, however, also instances where actinomycin D does not have much of an effect, even on processes which are inhibited by cycloheximide, suggesting that (1) actinomycin D is not reaching the tissues where RNA synthesis is occurring (JACOBSEN 1977), or (2) some ethyleneregulated phenomena do require RNA production (and subsequently probably also protein synthesis) whereas others do not (even if these, too, involve the manufacture of new proteins). The latter may be the reason why swelling and straightening of columns as well as stigmatic closure, although blocked by cycloheximide, are not strongly affected by actinomycin D.
WILTING.-Senescence and wilting of the perianth can be induced by either NAA or ethylene, are initiated at approximately the same time, and are difficult to separate visually (ARDITTI et al. 1973). Wilting is the result of water and dry weight losses by Cymbidium orchids and other flowers (MAROUSKY 1973;ROGERS 1973;ARDITTI and HARRISON 1979). Senescence, which in Cymbidium flowers accompanies the wilting, is undoubtedly no different from aging in other plant systems and may, therefore, require the production of new RNAs or proteins. This is indeed so in Nicotiana (TuPY and RANGASWAMY 1973) and Phalaenopsis (ARDITTI and FLICK 1976). Hence, it is not surprising that inhibitors of protein and RNA svnthesis can also inhibit wilting and aging (table 1).
The initial responses of orchid flowers to pollination, auxin applications, and ethylene treatments are relatively rapid. Some postpollination phenomena are sensitive to inhibitors of protein and RNA synthesis, whereas others are not. This sensitivity changes with time of application of the inhibitor, suggesting that postpollination phenomena are brought about by two sets of proteins and RNA: one preexisting, the other newly produced. | v3-fos |
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} | s2 | The composition of the faecal microbial flora of milking cows on feeds containing urea as the sole or partial source of nitrogen
Using a variety of culture media, counts of live bacteria in the faeces of milking cows on purified, protein-free feed in which urea was the only source of nitrogen (0-cows) and on urea-rich, low-protein feed (ULP-cows) were made. Samples were taken from 3 0-cows and 3 ULP-cows weekly. Counts of lactobacilli, micrococci, propionic acid bacteria and lactate-utilising butyric acid bacteria were made during a period of 41 weeks, and counts of anaerobic and aerobic bacteria, enterococci and coliform bacteria for 26 weeks. Counts are given as log10 number/g fresh faeces. In the 0 cows anaerobic bacteria counts were 5 9, ULP cows 6 9; aerobic bacteria were 5 7 and 5 8 respectively. »Propionic acid bacteria» counts were 4 7 with both 0and ULP cows, lactobacilli 3—B in 0 cows and 4—B in ULP cows, and micrococci 2—7 in both groups. Enterococci were 2—6 in 0 cows and 37 in ULP cows, and coliforms 3—6 and 2—6 respectively. Lad ate utilising butyric acid bacteria were o—4 600/g fresh faeces, being fewer in 0cows than in ULP cows. There was large variation in the counts of all bacteria from one week to the next. In a single cow almost all the bacterial counts could be low at the same time and the following week they could all be high. In different animals the timing of these variations was not the same. The counts of bacteria growing under anaerobic conditions were the main exception to the above variations. The ratio between gram-negative and gram-positive bacteria in faeces was measured and it was found that with both types of test feed the gram-negative were more numerous. In addition the occurrence of bacterial spores was studied; their counts, as a proportion of the total number of bacteria reacting to stain, averaged 6.5 and 26.7 % respectively in 0and ULP-cows.
Introduction
In young animals the faecal microbial flora is uniform, that is speciesindependent, but in the adult animal each species has its own characteristic bacteria (Smith 1961). The uniformity, according to Smith, is due to the similarity of the diet (milk) and to the early stage of development of the digestive tract. However Medrek and Barnes (1962) found that in fullygrown beef animals the differences brought about by winter feed (hay, silage and cereals) and summer feed (grass and cereals) were small.
In feeding studies using urea as the sole or partial source of nitrogen (Virtanen 1967) protein production was based on actively-functioning rumen bacteria. Thus it was probable that the microbial population of the faeces would be different from that in cows given ordinary feed. For this reason the test cows' faeces were analysed, using a variety of culture media, for total count of bacterial colonies grown under aerobic and anaerobic conditions, and for total counts of live enterococci, coliform bacteria, microaerophilic lactobacilli, propionic acid bacteria, micrococci and lactate-utilising butyric acid bacteria.
Materials and methods
The composition of the test cows' feed in which urea was the only source of nitrogen is shown in Table 1. The names 0-cows and 0-feed have been used for these cows and their feed. For those test cows with urea as a partial source of feed nitrogen the term »urea-rich, low-protein» abbreviated to ULP-cows has been used; the feed is denoted ULP-feed. Details of the ULP-feed are given in Table 2. Both types of feed were maintained basically unchanged the whole time, the size of the daily ration varying with rate of milk production. With the exception of a I-2 hour period of excercise daily the cows were kept in the cowshed continually. Faeces samples were taken from the test cows once a week during the period January -November. The samples were taken about 1 hour after the start of feeding and brought to the laboratory for analysis within 3 hours. Pending dilution the storage was in sterile glass jars at 15-18°C. According Crowther (1971) there are no large changes in the bacterial counts of human faeces during transport at 15°, provided the period is less than 24 hours.
Ringer's solution was used for the dilutions. All bacterial groups were inoculated from the same series of dilutions. The media and methods were as follows.
Gram-and spore staining was performed as described by Norris and Swain (1971).
In examining the results it must be remembered that the predominant part (more than 97 %) of the faecal microorganisms are anaerobic organisms, some being obligate anaerobes (Moore and Holdeman 1972). In culture procedures only a fraction of the microorganisms form colonies. According to the studies of Maki and Picard (1965) only 1-10 % of the microscopicallycounted bacteria are revealed in culture. The microscope does not distinguish live bacteria one reason why its counts are higher. While some bacteria can pass undisrupted through the digestive tract, thereby taking stain in the normal manner, they are no longer capable of multiplying.
Results
Counts of lactobacilli, micrococci, propionic acid bacteria and lactateutilising butyric acid bacteria in the faeces of 3 0-cows and 3 ULP-cows were performed weekly for 41 weeks, using different culture media, and counts of aerobic and anaerobic bacteria, enterococci and coliform bacteria were made during 26 weeks.
The week-to-week variation in the number of bacterial colonies for each cow was very large (Figures I-4), such variation being evident in all bacterial groups. When the lactobacilli were numerous so too were the propionic acid bacteria and the enterococci. The total count of aerobically-cultured bacteria followed the same fluctuations. The group with the clearest deviation from this sequence of changes was the total anaerobically-cultured bacteria. The between-cow differences in bacterial counts of samples taken on the same day were considerable. Each cow's average count of bacterial colonies (log 10 /g fresh faeces) is shown in Table 3. Usually the count of anaerobic bacteria was greater than that of aerobically-cultured bacteria. In the 0-cows the difference between the counts of anaerobes and aerobes was greater than in the ULP-cows. Of the bacterial groups cultured on different media the so-called propionic acid bacterial group was the most numerous. The lactobacilli showed higher counts than the enterococci, and in the 0-cows the numbers of coliform and micrococci too were larger than those of the enterococci. As regards the single cow counts, the average for ULP-cow Euru was lower than those of the other ULP-cows. Lactate-utilising butyric acid bacteria were found occasionally in all cows, their numbers being very small; their frequency of occurrence and count in the ULP-cows were greater than in the 0-cows. At calving butyric acid bacteria were always present, even in exceptionally high numbers (4 600/g).
The ratio between the counts of gram-positive and gram-negative bacteria was determined on some of the samples, and it was found that the faeces of all cows contained more gram-negative bacteria than gram-positive. There was no appreciable difference between 0-cows and ULP-cows in these counts ( Table 4). The lowest counts of gram-positive bacteria were found in Euru's faeces. The mean proportion of bacterial spores in the total staining bacteria in ULP-cows' faeces was 26.7 %, in 0-cows' 6.5 %. The highest spore counts were found in Euru's faeces.
Discussion
In each animal there were large between-sample differences in numbers of bacterial colonies despite feed uniformity in quality and quantity. Examination of the mean counts shows that 0-cow faeces contained more anaerobic bacteria than ULP-cow faeces (P < 0.05) and fewer aerobic bacteria than ULP-cow faeces (P < 0.01). Mäkinen (1972) reported that the total count of rumen bacteria in 0-cows was 2.23 d: 0.36 X 10 10 and in ULP-cows 1.54 d: 0.29 X 10 10 . The number of colonies of microaerophilic lactobacilli in ULPcows' faeces was greater than in 0-cows' (P < 0.001). In both groups of test cows these counts were higher than those found by Smith (1961), who noted in fact than some cows had no faecal lactobacilli at all. They were present in every sample of 0and ULP-cow faeces, the counts ranging from 10 4 to 10 8 /g. Similarly, contents of coli bacteria too in faeces from both groups of test cows appear to be higher than in those cows studied by Smith (1961). The presence of micrococci in beef animals' faeces was reported by Wilssens and Buttiaux (1958); the average count in ULP-cow's faeces was greater than in 0-cow's (P < 0.05).
As regards total numbers, the faecal streptococci, one of the classes of enterococci, are regarded as the most important faecal microorganisms, just as they are in rumen fluid. However, the counts of enterococci were less than those of lactobacilli, particularly in O-cows and very frequently in ULP-cows. Moreover, the numbers of both coli bacteria and micrococci too were greater than those of the enterococci. Even so the counts of enterococci colonies obtained here are similar to those recorded in the literature. Average counts are 10 5 enterococci/g fresh cow faeces (Medrek and Barnes 1962) and about 10 6 streptococci/g (Smith 1961). Counts in 0-cow faeces were 10 4 -l0 5 and in ULP-cow faeces 10 s -l0 7 /g fresh material. Medrek and Barnes (1962) found that counts of enterococci during the winter did not differ greatly from those during the summer, the feeds containing hay, silage and cereals, and grass and cereals respectively.
Using Kambar lactate medium, the one generally employed for culturing propionic acid bacteria, the number of colonies from both groups of test cows was greater than those found with the other media used, with the exception of the anaerobic bacteria. The variations in the counts of these bacteria parallelled those of the lactobacilli and aerobic bacteria. According to Mäkinen (1972) rumen propionic acid contents of 0-cows in particular were higher than in cows on ordinary feed.
Lactate-utilising butyric acid bacteria were present occasionally in all test cows' faeces, in small numbers usually. Whenever the counts were high there were signs of disturbed function in the cows' rumen. The presence of butyric acid bacteria in faeces is regarded generally as resulting from the fodder consumed. Wilssens and Buttiaux (1958) reported the occurrence of Clostridium butyricum during the winter in cow's faeces, which they attributed to the feeding of silage. It has been observed (Kiuru et al. 1975, Ali-Yrkkö andAntila 1975) that counts of butyric acid bacteria are low provided the cows' feed is of high quality, even though it may contain silage. However, the studies of Ali-Yrkkö and Antila did reveal cows with faecal butyric acid bacteria despite the good quality of their feed and despite the absence of these bacteria from the other animals. Such was the case with those test cows used in the present study. Butyric acid bacteria were found even in 0cows, which were given feed of the highest possible purity. Faecal counts of these bacteria were elevated in both groups of cows during the time of calving.
By means of the spore stain it was found that the content of spores as a proportion of the other stainable microorganisms was 6.5 % in 0-cows' faeces and 26.7 % in ULP-cows'. Mäkinen (1972) gave the proportion of gram-positive bacteria as 35.9 ± 0.4 % of the total rumen bacteria of 0-cows, 16.7 ± 3.9 % in ULP-cows and 10.7 ± 2.1 % in normally-fed cows. In faeces too with both 0-and ULPcows the proportion of gram-negative bacteria was larger than that of the grampositive. Maki and Picard (1965) isolated intestinal bacteria from beef animals and found that most of them, with the exception of E. coli, were gram-positive; those bacteria which they were unable to identify were mainly gram-positive rods. The major portion of the microorganisms of the caecum consists of gram-negative rods and cocci (Orskov et al. 1970).
In the test cows the number of bacterial colonies in all the groups of bacteria studied was higher than those generally reported in the literature. According to Hungate et al. (1952) feed with a higher-than-normal content of carbohydrate alters the microbial flora of the rumen, and Orskov et al. (1970) state that addition of starch to feed increases bacterial numbers in the caecum.
The week-to-week fluctuations in the number of bacterial colonies in all the groups of bacteria were abrupt and of considerable size. In many cases the reason for the change could not be ascertained. Alterations in the amount of feed, which Smith (1961) found to result in changes in the microbial population of the faeces, appeared in the present study to have had no effect. However there were a number of factors which naturally affected the counts of live bacteria in the faeces. A decline in the quality of the drinking water supplied to the 0-cows brought about ruminal disturbances and so the numbers of live lactobacilli, micrococci and propionic-acid bacteria in the faeces also declined.
In addition, certain experimental operations, such as the sampling of rumen fluid and the lengthy (5 7 days) quantitative collection of urine and faeces, reduced the number of micro-organisms in the latter, probably a consequence of some functional abnormality in the rumen. On the other hand, the large weekly variations could have resulted in part from some stage in the work of taking and diluting the samples. Only one series of dilutions, from which all the cultures were inoculated, was made from each faeces sample. Parallel changes in several of the groups of bacteria from a single cow suggest that the factor in question was associated with the dilution. It is true that the anaerobic bacteria did not show this parallelism even though they were inoculated from the same series of dilutions as were the other bacterial cultures. In faeces from different cows the fluctuations occurred at different times, so that the factor in question could hardly have arisen from some part of the working procedure common to all the samples taken at the same time. | v3-fos |
2019-03-19T13:07:08.989Z | {
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} | s2 | MAIZE FOR SILAGE 11. The effect of urea and acid as preservative treament on rumen fermentations and on feeding values of silages
. The rumen fermentations and N-balances of rumen fistulated sheep were studied on diets of silages treated with urea and acid preservative. The digestibilities and feeding values of the silages were also calculated. The experiment was performed according to 5 x 5 Latin-square design. The digestibilities were determined by total collection the collection period lasting seven days. The rumen samples were taken on the last two days during the collection periods before and 1.5, 3.0, 4.5 and 6.0 hours after feeding. Besides the silages the animals received mineral mixture and water ad libitum. Urea or acid treatment had no effect (P > 0.05) on the consumption of silage DM. The consumption ranged from 1.7 to 1.9 kg DM/100 kg liveweight. Urea did not have a clear effect on the VFA production in the rumen. It tended, however, to decrease the proportions of C 3 and C 4 —C S acids in the rumen. Acid preservative decreased the production of VFA and the proportion of C 3 -acid (P < 0.05) in the rumen. The proportion of C 3 -acid was higher respectively, (P < 0.05). The rumen ammonia levels were extremely high, 60 —BO mg/100 ml, after feeding with urea treated silages. This was possibly partly due to the relatively short (14 days) adaptation time before collection period. The urea or acid preservative treatment gave no clear responses regarding the apparent digestibility values of the silages. The DCP values were highest (P < 0.05) in the urea treated silages, but no differences (P > 0.05) were found between the energy values, which varied between 0,12 0.14 f.u./kg of silage. There were no differences in the N-balances of the animals on different diets. The balances were positive on all diets.
Introduction
When the whole maize plant is made for silage at a proper vegetation stage, the silage contains a lot of energy and is suitable especially for growing cattle (Utley et al. 1973, Lelong 1976). Because of the low content of crude protein, maize silage should be supplemented at least by a protein concentrate. Urea may also be used, because rumen NH 3 liberated from urea degradation in the rumen could be efficiently utilized by rumen microbes owing to the abundance of sugars available (Huber et al. 1967, Fenner et al. 1970).
When silage is made fxom raw material in which the DM content is about 20 percent, the fermentation process is often stabilized with an addition of urea or acid preservatives. These substances could, however, cause some feeding problems in regard to the acidity of silages for instance (Wilkinson et al. 1976).
To throw light on these questions the effects of urea and acid treatments on the palatability and feeding values of the silages were studied. Rumen fermentation studies with sheep were also performed.
Materials and methods
The silages, their chemical composition, ensiling and quality have been described earlier (Setälä et ai. 1979 b). The experimental design is shown in Table 1. The digestibilities and feeding values of the silages and the rumen fermentations were studied according to 5 X 5 Lation-square design with five Finnish Landrace rams (liveweight 55 kg) fitted with rumen fistulas. Each period lasted 21 days including a collection period of seven days. The principle of total collection was used. The animals were fed twice a day. Besides silages they had free access to mineral mixture and water all the time. The composition of the mineral mixture was as follows: Ca 13.0 %, P 9.1 %, Mg 5.0 %, NaCl 22.1 %, Ca:P 1.4: 1. The animals received silages ad libitum during the first ten days ( ad libitum period). During the transition period and collection period the amounts of silages given were restricted to 90 percent of the ad libitum feeding so there were no refusals left. The amounts of mineral mixture and water consumed were determined during each collection period. The faeces and urines were weighed and sampled each day during the collection period. From the faeces 10 % of the total amount per day was taken after thorough mixing for the analyses. The urine of each animal was stored through one day in a bucket under the metabolic cage. To prevent microbial action 30 ml of 10 N H 2 S0 4 was added to the buckets. From the urines 5 % of the total daily amout was taken for analyses.
Silages for the experiment were taken from the silos twice a week and the doses for the experimental animals were weighed for 3-4 days at a time, after which they were stored in plastic bags in a cool place until feeding. The samples for the analyses were taken at the same time. Rumen samples were taken before feeding, and 1.5, 3.0, 4.5 and 6.0 hours after feeding during each of the last two days of the collection period. The samples were treated as described by Syrjälä (1972).
The composition of silages and faeces were analyzed according to standard methods. The nitrogen content of the urine samples was determined by the Kjeldahl method.
From the rumen samples the pH was measured with a Beckman meter Model H 2 GWB. Ammonia was determined according to the method of McCullough (1967) and the VFA determinations were made by the gas chromatographic method (Huida 1973).
The statistical analyses were made with a Monroe 1960 computor and its statistical programs.
Results and discussion
The palatahility of the silages In spite of the low pH values (3.5-3.6) the animals consumed willingly the silages so that the energy requirements at the maintenance level according to Kellner and Becker (1966) could be met. The average amounts eaten for silages A, B, C, D and E were 1.93, 1.73, 1.84, 1.88 and 1.81 kg DM/100 kg liveweight, respectively. These values are much higher than those found earlier (Setälä et al. 1979 a) for corn silage.
Urea or acid treatment did not have any significant effect (P > 0.05) on silage DM consumption. A positive response with urea treatment can only be attained if the crude protein content of the silage remains below 9 % in the silage DM after urea addition (Andrieu and Demarquilly 1974).
The effect of urea and acid preservative on rumen fermentation
Total VFA production in the rumen has been highest ( Fig. 1, Table 2) 1.5 hours after feeding, when silage with 0.5 % of urea included was fed. The yield of VFA has been most stable on diets with B-silage included. Urea has increased the total VFA production in the rumen on maize silage diets with lambs (Schaadt and Johnson 1964), but no response has been obtained with cows (Huber et al. 1967) or calves (Thomas and Wilkinson 1975).
The use of acid preservative decreased VFA production in the rumen the production being lowest when D-silage was fed. There was, however, a significant difference (P < 0.05) only between the A-and B-silages and the Dsilage 1.5-3.0 and 4.5 hours after feeding, respectively ( Table 2). It is possible that the formaldehyde in the preservative decreased the fermentation in the rumen as found by Vanbelle et al. (1978). This has been noted also on grass silage diets with sheep, when preservatives containing formic acid or a mixture of formic acid and formaldehyde were compared (Syrjälä 1972). Because Table 2. The average total VFA and NH 3 concentrations in the rumen (A -E, see Table 1). of the high amount of readily available carbohydrates and the succulent nature of the maize silage, the proportion of propionic acid in the rumen VFA is usually quite high, the ratio of C 2 :C 3 being 2.1-3.2 (Balwani et al. 1969, Schaadt and Johnson 1969, Fenner et al. 1970). Compared to those results, the proportion of acetic acid in the rumen is high (Table 3). In their experiments the proportion of ears in silage DM must have been quite high which was not the case in the present trial. Verite and Journet (1975) have shown that the proportion of grains can greatly influence the acetic (lower) and butyric (higher) acid production in the rumen. Possibly the chop length of the silage has also been different. In this trial the average chop length was 2-3 cm and according to Ely and Sisk (1977) already it can cause the higher proportion of acetic acid in the ruminal VFA. Urea has not affected the molar proportions of different VFA in the rumen.
It appears that the proportion of C 3 and C 4 acids have decreased. The proportion of C 4 C5 acids is very low when silage treated with 1.0 % of urea and Viher-acid has been fed.
The acid treatment of the silage increased the production of C 2 and decreased the production of C 3 -acid in the rumen. These changes were significant (P < 0.05) 1.5-4.5 hours after feeding. It was also evident that the proportions of the C 4 C 5 -acids had decreased.
Rumen ammonia concentrations after feeding have been extremely high ( Table 2, Fig. 2). This was so especially when silage treated with 1 % of urea and Viher-acid was fed. The effects of the different NH 3 -levels could also be seen in the pH values in the rumen (Fig. 1).
The ammonia levels of diets that included urea are much above the desired levels suggested for microbial protein synthesis (Satter and Roffler 1975) and for optimal feed digestion in the rumen on diets rich in carbohydrates (Mehrez and orskov 1975). Levels are already too high when diets not including urea are considered. According to Thomas andBrown (1968, ref. Huber et al. 1968) there is no response for instance in heifer gain from urea addition if the CP content in the silage DM is already 10 percent.
These high ammonia levels could be accounted for by the relatively short adaptation period (14 days) for urea feeding when the feeding was changed according to the Latin-square design. According to Ettala et al. (1977), an adaptation period of 28 days was not long enough in the feeding of lactating cows. It should also be noted that urea was not degraded severely to ammonia during ensiling (Setälä et ai. 1979 b).
When comparing the total VFA production in the rumen (Fig. 1) and the digestibility values (see later) it can be seen that the rumen microbes have not been able to utilize efficiently the »extra» ammonia liberated from the urea.
The slight response of the Viher-acid treatment to the lower ammonia and fermentation levels could be explained by the formaldehyde of Viher-acid. Syrjälä (1972) e.g. has shown that the silage protein could be protected by the formaldehj'de treatment.
The digestibilities and feeding values of the silages
The use of preservatives has not affected noticeably the apparent digestibility values (Table 4). The differences have only been significant in the digestibilities of crude fat (P < 0.01) and N-free extracts (P < 0.05). It is somewhat difficult to explain these results, but a possible explanation for the better digestibility of the NFE-fraction in D-silage could lie in the higher concentration of sugars in silage DM (see Setälä et ai. 1979 b). Generally the digestibility of the NFE-fraction has been lower in urea-treated silage, which has also been noted by Fornaroli and Perotti (1972) with sheep. Figure 2. Rumen ammonia levels before and 1.5 -6.0 hours after feeding (A -F see Table 1 The use of urea as a preservative can increase the apparent digestibilities of crude protein and crude fat in maize silage (Polan et al. 1969 andKabuga et al. 1973).
The use of acid preservative has tended to decrease the digestibility values in urea treated silages. This could be due to the lower fermentation in the rumen (see Fig. 1). When used with propionic acid, 0.5 % of urea can improve the digestibility of silage DM, but there has been no response when urea has been used alone as a preservative (Otterby and Murphy 1973). The use of propionic acid or formic acid alone has improved slightly the digestibility values (apart from crude fat) of maize silage (McCullough 1972, Leaver 1975. The effect of urea on the feeding values of silage has been taken into account according to Lampila (1968). There are no significant (P > 0.05) differences in the energy values of the silages. Because of the low proportion of mature ears in the fodder mass ensiled, the energy values are quite low. However, when fed as a sole roughage ad libitum to a dairy cow, liveweight 500 kg, the silages treated with 0.5 % of urea would provide the energy and DCP requirements for maintenance and production of 10 kg FCM/day. The urea treated silages contained significantly (P < 0.05) more digestible crude protein/f.u. These values are approximately the same as in grass silage made from N-fertilized grass. Because of the possibility of high ammonia levels in the rumen and the danger of urea-rich layers of silage in tower silos (Böttger 1969), it is not reasonable to use urea treatment of 1 % in silage making.
The nitrogen utilization of the animals The nitrogen balances have been positive in all diets (Table 5). The use of urea has not given a clear response in nitrogen retention (P > 0.05), because the DCP requirements of the sheep could already be met by feeding maize silage without urea (Kellner and Becker 1966). The effects of the high NH 3 levels in the rumen could also be seen from the N excretion in the urine of the animals fed with urea treated silages. The use of urea may improve the Table 5. Nitrogen balances and N excretion in urine and faeces of the animals (A -E, see Table 1). N retention with sheep on maize silage diet, if the CP content of the silage without urea is about 7 % in DM (Fornaroli and Perotti 1972). The nitrogen excretion in fa.eces has been largest (P < 0.01) on the B-silage diet. It is possible that large amounts of urea have reached the duodenum and have been degraded to ammonia which has been partly utilized by microbes in the caecum. It is possible that the proportion of endogenous nitrogen in faecal N could be larger with this diet compared to other diets. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | 0 | [] | 1981-01-01T00:00:00.000Z | 11963517 | {
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} | s2 | Effects of exogenous materials on pollen tube growth in Lilium longiflorum pistils.
With the use of stigmatic exudate or distilled water as carriers, various antimetabolites, inhibitors, and miscellaneous materials were injected into the hollow styles of detached Lilium longiflorum pistils before, at, or after compatible or incompatible pollination. Pollen tube lengths were measured 48 hr after pollination with pollinated styles incubated at 22-23 degrees C. Substances considered inhibitors of protein synthesis in microbial systems significantly retarded both compatible and incompatible pollen tube growth while inhibitors of RNA synthesis tended to significantly inhibit compatible pollen tube growth with less or no effect on incompatible pollen tubes. Application of the inhibitors in stigmatic exudate at or after compatible pollination produced significant results at the lowest concentrations. Significant retardation of pollen tube growth also occurred after injection of 2,4-dinitrophenol, mercaptoethanol, indoleacetic acid, naphthaleneacetic acid, benzyladenine, dimethyl sulfoxide, or potassium or sodium iodide. Pollen tube growth in detached pistils of L. longiflorum may be useful as a bioassay in situ for screening biologically active materials.
Introduction
Lilium longiflorum Thunb., the Easter lily, is a major floricultural crop in the United States with unique value as a research tool. Easter lily bulbs are asexually propagated (cloned) from bulb scales by specialized growers. Clones available in the United States include Ace, Nellie White, and less frequently Croft, used commercially for production of potted plants; Georgia Belle for cut flowers; and Arai, which is imported from Japan and used as a cut flower. Since the market demands a limited number of cloned genotypes, the researcher is assured of a large supply of a particular genotype from year to year. For research purposes, the Easter lily can be brought into flower on a year-round schedule. L. longiflorum bulbs dug in the autumn require a cool period for rapid floral initiation. Commercially, this is supplied by 6 weeks at 4°C applied to bulbs either in the packing case or after potting. Bulbs can be cooled longer to delay flowering, although excessive cold storage reduces flower number and creases pollen tube staining and reduces background. In addition, stained pistils can be stored at 4°C for several weeks without loss of freshness, so long as they are not allowed to dry. To determine pollen tube length, the style is slit with a triangular needle and the stylar canal held open with another needle under a dissecting microscope, beginning at the ovarian end and progressing toward the stigma. When pollen tube tips are visible, the distance to the stigmatic surface is measured with a ruler. Data may be taken as the length of the longest one or more pollen tubes, depending on statistical design. Because the Easter lily is self-incompatible, two types of pollen tube growth occur from intraspecific pollination. Self or intraclone pollination yields incompatible pollen tubes which reach 40-50% of the length of the style in 48 hr at 22-23°C, while interclonal pollination yields compatible tubes which penetrate 80-90% of the length of the style under the same condition (9). Of the clones commonly grown in the United States, Georgia Belle and Arai are cross-incompatible (bear the same S genes for incompatibility) but both are reciprocally compatible with Ace, Nellie White, and Croft which are, in turn, reciprocally cross-compatible in all combinations (10). Pollen tube growth rate in L. long4loruni pistils is sensitive to temperature since elevated temperatures (30°C or higher) destroy the selfincompatibility reaction (11). Therefore, incubation temperature following pollination must be standardized. Pollen tube growth in pistils detached from the plant and incubated on moist filter paper in petri dishes does not deviate from growth on the plant, so long as temperatures are equal. The use of detached pistils and controlled temperature incubation after pollination provides reproducibility.
In addition to facilitating pollen tube staining and measurement, the hollow lily style provides an opportunity to introduce exogenous materials into the pollen tube environment in vivo. Mature Easter lily flowers secrete exudate from the stigma. This exudate serves as a postpollination carrier of exogenous substances into the stylar canal (12). Repeated freezing and thawing and prolonged storage at -29°C has no measurable effect on the biological activity of stigmatic exudate as a carrier. Both compatible and incompatible pollen tube growth is slightly enhanced when styles are filled with stigmatic exudate at or 6 hr after pollination, although the incompatibility reaction is not obscured (13). Exudate injection 12 hr after pollination produces pollen tube lengths not significantly different from noninjected controls while injection 24 hr after pollination significantly retards the growth of both compatible and incompatible pollen tubes. Stigmatic 108 exudate remains in the stylar canal throughout the pollen tube growth period and appears as bubbles from the ovarian end of the style when stain is injected (10). Therefore, using stigmatic exudate as a carrier constitutes a combined stylar-canal, pollentubes treatment.
Prepollination treatment of the stylar canal alone can be accomplished using distilled water as a carrier (14). Soluble materials injected into the lily style in water appear to be taken up by the cells lining the canal 4-6 hr after injection (15). However, if there is a question of material remaining free in the stylar canal, the pistil may be flushed with distilled water prior to pollination without adverse affect on pollen tube growth (14).
Using various inhibitors of RNA or protein synthesis and either stigmatic exudate or distilled water as carriers, we found that both compatible and incompatible pollen tubes require protein synthesis for normal growth, that only compatible tubes require RNA synthesis, and that the style requires RNA synthesis to produce the selfincompatibility reaction in L. longiflorumn (14,16).
The purpose of this presentation is to report the effects on pollen tube growth of a number of exogenous materials injected into L. longitforum styles. Although the screening of biologically active materials was not the purpose of the experiments, these data should provide a background from which to draw conclusions as to the sensitivity and suitability of the system.
Material and Methods
Lilium longiflorum cultivars Ace, Nellie White, and Arai provided pistils and pollen for all of the experiments. The plants were glasshouse grown and flowered throughout the year. To minimize contaminating pollinations during harvest without having to emasculate each flower before anther dehiscence, flowers were cut individually early on the day of opening and placed in water in containers in the laboratory. Pistils were harvested one day later by cutting through the ovary with a scalpel, leaving the stamens and the rest of the flower intact. These flowers served as sources of pollen.
Exudate to be used as carrier was collected twice daily with a syringe from the stigmas of flowers cut early on the day of anthesis and maintained in containers of water in the laboratory (22°C) or cold room (4WC). After collection, the exudate was frozen (-29°C) until used. Stock solutions were made fresh for each experiment. Stocks for experiments using stigmatic exudate as a carrier were made 10 x final concentration in distilled water and the test solution made by adding 1 part stock solution Environmental Health Perspectives to 9 parts thawed stigmatic exudate. Control for these experiments was 1 part distilled water in 9 parts stigmatic exudate. Material injected into the style at or before pollination was carried in distilled water. In some experiments styles were filled with exogenous material in water 6 or 12 hr before pollination and flushed with 10 drops distilled water just before pollination. Material insoluble in water but soluble in ethanol or alkali (KOH or NaOH) were dissolved in the appropriate solution and diluted in distilled water. Exudate or water controls were made with the same dilution of ethanol or alkali. Pistils were prepared for injection by snapping the remaining ovary tissue from the style. However, the ovary of L. longiflorum is also hollow so that materials and stain can be injected without removal of the ovary. Disposable hypodermic syringes with 22 gauge needles were used to administer the treatments. The Easter lily stigma is triangular, with 3 sutures which converge in the center. Inserting the needle into a suture often caused cracking of the suture and the material to be injected spilled out of the damaged stigma rather than filling the stylar canal. Therefore, care should be taken to place the needle between sutures. Solutions were injected until a drop appeared at the ovarian end of the style. In experiments involving water as carrier, the filter paper in the petri dishes was folded into ridges to prevent contact between the ovarian end of the style and the moist filter paper. Such contact caused the style to drain.
Incubation of all experiments was in the dark at 22-23°C in controlled temperature cabinets. Various prepollination incubation times were used but postpollination incubation lasted 48 hr for all experiments. At this time aqueous aniline blue was injected into the styles and the styles were refrigerated for pollen tube measurement as reported previously (8).
Two experimental designs were employed, both completely random (17). One design involved compatible versus incompatible pollination as one variable and carrier versus three concentrations of exogenous material as the other. Concentrations usually differed by a factor of 10. These treatments were replicated four times giving a total of 32 lily styles per experiment. ThE other design involved compatible versus incompatible pollination as one variable and carrier versus one concentration of material to be tested as the other. These treatments were replicated 8 times, again resulting in 32 styles per experiment. Both designs incorporated two observations per style, treated as subsamples. The two observations were the length of each of the two longest pollen tubes. Data were subjected to January 1981 standard analysis of variance and F tests and means were separated with Duncan's new multiple range test (17).
Experiments were performed over a 7-year period and involved a large number of metabolites, antimetabolites, inhibitors and miscellaneous materials. For data presentation, these have been grouped into somewhat arbitrary classifications. The substances tested are listed in Tables 1-9.
Results and Discussion
Data from examples which typified the 76 experiments carried out to determine the effect on pollen tube growth in Easter lily of materials considered inhibitors of protein synthesis in microorganisms appear in Table 1. Compatible pollen tube growth in L. longiflorum was most sensitive to cycloheximide administered in stigmatic exudate at pollination; 1 x 10-6 M caused retardation. Combined pollen tube and stylar canal treatments, that is injection at or after pollination using stigmatic exudate as carrier, tended to retard both types of pollen tube growth, suggesting that protein synthesis is necessary for development of the male gametophyte in L. longiflorum (16). Interestingly, prepollination injection of these inhibitors in water decreased compatible pollen tube lengths at concentrations which had no effect on incompatible tube growth. Table 2 summarizes all experiments conducted with these inhibitors. Tetracycline did not appear stable in water since precipitation usually occurred during injection. On the other hand, none of the four experiments with puromycin at 1 x 10-3M in exudate exhibited significant change in pollen tube growth although this concentration significantly retarded both types of pollen tubes when carried in water. Chloramphenicol could not be test in that the KOH used to dissolve it affected the control sufficiently to prevent all pollen tube growth ( Table 2).
The results of selected experiments involving materials considered to be inhibitors of RNA synthesis are shown in Table 3. When carried in stigmatic exudate and injected at or after pollination, these substances tended to significantly retard compatible pollen tube growth at concentrations which did not affect incompatible tube lengths. However, injection in water before pollination often significantly increased the length of incompatible pollen tubes. In the 26 experiments conducted with these materials (Table 4), compatible pollen tube growth was more sensitive than incompatible to RNA synthesis inhibition, suggesting that compatible pollen tube development requires RNA synthesis unnecessary for normal incompatible pollen bExperiments with treatments reducing pollen tube growth to near zero cannot be subjected to standard analysis of variance since the variance is correlated to treatment. tube growth (16). The stimulation of incompatible pollen tubes after prepollination treatment with water as a carrier suggests that stylar RNA synthesis is necessary for the elaboration of the selfincompatibility reaction (14). Mercaptoethanol and 2,4-dinitrophenol significantly retarded both compatible and incompatible pollen tube growth, while dimethyl sulfoxide (DMSO) was the only substance tested which significantly retarded incompatible pollen tubes at a concentration that had no significant effect on compatible tube growth (Tables 5 and 6). Cyclic AMP up to 1 x 10-2 M produced no significant change in pollen tube growth. DMSO at a concentration of 5% in an in vitro culture medium has been reported to reversibly inhibit pollen tube growth of L. longiflorum (18). The significant retardation of only incompatible pollen tube growth by 5% DMSO injected into the lily style (Table 6) points to metabolic parallels between incompatible pollen tube growth in vivo and L. longiflorum pollen tube growth in vitro 110 which are not shared with compatible tube growth in vivo (19).
The plant hormone indoleacetic acid (IAA) and growth regulator naphthaleneacetic acid (NAA) significantly retarded compatible pollen tube growth when injected into the lily style at relatively high concentrations in water while the cytokinin 6-furfurylaminopurine (kinetin) stimulated incompatible pollen tube growth without affecting compatible, and the cytokinin benzyladenine retarded both types of pollen tubes (Tables 7 and 8). Gibberellic acid (GA), succinic acid 2,2-dimethylhydrazide SADH), and (2-chloroethyl)phosphonic acid (Ethaphon), the first a plant hormone and the others growth regulators, had no significant effect. All of these plant hormones and growth regulators were tested at concentrations considerably higher than those considered physiologically active in the plant. Stimulation of incompatible pollen tube growth without effect on compatible tubes by kinetin ( Inhibitor experiments tested Incompatible Compatible or distilled 6-methylpurine (Table 3). Since both are substituted purines, the kinetin effect is probably one of interfering with RNA synthesis rater than one of hormonal activity. As a prelude to radioactive labeling experiments, 1-amino acids were tested for activity in the Easter lily pollen tube growth system. Amino acid analogs were tested as potential inhibitors of protein function. Table 9 lists the amino acids and amino acid growth. A total of 13 experiments were performed. Treatment with KCl had no effect in the seven experiments completed.
Conclusions
The hollow style of Lilium longiflorum which facilitates the application of exogenous materials into the pollen tube environment and the fact that pollen tube growth in detached pistils reflects growth in situ make this system intriguing as a potential bioassay for biologically active materials. Materials injected into the lily style can be screened against two metabolisms. Prepollination injection of the material carred in water with a stylar flush immediately before pollination offers a test of the exogenous substance on the metabolism of the cells lining the stylar canal. The function of these cells is secretory; they nourish the growing pollen tubes. Postpollination injection of the material carried in stigmatic exudate exposes both the growing pollen tubes and the cells lining the stylar canal to the exogenous substance. Our data suggest the greatest sensitivity occurs when exogenous materials are carried in stigmatic exudate and injected into detached pistils at or after compatible pollination. | v3-fos |
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} | s2 | Advances in Nutrition and Management of Calves and Heifers
Strides have been significant in the knowledge of calf and heifer rearing during the last 25 yr. Much information has been gathered on digestive enzymes, development of the digestive system, and metabolism. Investigations have clarified further the role of colostrum in immunity and nutrition of the young calf. Several sources of nutrients have been tested for their suitability in formulation of acceptable milk replacers and calf starters. Once-a-day feeding of milk, colostrum, or milk replacer and early weaning are practical management procedures. Labor and cost efficient methods of feeding and caring for young calves have developed. Extensive work on rearing rates and methods of rearing was published during these 25 yr. Successful schemes have evolved for feeding heifers to freshen at an optimal age and to occupy a respectful position in the milking line. Developments in housing have been phenomenal – from the calf hutch to environmentally controlled nurseries for calves. Heifer housing has ranged from relatively simple, but labor-efficient housing, to complete confinement systems. Herd health programs have developed to minimize many disease problems that can be particularly disastrous in large herds. Contract rearing of herd replacements has become a more frequently chosen option in this period. Budgets for rearing calves from birth to freshening have appeared in recommendations for raising heifers.
INTRODUCTION
This paper is to review advances in knowledge of nutrition, physiology, and management of young dairy animals. Because of the large amount of research and developments, reviews and reports from proceedings are cited in most instances rather than original references. Complete coverage has not been attempted. Many scientists, producers, and others have been responsible for progress in calf rearing. The reader is referred to the several reviews used for citations of individual studies and developments and to other papers in this 75th Anniversary issue.
Events in the Abomasum
Although considerable knowledge had accumulated on digestion of liquid feed by young calves before 1956, investigations on the digestive process have continued. Upon the feeding of milk or a milk replacer, the esophageal groove closes, causing liquids to flow from the esophagus to the omasal-abomasal canal. Coagulation of milk in the abomasum occurs quickly after feeding. However, clotting does not occur with plant proteins or milk products dried at excessive temperatures. Gastric secretions are stimulated by feeding and together with saliva increase the volume of material to twice that fed. The pH of the abomasum is 1 to 2 before feeding, increasing to 6 after feeding, and then decreasing to prefeeding pH. The amount of acid secreted into the abomasum increases with age (5 5).
After coagulation, the liquid portion, whey, moves into the small intestine. Whey appears in the duodenum within 5 rain after feeding (57). The largest flow from the abomasum occurs shortly after feeding, but the overall rate is half the volume every 2 h. Transit time through the small intestine is about 3 h (55,63).
Lipolytic Enzymes
An oral lipase was known prior to 1956, but information concerning its site of secretion, characteristics, and physiological significance to animals was not established. This group of salivary lipases, collectively known as pregastric esterase, is secreted from the region of the vallate papillae, the glosso-epiglottic area, and the pharyngeal end of the esophagus (47). Although not isolated, at least six esterases have been detected electrophoretically. Evidence has accumulated to show that these enzymes preferentially hydrolyze short-chain fatty acids from triglycerides. Considerable lipolysis occurs with milk fat and coconut fat, buy activity is limited with lard, tallow, and vegetable oils. Secretion of salivary lipase is not affected by age or diet, although its significance appears to be with the milk-fed animal. Most of the lipolysis of milk fat by pregastric esterase occurs in the abomasum with little or no action by gastric lipase. Recent studies (17) show greater release of longchain fatty acids by pancreatic lipase when milk-fat is subjected to pregastric esterase before pancreatic lipase. Pancreatic lipase activity is low at birth and during the 1st wk of life (27); hence, some importance may be ascribed to pregastric esterase during this early period of life even though its exact physiological significance has not been elucidated fully.
Carbohydrate Enzymes
In the late 1950's and early 60's evidence showed that newborn calves possessed large quantities of lactase. Enzymes decrease with increasing age and changing diet, but intestinal lactase can be increased or maintained by feeding lactose (27,57).
Preruminant calves under 100 days of age make insignificant use of starch (44). Salivary amylases and other carbohydrate digesting enzymes are either in insignificant amounts or absent. Pancreatic amylolytic activity and juice flow rates increase with age (27). The virtual absence of sucrase has been established, and metabolism of sucrose is primarily a consequence of microbial activity in the lower tract.
Use of this carbohydrate in diets for young calves is not recommended because of diarrheic problems. Monosaccharides, glucose, galactose, and xylose, are absorbed readily from the small intestine. Fructose is not absorbed, however (57).
Protease
Rennin is responsible for coagulation of milk in the abomasum. Evidence collected during the last 25 yr indicates that casein increases secretion of rennin, but soy, fish, and whey proteins decrease secretion (20). Pepsin secretion is not affected by protein substrate. Protease activity of pancreatic tissue increases during the 1st wk of life (27). The increased activity plus increased flow rate of pancreatic juice probably accounts for the increased digestibility of many proteins as the calf grows older.
Rumen Development
Knowledge of rumen development in 1956 (60) included the rapid increase in tissue weight and papillary size with age when dry feeds were fed. The suggestion was made that the chemical nature of the rumen ingesta stimulated rumen development as inert materials did not elicit a response. Also, "cud inoculation" of calves was studied with inconsistent results.
Research during this 25 yr showed that papillary development and absorptive capacity of the rumen are responsive to volatile fatty acid production, Structural development of the rumen is related to increased metabolic activity of rumen epithelial tissue (27). Other work (83) showed that young milk-fed calves do possess the metabolic capacity for utilization of rumen fermentation end products and can adsorb volatile fatty acids from the small intestine. Rumination in calves is functional as early as 2 wk of age and in all calves by 6 to 8 wk if they are offered solid feed (14). Studies of rumen microbes from calves inoculated with rumen contents and from isolated calves showed different bacterial populations, but some groups of typical tureen bacteria were in contents of isolated calves. If offered solid feed, rumen of calves will develop adult characteristics by 3 mo of age. Blood glucose decreases with age and reaches constancy by about 8 wk (27). Reid (60) reviewed the status of colostrum to 1956. It had been established that the calf had little circulating antibody at birth and was dependent upon colostrum for passive immunity. The absorption period for immune globulins was during the first 24 h of life. Calves were not protected necessarily by colostrum from microbial challenges outside the dam's environment. The nutritional value of colostrum as well as its immunological properties was recognized.
COLOSTRUM
Since then three classes of immune globulins have been identified in bovine colostrum. The IgG comprises the greatest fraction, 85 to 90% of immune globulins, and is divided into two subclasses, IgG1 with 163,000 molecular weight and lgG2 with 150,000 molecular weight. The IgM with a molecular weight of 900,000 makes up an additional 10%. An IgA is the other immune globulin that has been identified. Butler (11) prepared an extensive review on characteristics and properties of immune globulins.
Considerable research effort has been on absorption of immune globulins, much of it within the last 5 to 10 yr. The ultrastructure of cells of the jejunum and ileum have been described (10). Pinocytosis is the method of absorption of "y-globulin and is greatest in the distal portion of the small intestine with little in the duodenum. Colostral whey proteins are absorbed without selectivity in newborn calves. Also of importance is that cells of Escbericbia coli are absorbed through ileal cells of calves not fed colostrum. Colostrum-deprived calves exhibited large numbers of E. coli in the ileal mucosa and mesenteric lymph nodes, but no bacteria were in cells and lymph nodes of colostrum-fed calves. Ability of the calf to absorb immune globulins continues for 24 to 36 h; however, intestinal closure occurs at 24 + 4 h after birth. Intestinal closure to immune globulins begins spontaneously after 12 h of life in the absence of feeding. If calves are fed colostrum shortly after birth, more time will be available for absorption than if fed several hours later, even though closure time is delayed with later feeding.
Total amount of immune globulin consumed appears to be a more important factor influencing serum than concentration of immune globulins in colostrum (10). Roy (61) has suggested 1.7 kg/feeding at four intervals during the first 36 to 48 h of life; others suggest 2 liters at first feeding (10). Concentration of immune globulins varies considerably in colostrum from different cows, but older cows have greater concentrations than younger cows. Efficiency of absorption of IgG and IgA does not change as intake increases, whereas absorption of IgM increases with decreasing concentrations and amounts (10). Immunological activity of IgM is greater than that of lgG.
Absorption of immunoglobulins appears to be superior for calves that suckled their dams as compared to those that were separated from their dams and hand-fed pooled colostrum at birth. There is suggestive evidence that breed affects absorptive ability of calves, but further clarification appears warranted. Chemical additives such as salts of organic acids, glucose-6phosphate, inorganic phosphate, and low molecular weight proteins increase absorption of 3'-globulin experimentally, but there was no effect when the additives were included with colostrum and fed to intact calves (10). Absorption of colostral proteins is reduced when colostrum is fermented, but this can be inproved through additions of sodium bicarbonate to colostrum (19). Feeding of glucose solutions at birth did not alter closure time, but inoculations with duodenal fluid reduced absorption (10).
Longevity of acquired immunity is 14 to 67 days (11). Half-lives up to 20 to 25 days for IgG were reported with 4 days for IgM and 2 to 2.8 days for IgA. The calf is capable of endogenous production of IgG at about 1 g/day during the first 3 wk of life (16). The bovine fetus is capable of producing immune globulins if antigens are introduced into amniotic fluid (13).
Turbidity tests measure the immunological status of the calf (61). A colostrometer has been developed (18) to measure specific gravity of colostrum which is correlated with its immune globulin content. Blood assays of marketed young calves show many are hypogammaglobulinemic. Brignolle and Stott (7) found that 42% of 983 calves left with their dams 24 h after birth failed to suckle or absorb immunoglobulins. Bottle feeding of colostrum at 24 h resulted in 70% of the calves absorbing immunoglobulins. Colostral immunoglobulins also may provide local immunity within the in-OTTERBY AND L1NN testine as survival of calves with low systemic immunoglobulin concentrations is good.
Whole Milk
Milk was predominately the liquid feed before 1956. Feeding rates were liberal. Most reports now suggest that milk be fed at 8% of birth weight. Whole milk often is a standard of comparison in experiments with other liquid feeds.
Milk Replacers
Usage of milk replacers has increased tremendously during the past 25 yr. The first true milk replacers were developed about 1950 (66). Previously milk replacers were really milk extenders and consisted of linseed oil meal, wheat middlings, wheat red dog, and oat flour. These were fed as gruels and were successful only if supplemented with milk (15). During the late 1950's and early 1960's, milk replacers were formulated from dried skim milk, dried buttermilk, dried whey, and animal fat. Dried skim milk was the principal source of protein and carbohydrate because of its low cost. Fat was added directly to milk products and batchmixed -a process that permitted incorporation of fat to less than 10%. Consequently, energy content of milk replacers was not much higher than that of skim milk (66). Methods for inclusion of fat were improved by homogenization of the mixture before spray drying, and proportions of fat in replacers were increased to 10% or more. As a result, high quality replacers were available, but high heat in drying of skim milk produced an inferior product (61). Use of such replacers containing large amounts of heated skim milk often induced severe diarrhea. Lister and Emmons (34) showed severe diarrhea in calves fed milk replacers formulated with powders from skim milk heated for 30 min at 85°C. Much less diarrhea occurred when replacers were manufactured from skim milk preheated to 60°C or 74°C for 30 rain before spray drying.
In 1966 prices of skim milk increased, and substitutes for dried skim milk were sought (66). Casein and whey were available at moderate cost, and the industry shifted to these products with little reduction in quality.
Later, prices of casein increased because of subsidized slaughter of dairy cows in Australia and New Zealand, the major casein producing countries, and industry looked to alternate sources of protein for replacers (66). Some replacers containing protein other than milk were marketed, but growth of calves was restricted. Several studies on improving nonmilk proteins for replacers occurred during the 1960's and early 1970's. Meat solubles composed largely of collagen, soy flour, distillers dried solubles, brewers dried yeast, wheat flour, and fish protein concentrate generally have not supported performance when incorporated into replacers in large amounts. Soy protein concentrates and soy isolates have been used in milk replacers with good results and are in large amounts in present day formulations. Chemical treatment of soy flour has been studied extensively, and the product shows promise in milk replacers (58). Soy protein concentrates are prepared by treating oil-extracted soybeans with water and alcohol to remove soluble carbohydrates and heating to inactivate inhibitors. The concentrate is 71% protein and has been a replacement for milk protein in several studies. Polzin (54) concluded that at least 50% of milk protein can be replaced by soy protein concentrates. Fish protein concentrates as the principal source of protein in milk replacers have not yielded good results, but recently Opstvedt et al. (49) successfully raised calves to 63 days of age on replacers containing fish protein concentrate. Digestibility of the milk replacer containing fish protein was lower than that of allmilk replacer, particularly during the first 3 wk. Performance of calves fed replacers of fish protein concentrate and dried whey was equal to calves fed an all-milk replacer. In the abomasum, the fish protein concentrate swelled and resembled the coagulation of casein.
Other protein sources for milk replacers are fababean, rapeseed, and alfalfa protein concentrates. These protein concentrates can be included in milk replacers up to 25% of the protein for fababean (82), 30% for low glucosinolate rapeseed (21), and 50% for alfalfa (1) for acceptable calf performance.
Fats in milk replacers include animal fat, hydrogenated vegetable oil, and coconut fat (59). Tallow and choice white pork grease have been used extensively, but recently milk fat, palm oil, sunflower oil, soybean oil, hydro-genated soybean oil, and partially hydrogenated fish oil all have been used successfully (65). Soy lecithin improved fat utilization and mixing qualities of replacer. Fat in milk replacers reduces incidence of diarrha (15). Homogenization of fat improves digestibility and retention of nitrogen from milk replacers (57). The amount of fat in milk replacers is 10 to 20% (57). Additions beyond 20% do not seem to be warranted.
Lactose is the primary carbohydrate in milk replacers. Glucose generally has been uneconomical. Other carbohydrates have not produced satisfactory responses in calves (9), probably a result of the lack of specific enzymes for digestion. Supplementation of milk replacers with amylolytic and proteolytic enzymes (15,50) has not improved calf performance, nor have products of lactobacillus fermentation. Based on work previous to 1956, milk replacers are supplemented with vitamins, minerals, and frequently antibiotics.
Reconstitution of milk replacer powder with water was studied by Pettyjohn et al. (53). Their trials indicated that liquid diets containing 15% dry matter elicited optimal performance. Most recommendations call for powders to be reconstituted to 12 to 18% solids (3).
Colostrum
Colostrum as a liquid diet beyond 3 days of age had been advocated prior to 1956, but little was used. Work during the 1960's and early 1970's stimulated dairy producers to include surplus colostrum in feeding programs. Enough surplus colostrum is produced in most dairy herds to feed heifer calves born into the herd from birth to weaning at 28 to 35 days of age (19). However, suitable storage of the surplus must be provided. Freezing of colostrum is an acceptable storage method, and calves fed colostrum stored by freezing show equal or better performance to weaning than those fed whole milk (3). However, prior to the late 1960's freezing of colostrum was not a feasible alternative, and it was common to discard excess colostrum.
One of the first reports of feeding nonrefrigerated colostrum was by Swannack (72). He discovered an English farmer who successfully had fed colostrum stored at ambient temperature to calves. Swannack's subsequent research stimulated interest among dairy operators and scientists, and use of fermented colostrum became increasing popular. Studies on effects of time, and temperature fermentation, microbial composition of fermented colostrum, dilution rate, changes in composition during fermentation, amounts of colostrum to feed, acceptability, feeding value, and methods were conducted during this period. Chemical agents including organic acids, formaldehyde, and others have been tested as preserving agents. Those used most frequently are propionic, acetic, and formic acids. Some problems occur with acceptability at larger amounts of acid, but these can be alleviated with neutralization with sodium bicarbonate to pH 6. Recommendations developed from these studies were reviewed (19).
Mastitic Milk
Until recently, limited controlled studies of feeding mastitic milk to calves had been reported. Kesler (30)summarized 10 experiments that included mastitic milk fed as fresh, fermented, or acidified milk. Gains of calves fed mastitic milk were similar or superior to those of calves fed control diets. In general, health problems did not differ between groups. Limited evidence suggests that incidence of mastitis in lactating animals that were fed mastitic milk as calves is no greater than that in cows fed other liquid feed as calves. Separate pens for calves fed mastitic milk was recommended to prevent introduction of mastitic organisms into udders of calves nursing each other.
FEEDING METHODS AND WEANING
Since 1956 once-a-day feeding of liquid has become acceptable management. A review (3) of several experiments indicated good performance of calves fed milk, colostrum, or milk replacer once daily. Quality, amount, and concentration of milk replacer require careful attention, but labor requirements for feeding can be reduced as much as 40% (45). Reduction of liquid feeding to six times per week also has been reported. Although this procedure resulted in satisfactory gains and health, it has not been adopted by calf growers.
Feeding milk or milk replacer by open pail is common, although nipple feeding by pail or bottle is chosen by many calf raisers. No advantage in calf health or performance has been shown for either method. Feeding large numbers of calves by either of these methods is slow and tedious, and producers have been interested in automated feeding. Mobile units with refrigerated storage of feed that automatically dispense feed at given intervals have been used where large numbers of calves are fed. Stationary automatic dispensers also have been used. Pelissier (52) reported that many large operators dispense milk by hose from mobile thermos tanks into individual pails rather than to use automated systems. Others have organized feeding with equipment for mixing and transporting liquid feed in nipple bottles or pails. Time for clean-up of equipment also has been reduced through organization and provision of good facilities. The offering of liquid diets at cold rather than warm temperatures was investigated. Cold milk or milk replacer fed in moderate environmental temperatures generally has supported gains equal to or superior to warm liquid. For veal calves fed ad libitum, warm liquid is preferred. There was some indication of increased diarrhea with cold milk feeding, but growth was not suppressed (3,67).
Abrupt weaning of calves at an early age is an acceptable practice (3,45). If weaned at 21 days, calves may suffer from slightly depressed growth rates. However, by 12 wk of age, earlyweaned calves and later-weaned calves (6 wk) are of similar weights. Weaning according to starter intake has found some acceptance, but abrupt weaning usually stimulates dry feed consumption. Starter intake can be stimulated by placing the dry feed in the pail immediately after liquid has been consumed. In general, weaning age has been reduced sharply and good results can be obtained with weaning at 21 to 35 days of age. peared in publications of the National Research Council (46). Ingredient composition of starters has been studied (42) and, in general, simple well-balanced preparations have supported good growth as well as more complicated formulations. Various sources of protein such as soybean, linseed, cottonseed, rapeseed, peanut, sunflower, meat, and fish meals were satisfactory in starters, and dried skim milk was not superior to vegetable protein. Dried poultry manure also has been used with success, but nonprotein nitrogen in calf starters has produced variable results and usually is not recommended (45). In addition, supplementation of starters with amino acids has not proven beneficial.
Extremely low crude fiber does not support gains as well as moderate fiber. The exact percentage has not been ascertained and probably depends on other factors such as physical form of fiber (77). Fiber below 5 to 6% probably will not produce best growth, and some bloating may occur (42). Simple mixtures with 13.9% crude fiber supported growth as well as a complex mixture containing 6% crude fiber. Pellets are not accepted as well as coarsely ground mash (78). Mashes generally have a higher proportion of larger particles, suggesting that 50% of the particles should be larger than 1190/.tm (16 mesh) (78).
Complete or all-in-one rations have been developed for calves. These rations have reduced the labor requirement in rearing and assure a uniform intake of nutrients. Various sources of forages have been used with amounts depending on source (25), but most results have been satisfactory with 25 to 35% roughage. Often the diet has been pelleted, and Bartley (4) found .48 cm pellets preferable to .95 cm pellets. Warner et al. (78) developed formulations of all-in-one diets for calves from birth to 32 wk. Starters vary in protein and fiber concentration according to age of calf.
CALF STARTERS
Work on calf starters during the past 25 yr probably did not bring dramatic breakthroughs, but there was steady progress in this area of nutrition and management of calves. Quality calf starters that are consumed readily are essential to early weaning programs. Recommended nutrient content for starters has ap-
NUTRITIVE REQUIREMENTS
The 1978 Nutrient Requirements for dairy cattle (46) contain the most recent recommended nutrient requirements for calves and heifers. Much of the information on calves' nutrient requirements is discussed elsewhere in this paper or issue. The most recent studies since Jacobson's review (28) of energy and pro-tein requirements and accepted recommendations on nutrient requirements (46) have been concerned with amino acid requirements. The requirements for DL-methionine range from .17 to .23 g/day per kg of body weight (76) and lysine from .27 to .31 g/day per kg of body weight (81). Amino acid and mineral composition of preruminant calves has been determined by Williams (80).
REARING RATES OF HEIFERS
Prior to 1956 information was limited on rearing rates of heifers, and it was general practice to calve heifers well beyond 24 mo of age. Reid (60) reported that several experiments on rearing rates were in progress or being completed, but little of the information had been summarized. Most of the research involved overconditioning or liberal feeding of heifers; however, some work suggested that heifers raised on low or medium nutrition produced slightly more milk than heifers raised on high nutrition. Since then objectives of several studies (8,62,68,74) have concerned a rate of development that allows for calving at 24 mo, efficient use of feed during rearing, delivery of a healthy, vigorous calf, few calving problems, production at maximum genetic potential in first lactation, resistance to metabolic problems and infections, enough size successfully to compete for feed, and longevity.
A considerable body of data indicates that excessive fattening of dairy heifers is detrimental to conception rates, calving ease, production, and longevity. Swanson's work (75) showed that udders of heifers fed liberally throughout rearing did not develop normally. Milk production of fat heifers was substantially less than that of heifers reared "normally"; however, extreme underfeeding during growth delayed onset of estrus. Heifers within a breed appear to reach puberty at approximately similar weights regardless of age (68,74).
Underfeeding of bred heifers resulted in calves only slightly smaller than those from well-fed heifers. More calving difficulties are encountered with undersized heifers than with those that are well-grown. Swanson (74) recommended 567 kg for precalving weight and 500 kg for postcalving weights of heifers from large breeds. Recommendation for age of heifers at first calving is approximately 24 mo. Suggested growth rates (46) are less than the maximum that could be expected and are based on the work of several investigations during this 25 yr. These allowances were developed to achieve goals mentioned previously. To calve at 24 mo at desired size, heifers of large breeds must gain approximately .7 kg/day (74). Heifers with average daily gain less than this will require longer to attain desired calving weights. However, heifers fed to gain .8 kg per day could calve at 22 too. Heifers that are fed less than "normal" produce well and grow if fed liberally after calving.
First estrus occurs earlier and at lower weights for small breeds than for large breeds. If heifers are fed to gain .9 to 1.0 kg/day, first estrus may be as early as 6 to 8 mo for Holsteins (62). Swanson (73)has predicted puberty at 10 mo with .82 kg gain/day, 11 mo with .68 kg gain/day, and 14 mo with .54 kg gain/day. Norman et al. (48) showed that up to 23 or 24 mo of age each month delay in freshening yields 35 to 180 kg more milk for Holsteins and 90 kg for Jerseys, but only 18 kg could be expected for each month delay after 27 mo for Holsteins and about 9 kg for Jerseys.
Swanson (73) reviewed several experiments in which feeding was below recommended standards for all of gestation except for the last 2 or 3 mo before calving. Feeding rate was increased for rapid growth through the last part of gestation. Production was greater for these heifers than for controls, indicating a stimulatory effect on lactation. Swanson (73) proposed several schemes of feeding: 1) rapid growth during the 1st yr followed by roughage feeding; 2) uniform rate of gain at .68 kg/day throughout rearing; 3) slow growth of calves through use of low quality feeds followed by grain feeding during last 4 mo of gestation; 4) slow growth, delayed breeding for calving at 27 or 28 mo; 5) underfeeding with breeding at 20 to 21 mo; grain-feeding during last 3 or 4 mo. Scheme 1 may tend to overfatten heifers, schemes 2 and 3 apparently work well, scheme 4 would be useful for heifers calving late for seasonal reasons, and scheme 5, while economical in use of feeds, generally would require careful management to avoid problems associated with underfeeding.
HOUSING
Prior to 1956 few research projects had been on housing of calves and heifers. Calves and hei-fers often have been housed in the same building as the milking herd or in sheds. As herd size increased, more dairymen began using separate facilities or attempted to extend use of existing facilities. Problems associated with inadequate housing emerged, particularly increased incidence of respiratory ailments and diarrhea. Research on and development of facilities consistent with good health, optimal growth, labor efficiency, and low construction or remodeling costs increased. Reports of research and field experience (6, 22) set forth criteria for satisfactory housing of dairy animals. Cleanliness, isolation of small calves from the milking herd, low humidity, freedom from drafts, dry beds, and provisions for ventilation and shade were vital for calves and heifers. Equipment for handling heifers during breeding and treatment was recommended. Individual pens or stalls were advocated for calves fed liquid diets. Group pens with limited numbers of animals of similar size and age were recommended for recently weaned calves. Older heifers could be kept in large groups so long as access to feed and water was satisfactory.
Although many dairymen in certain areas continue to house calves with the milking herd, separate buildings or rooms are becoming more popular as herd size increases. Buildings have been cold uninsulated buildings with open eave and open ridge type of ventilation or warm, insulated, mechanically ventilated units. For calves fed liquid diets, individual stalls or pens often are recommended. The elevated stall of steel or wood construction has become commonplace. These stalls help to keep calves clean and dry. The separate pen or stall prevents nursing. The elevated stall with slotted flooring also eliminates the need for bedding. Housing for calves can be either a controlled or climatic environment. In cold climates the most successful buildings are those that remove moisture and stale air. Incidence of respiratory problems is greater in barns with insufficient ventilation than in those adequately ventilated (5). At least four air exchanges are required per hour to remove aerosol contaminants and moisture. More exchanges are needed in warm weather. Temperature in calf barns is not as critical as humidity and air exchange. Calves tolerate temperatures to 5°C or lower with no discomfort (79), provided adequate shelter, bedding, and feed are available. In very hot environments, higher mortality may occur. Under these conditions calves may have higher serum corticosteroid concentrations and lower IgG concentrations than controls (71).
Because of increased and continued incidences of problems in conventional housing, many dairy producers have abandoned traditional housing (2) and have switched to hutches for young calves. Many experiment stations have developed plans for these individual calf units. Hutches have improved calf health and growth where problems, seemingly unsolvable by other means, existed. Although successful even in cold climates, caring for and feeding of calves in hutches may be uncomfortable for the calf caretaker. Jorgenson et al. (29) compared good indoor facilities with hutches and found no differences in growth, health, or weaning age of calves. Calves in hutches may be stressed during extreme cold, especially those less than 1 wk of age. More feed is consumed, and more labor is required by calves in hutches (40) as compared to calves in conventional housing.
Housing for older heifers has been in open lots, pole sheds, confinement buildings with slatted floors, and barns with free stalls. Openside sheds continue to provide comfortable housing for heifers if adequate bedding is used. Free stall layouts for heifers have been developed and are satisfactory if stall size is correct. The newest design in loose housing is the counterslope, where bedding and feeding areas are sloped in opposite directions to a common center manure alley (12). Grouping animals according to age and size is important in the success of heifer loose housing units (32).
High intensity housing for heifers has been developed and may reduce building space by 70%. In such units, either warm or cold, 3.6 m 2 of space has been used successfully for large bred heifers gaining .68 kg/day. Less space is used for smaller heifers. Floors are slatted in these barns with manure storage underneath, and free stalls may or may not be used. Details on calf and heifer housing are in many sources (6,12,32,35,43).
CALF HEALTH
Mortality rates of dairy calves averaged about 10% with ranges from 8 to 25% prior to 1956 (23,37,51). Reports from New York (24), Michigan (51,70), and California (37) indicate little or no advancements in reducing calfhood mortality during the last 25 yr. A survey of 477 Michigan dairy farms revealed total mortality rates of 17.7% with 11.3% of the deaths between birth and 2 mo of age (5•). Mortality rates ranged from 3.7 to 32.1% with an average loss of 20.2% of heifers born live on 16 farms in California (38).
Many factors are associated with calf mortality, but herd size seems to be most highly related to mortality. Table 1 lists the mortality rates of calves according to herd size for surveys in Michigan and New York. Speicher and Hepp (70) have hypothesized that: 1) in smaller herds stanchion barns often are the choice for housing both cows and calves; hence, sickness may be detected earlier as calves are observed more frequently; 2) expanded dairy facilities often neglect to provide adequate freshening and youngstock housing; and 3)management becomes diluted with expansion as it becomes spread over a greater number of animals.
The two major health disorders affecting calves are enteric and respiratory (61). Enteric disorders, in the form of neonatal diarrhea, primarily affect calves the first 2 wk of life. Respiratory disorders are more apparent in older calves and closely related to environmental conditions. Neonatal diarrhea or "scours" is the clinical appearance of hypersecretion of fluids into the alimentary tract in response to an irritant (56). Irritants can be either infections, chemical, or physical. Infectious agents include both bacteria and viruses. The major bacteria associated with enteritis are E. coli and Salmonella. Roy (61) has stated that the basic knowledge on E. coli infections was established in the 1920's and is being reconfirmed every 20 to 25 yr. Knowledge on isolating and identifying viral agents and their interactions with enteropathogenic strains of E. eoli has broadened during the last 25 yr. Mebus (41) found the rota and corona viruses increased the severity of enteric infections when accompanied by bacteria. McClurkin (39) also has indicated that diseases produced by viruses are not necessarily severe or fatal, but severities are increased greatly when accompanied by other disease organisms or stresses associated with most farm conditions.
Respiratory disorders are a greater problem than enteric diseases in calves 6 to 8 wk of age. High population density with inadequate venti- lation usually are associated with higher incidences of the problem. Respiratory disorders are accentuated during periods of low relative humidities at high environmental temperatures and high relative humidity at low environmental temperatures (64). California workers (36,38) suggest calf mortalities increase during cold, wet, and windy weather in winter and to less extent with hot dry weather in summer. Speicher and Hepp (70) indicated higher mortality rates in winter than in summer. Calves housed within the stanchion barn were shown (24,70) to have lower mortality rates than calves housed separately from the dairy herd. Perhaps one of the major advancements in health knowledge during the last 25 yr has been in the treatment of diarrhea in calves. While the effectiveness of broad spectrum antibiotics is unknown, replacement therapy of fluids with electrolytes has been valuable in preventing dehydration, metabolic acidosis, and subsequent death (56). However, the best control method is adequate intake of colostral immunoglobulins by calves shortly after birth (61). Enhancement of immune mechanisms through immunization programs should be in support of and not in place of colostral antibodies or good nutrition and managernefit. Vaccination programs (69) will vary according to geographical area, current knowledge of product immunogenicity, resistance developed by vaccinated animal, and risks of not vaccinating animals.
Rearing Options
Prior to 1956 most heifers entering the herd were either home-bred and reared or purchased. These two methods of replacing milking cows still are used extensively, but contract rearing of replacements also has appeared as an option. This is especially true in the case of extremely large herds where labor and management focus is on the milking herd. Appleman (2) described two types of contracts: 1)the contractor raises the dairy heifers for a fee, but the dairy operator retains ownership of the heifers; 2) the heifers are purchased by the contractor and the dairy operator has an option to buy back the heifers before freshening.
Costs in Rearing Replacements
Budget guides have been developed for estimating costs of heifer rearing. Expenses vary, but 55 to 65% of the cost is feed (2) with 95% of the cost postweaning.'Labor requirements, the next largest item in heifer rearing, can be extremely variable and are dependent upon size of operation, convenience of facilities, and incidence of health problems. Appleman (2) estimated 912.5 h/yr are required for care of heifers in a 100 cow herd. Housing needs and costs vary considerably depending on climate. Sick calves require, on the average, 53 min extra care. Breeding fees, power, miscellaneous supplies, interest, insurance, and taxes will account for 6 to 10% of total expense in raising replacement heifers.
CONCLUSIONS
Research should continue on efficiency of producing dairy replacements, an objective of NC-119 (31). Work by this committee has yielded much information. This group has developed guidelines (33) for uniform measuring and reporting of research on calves and heifers which should be useful for publishing data in years to come.
What the next 25 yr will bring is, of course, speculative, but new information appears to be unfolding in the areas of immunity, amino acid requirements, disease control, and environment. Alternate sources of nutrients and modification of feedstuffs for economical milk replacers and growing rations probably will result from ongoing and future research. Continued research will be needed to define nutrient requirements under different conditions of rearing and to add to present knowledge on digestion and metabolism in growing animals. Certainly, studies to use energy more efficiently in rearing calves will have a place in research priorities.
With advances in genetics and reproductive physiology, genetically superior animals will be available to producers. However, the livability, growth, and health of these animals from calves to freshening will continue to be a major problem. Continuation of educational programs with existing and future knowledge will be an important aspect of dairying and the successful, practical, and economical rearing of replacement animals. | v3-fos |
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This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1981 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.
In 1974 we evaluated how irrigation amount and timing influenced corn and grain sorghum yields at Man· hattan and Tribune. The results should help irrigators who want to use limited or less water because of limited pumping capacity, limited time, limited water supplies, increased cost of fuel for pumping, or other reasons.
At Manhattan, the study was on the Ashland Research Farm, approximately 8 miles southwest of Manhattan. The soil is Muir silt loam, which developed from river sediments .
The Tribune data were collected on the Tribune Branch Experiment Station Irrigation Field on Ulysses silt loam; a soil developed from windblow n deposits. A brief description of the field plots is given in Table 1. Available water stored in the 5-foot soil profile exceeded nine inches at plant emergence at both locations. Figure 1 presents the 30-year average rainfall pattern and the ra·infall received during the warm season at both Manhattan and Tribune. Rainfall at Tribune was 8.3 inches compared to Table 2 gives 1974 corn g rain yields at Manhattan and Tribune. Treatments consisted of no in-season irrigation; one irrigation at either one week before tasseling, during silk emergence, or at blister stage; and three irrigations, one at each of the three growth stages mentioned. Corn plots at Tribune received a pre-plant irrigation.
AGRICULTURAL EXPERIMENT STATION
rable 3 g ives sorghum grain yields at Manhattan and Tribune. Treatments consisted of no
1974.
Note: A ll plots at Tribune were damaged by hail August 10 and by frost Sept ember 3. in-season irrigation; a single irrigation at either boot stage, half-bloom; or soft-dough; and three irrigations, one at each of those g rowth stag€ Grain sorghum plots at Tribune received a preplant irrigation.
The corn data indicate that a single irrigation applied near tasse ling or at early silking increased yields greatly over one irrigatio n at blister stage or no in-season ·irrig ation. Due to grain sorghum's drought resistance, and timely rainfall, no definite trend between irrigation timing and gra in sorghum yield w as determined in 197 4. limited in-season irrigation is most practical in soils that contain nothing to restrict extensive root . developme nt, that have large water~holding capacity, and if a moderate to large amount of available water is stored before planting.
Info rmation in this report is for fa rmers, producers, colleag ues, industry cooperators, and other inte rested pe rsons. It is in tended as a n aid in irriga tion ma nageme nt decisio ns and not as an irrigation gu ide. It is not a recommendation o r endorsement and represents one year's research at two locations.
Contribution no. 160, Kansas Water Re sources Research Inst itute, Manhattan. The U. S. Department of Interior, Office of Wate r Resea rch and Technolog y p rovided partial support. Con tribution no. 1492, Agronomy Department, Evapotranspirati o n Laboratory; and no. 26, Tribune Branch Experiment Station, Kansas Agricultural Ex periment Station, Manhattan, Kansas 66506.
Publ ications and public mee tings b y the Kansas Agricultura l E?<perime nt Statio n are avail ab le and open to the public regardless of race, color, national o rigin, sex, or religion.
This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. | v3-fos |
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} | s2 | Veterinary Use of Antibiotics
mary glands that yield many times more milk than is needed to feed a calf, and we abuse the teats twice a day with the milking machine, which is passed unsterilised from cow to cow. It is no wonder that mastitis, usually either streptococcal or staphylococcal, is the major infectious disease of the dairy industry. Resultant tissue reaction leads to clots in the milk comprised of epithelial cells, leucocytes and bacteria. Indeed, modern milk has been described as 'dilute pus produced in a public lavatory'. Intensification has proceeded furthest in the case of poultry. For egg production, hens are kept, many hundreds to the house, in groups of four to five crammed into tiny cages, but the mesh floor minimises faecal
In the layman's eyes, vets may still be regarded as animal doctors, but what has happened to agriculture in recent decades has led to a major change in our role. Increasingly, we have become specialised technologists in a rapidly intensifying livestock industry, and it is this intensification that determines the problems of infection with which the veterinary surgeon in farm animal practice is faced today.
Under the pressure of society's demand for cheap protein, mankind has changed farming from being a vocational way of life into something more like a manufacturing assembly line ?the term 'factory farming' is excellently explicit?and in so doing has created a whole new range of man-made endemic disease problems. We call these 'diseases of production and breeding': more frankly, we should call them 'diseases of animal abuse'.
In intensifying beef and pork production, the neonatal and juvenile management is now widely removed from what could be regarded as 'natural'. Major features are: extremely early weaning, with concomitant maternal deprivation, colostral insufficiency and the use of artificial milk substitutes, translocation and close confinement with lack of bedding, leading to all-body contact with own and peer group faeces and urine, and dependence on inadequate and fallible ventilation systems with a high level of aerosol re-inhalation. The result of this is a severe challenge with pathogenic viruses, mycoplasmas and bacteria, particularly coliforms, which leads to a heavy dependence on antibiotics.
In the dairy industry, the milk cow is an expensive item of plant that must be kept in constant production, which means constant reproduction. Cost-effectiveness demands that she must bear a calf every year, so it is not surprising that infections of the genital tract are a major part of the veterinary surgeon's work. Not only must the cow calve, but she must produce milk for 10 months in the year. We have selected them for vast pendulous mammary glands that yield many times more milk than is needed to feed a calf, and we abuse the teats twice a day with the milking machine, which is passed unsterilised from cow to cow. It is no wonder that mastitis, usually either streptococcal or staphylococcal, is the major infectious disease of the dairy industry. Resultant tissue reaction leads to clots in the milk comprised of epithelial cells, leucocytes and bacteria. Indeed, modern milk has been described as 'dilute pus produced in a public lavatory'.
Intensification has proceeded furthest in the case of poultry. For egg production, hens are kept, many hundreds to the house, in groups of four to five crammed into tiny cages, but the mesh floor minimises faecal contact and disease in these battery houses is predominantly respiratory. But for meat production, broiler houses each containing ten to twenty thousand birds are common, and commercial considerations demand that birds shall grow from day-old chick to market weight in eight weeks. The houses are sealed, with automatically controlled ventilation and dim lighting. The floor cover of wood shavings or similar material is not changed during the eight week period: thus an increasingly severe problem of inhaled faecal dust develops, causing a characteristic E. coli respiratory infection and septicaemia.
In handling these problems, the veterinary surgeon finds the system immutable and he is forced to resort to the use of antibiotics to make up for the system's defects: these antibiotics all too readily become an accepted in-put into the system, and regarded as no different from food, water and fuel.
This article is only concerned with farm animals as a source of antibiotic-resistant strains of bacteria for humans, because, in terms of biomass ?the actual quantity of such bacteria generated and released into the environment through the food industry to human kitchens ?not to mention the very high rate of exposure of farm workers, farm animals are outstandingly important. However, we know that dogs and cats can carry resistant staphylococci and coliforms but their exposure to antibiotics, as a population, is far less intense than it is with farm animals. Veterinary Use of Antibiotics Therapeutic Use The veterinary surgeon acts like a medical general practitioner when consulted over a single animal, as an advisory good husbandman when consulted over the small herd problems of traditional farming, but as an agribusiness technologist when retained by big companies engaged in large-scale factory farming. The numbers of 'patients' involved, their cash or sentimental value, and the cost and practicability of medication, drastically dictate how he may proceed and are shown in Table 1, which shows that therapeutic antibiotic usage varies over a spectrum: at one end there is no limit to the care and expense of medication, at the other end, antibiotics may be used overtly or covertly to cover up or insure against the deleterious consequences of modern production methods.
In between these extremes, there is a wide field of strategy. When infection is recognised in one or two members of a small group ?for example, a batch of calves from a market, or a single litter of piglets or puppies ?it may be wise to assume that the whole group is at risk or even already incubating the disease, so all are put on to a full therapeutic regime. When larger groups are involved, such as 30-40 over-wintering young steers in which one or two are showing pyrexia and coughing, the vet may begin treatment of the clinical cases, leaving the farmer to continue the injections and to start it on his own initiative in any further cases that declare themselves. This is a practical compromise in a situation in which it would be economically quite unjustifiable for the vet to make constant return visits. But it can undoubtedly lead to quantities of quite legally obtained antibiotic lying about on farms and to a grey area of 'do it yourself medication by farmers, where the animals are scarcely legally under the proper care of a veterinary surgeon. It is only a small step from this to illegal 'black market' unprescribed antibiotic usage. Ruminant animals and other herbivores are usually treated parenterally, because oral use can have a deleterious effect on the vital microflora of the gut. But pigs, preferably, and poultry, always, are treated by mouth. In big units this is only possible by incorporating medication in either the food or water. In this case, there is no possibility of distinguishing between clinical, sub-clinical, incubating and already recovered cases: the whole lot must go on to the full regime on a computed basis likely to give each individual an effective dose, but the actual dose will, of course, vary with individual consumption. With water medication, the farm operative will add the appropriate water-solubilised formulation to the header tank. But if the antibiotic is to be given in the feed, tons of feed may be involved, in which case the vet's prescription will be passed to the food compounding mill, which will produce the appropriate consignment of medicated meal. This in itself can lead to problems of contaminating machinery and exposure of the mill workers to antibiotics.
Even in such mass medication, the decision is a veterinary one, based on clinical identification of the disease and a knowledge of antibiotic sensitivity. How-ever, under the intense economic pressures of factory farming, other criteria may be invoked. Thus the decision to put a whole broiler house of 20,000 birds on to a therapeutic antibiotic course may be decided solely on the age of the birds and changes in the daily house mortality and food consumption. These can be used quite precisely to indicate, for example, an outbreak of E. coli septicaemic air sacculitis in five-week-old birds, and the veterinary adviser to the company may well have provided appropriate prescriptions for such an eventuality, while the compounder may routinely hold readymedicated rations.
It is not widely realised that British legislation provides substantially greater rights to veterinarians than to doctors. All treatment of animals (with the exclusion of first aid and some simple farming practices) is absolutely restricted to the qualified veterinary profession: there is no equivalent to the right of humans to choose unqualified and paramedical treatment. The law recognises that animals cannot make the choice for themselves, and protects them against their owners making it for them.
However, actual medication, once the treatment has been prescribed, is often, for purely practical reasons, left in the hands of trained animal nurses or lay owners. The law allows this, provided such animals are recognisably 'under the care of a named veterinary surgeon. The veterinary surgeon also has no restriction on his freedom to prescribe. He has full use of the whole human pharmacopoeia and more, because the veterinary pharmacopoeia also gives him access to a range of potent products designed specifically for euthanasia and the immobilisation of domestic or wild animals, as well as compounds that exert a profound effect on the growth rates of animals.
In practice the range of widely used anti-microbial substances is somewhat restricted compared with human medicine. Those most commonly used in Britain are shown in Table 2.
Two associated reasons account for the list being so short. The first is cost. With the exception of highly priced or sentimentally valued animals, the cost of treatment is a major factor in choice of medication; gentamycin may have a place in the racing stable, but not in the farmyard. The second reason is the high cost to pharmaceutical companies of obtaining veterinary product licences, even for well-established substances. Thus, it has never been thought commercially worth while to Dimetridazole seek veterinary labels and data sheets for most of the cephalosporins, or for penicillins such as carbenicillin or ticarcillin. The limited extra usage would not justify the cost of the special formulatory, pharmacokinetic, toxicological, clinical and tissue residue studies which the Veterinary Products Committee would require. If the vet wishes to use such substances, he simply assumes the responsibility of prescribing the human products.
Worth a special mention is the tissue residue problem, which may affect human health but has no correlation in the development and use of human medication. When animals may be used for food (meat and poultry), or to produce food (dairy products, eggs), precise withholding times (the period that must be allowed to elapse between last dosage and slaughter or resumed production for human consumption) must be determined by approved studies, be printed on containers and literature, and be verbally explained to owners by the vet. This is obviously to protect the public from consuming adulterated food with possible toxicological and allergic sequelae, and less obviously, to reduce the contamination of the environment and the consequent engenderment of resistant strains of bacteria.
While the UK laws are second to none in restricting the pharmaceutical industry in this way, we are far behind the leading nations in monitoring actual residues reaching the public. With the exception of the Milk Marketing Board which monitors antibiotics in milk primarily to prevent damage to cheese fermentation processes, there is no statutory routine antibiotic assay of dairy or meat products reaching the consumer ?a grave source of criticism of our food inspection system, and a wide open lacuna strangely neglected by the consumer lobby.
With regard to the important subject of antibiotic residues in milk (drunk in large quantities in relation to bodyweight, especially by children), a unique feature is the so-called 'dry cow' therapy of milk cows. Chronic or sub-clinical infection of udders with staphylococci and streptococci affects approximately half our national herd for some period during each lactation. It is a major disability of the dairy industry, associated with machine milking. Intramammary infusion of antibiotics during lactation, while valuable in suppressing clinical symptoms, leaves many sub-clinical infections and can clearly pose serious residue problems if withholding times are not scrupulously observed. These cases may be more radically treated by infusing larger doses of a persistent formulation after the last milking of a lactation?a procedure known as 'dry cow therapy'. Ideally, only udder quarters bacteriologically identified as infected would be treated, but economic considerations make this impracticable. Thus, the official recommendation of the Ministry of Agriculture is to infuse all quarters of all cows at drying off, regardless of infection status. The intention is therapeutic but, in the process, innumerable uninfected cows are treated. However, the cows are given some protection against new infections during their dry period.
Routine Feed Additives and Growth Promoters
Soon after the first antibiotics came into use, notably benzyl penicillin and oxytetracycline in the early and late 1940s respectively, it was discovered that quite low subtherapeutic doses could enhance the growth performance of meat-producing animals, either by enabling them to reach market weight earlier, or by improving feed conversion efficiency, in either case increasing profit margins. The mechanisms by which this is brought about are not understood, but the presumption is that the natural balance between the host, its complex benign dominant commensal population, and its minority of unhelpful or overtly harmful flora, is advantageously shifted in favour of lifting the ceiling on potential growth. The more sceptical view is that antibiotics are doing no more than suppress the sub-clinical and incipient diseases that are an inescapable aspect of modern intensive systems.
Be that as it may, in most countries of the world, notably including the USA, but notably excluding the UK, therapeutic antibiotics, particularly benzyl penicillin and oxytetracycline, are routinely used as feed additives given to overtly healthy stock for this purpose. The US Food and Drug Administration defines this subtherapeutic use as the administration of doses of less than or equal to 200 g of anti-microbial per ton of feed for two weeks or longer. In Britain too, these two antibiotics were originally used in this way, but public and medical disquiet, following the discovery of transferable antibiotic resistance in the 1950s and the phage type 29 Salmonella typhimurium incident in the 1960s, led to the Swann Commission report[l] of 1969. As a result, the use as routine feed additives of all antibiotics useful in human or veterinary therapy was prohibited. This report did approve the use of non-therapeutic anti microbial substances for feed additive purposes, and at the present time large quantities of such antibiotics, notably bacitracin, flavomycin, virginiamycin and avocarpin, and synthetics such as nitrovin, halquinol and carbadox, none of which have any appreciable use in human or veterinary therapy, are used for this purpose, generally giving an economically useful growth rate return of between 5 and 10 per cent over non-medicated stock. The chemotherapeutics listed above characteristically have a predominant activity against Gram-positive organisms. Journal In the poultry industry there is also the almost universal process of adding suppressants for coccidiosis to diets. Anti-protozoals such as the antibiotic monensin and the synthetic purine amprolium are used, but none of these play any part in human or veterinary therapy. .
While often loosely referred to as 'growth promoters', the substances mentioned above do not qualify as 'true' growth promoters. The latter are claimed, probably justifiably, to function in completely disease free and correctly fed animals, although, of course, in the case of ruminants, such animals necessarily have a complex benign commensal population in the fore-stomachs and caecum. Indeed, it is in relation to this natural microflora that one of the most successful and ingenious growth promoters, rumensin (analogous with the poultry coccidiostat monensin) works. Ruminant digestion functions by the enzymatic breakdown of plant cellulose to volatile fatty acids, the enzyme-producing mediators being the bacteria and protozoa of the rumen. The natural process functions at the cost of a massive production of methane and hydrogen, which are totally lost by eructation. Rumensin partially inhibits this methane/hydrogen energy loss, and also shifts volatile fatty acid production from the less useful butyric to the more valuable propionic range.
To complete the list of 'growth promoters', I should mention anabolic steroids, the widespread use of which is a subject of public health controversy. Natural steroids (e.g. oestradiol/progesterone combinations); synthetic androgens (e.g. zeranol); and synthetic stilbenes (e.g. trenbolone), usually given by subcutaneous depot implantation, are claimed to improve the efficiency of nitrogen usage from dietary protein in cattle. That they can lead to increased body weight seems to be proven, but it has been reported that this is primarily due to increased muscle water retention, and, on the Continent at least, there is growing objection to such substances, as they offer no benefit to the consumer and are unacceptable food adulterants.
Veterinary Use and Antibiotic Resistance
Staphylococcal resistance to benzyl penicillin was an early feature of antibiotic use in humans. This was paralleled by the development of resistant mastitic staphylococcal populations in cows' udders, to the extent that some 70 per cent of such isolates in the UK are now penicillinase producers; perhaps, because nearly all milk in this country is pasteurised, there has been no suggestion that this situation poses a public health risk. All staphylococcal isolates in this country have remained highly sensitive in vitro to cloxacillin and other (i-lactamase stable antibiotics. Nonetheless, there is an in vivo cure rate problem that may be related to such factors as tissue reaction impeding penetration, intraleucocytic dormant sequestration of bacteria and perhaps autolysin inhibition.
The remaining veterinary resistance problems mainly relate to enteric infections. Treponema hyodysenteriae, the causal organism of swine dysentery, an important disease of intensive pig farming, has an erratic history of antibiotic and dimetridazole resistance, but outstandingly important, both economically and from the zoonotic point of view, is antibiotic resistance in E. coli and Salmonella infections. The present situation is best explained in historical terms.
The explosive intensification of livestock production (quite properly called factory farming) has created conditions in which these animals, from birth to slaughter, are constantly and massively in contact with their own and their peer group's faeces and urine and constantly cross-inhaling air and its aerosol contaminants. This and associated deleterious aspects of intensification combine to create a high incidence of neonatal colibacillosis and respiratory disease in calves and pigs, and colisepticaemia in poultry.
The reaction to this has been to use anti-microbial drugs, particularly tetracycline, neomycin, ampicillin, streptomycin, furazolidone, chloramphenicol, and sulphonamides with or without trimethoprim. Tetracycline was also used as a feed additive and as early as 1956, Williams Smith, in his now classical study [2][3][4] on a pig catchment area in East Anglia, showed the existence of tetracycline resistant E. coli populations, although there was little or no resistance at that time to other antibiotics.
In spite of the concurrent awareness of the possibility of transferable resistance, disquiet about the situation remained muted until in 1965 there was a development in respect of salmonellosis in calves. Classical calf dysentery had been caused by Salmonella dublin, a serious pathogen that does not colonise human beings. In 1965, probably as a result of changes in traditional calf husbandry, Salmonella typhimurium began to replace S. dublin. This organism of human food poisoning, which is usually non-invasive in humans, causing only a mild self-curing enteritis, is highly virulent in calves, making antibiotic treatment essential, if only on humane grounds. However, veterinarians increasingly met antibiotic resistance with this organism and in the same year the Central Public Health Laboratory characterised phage type 29 as carrying plasmid determined resistance to streptomycin, sulphonamide, tetracycline, neomycin and ampicillin, and, occasionally, to chloramphenicol.
Medical attention was now called to the possible dangers of the chloramphenicol-resistant plasmid, either by E. coli or S. typhimurium being transferred to a human invasive strain such as S. typhi, when human life might be endangered through chloramphenicol failure.
The Swann Committee[l] eventually reported on this situation and its main recommendations were as follows 1. All antibiotics of use in human or veterinary therapy should be available only on prescription, only for therapeutic use, and only for animals actually under the vet's care.
2. Other antibiotics, not of therapeutic value, should be available to farmers without prescription. 3. The veterinary profession was asked to use chloramphenicol only as an exceptional measure, reserved for special situations, but the suggestion that it should be proscribed was turned down. However, the Committee recommended that the prevalence of chloramphenicol resistance in organisms isolated from farm animals and healthy humans should be monitored continually 'so that prompt action can be taken on reliable evidence if either increases significantly'. The Swann philosophy was based on the then prevalent idea that it was the continuous addition of low doses of antibiotics to food that caused the persistent antibiotic resistance. The Committee's report was generally welcomed and endorsed by the veterinary profession but ironically, before its recommendations could be brought into effect, phage type 29 disappeared from the scene of its own accord, so taking some of the wind out of the sails of the protesters. It should be noted that, however virulent it may be, S. typhimurium in calves is selflimiting in that in an undisturbed calf population all the animals will have become non-excretors about six weeks after infection is introduced.
Swann was implemented in full, but as the years went by, Williams Smith, continuing with his East Anglian survey, was unable to show any reduction in tetracycline resistence in E. coli (Table 3), while the incidence of which was resistant to streptomycin, sulphonamide, tetracycline and chloramphenicol, and a variant, phage type 193, which, in addition, was resistant to ampicillin and kanamycin.
The farm on which this isolation was made was in Leicestershire and it functioned as a dealer's centre to which calves were brought from diverse origins and then distributed according to commercial considerations throughout the country. Subsequent investigation revealed that chloramphenicol had never been used on this farm and it remained an open question as to whether the origin of the strain was bovine or human. However, the consequences were not in doubt; within a short time, phage type 204, and in due course 193, were scattered all over Great Britain by the mechanical process of the calf trade, and indeed, calf exports took it to the Continent. Furthermore, Threlfall and his colleagues [7] have now reported a new variant, phage type 204c, which is trimethoprim resistant as well (Table 5), and they have shown how, in parallel with the animal situation, these three types have been occurring in the human population ( Table 6). This is the potentially serious situation as it is today. What those decisions should be is a matter for debate. I am in favour of prohibiting the use of chloramphenicol in farm animals, if only to avoid the possibility of blame for a single tragic human incident. But one must give weight to Professor Richmond's recent views [8], that it may now be too late ?indeed was too late at the time of Swann ?to reverse the incidence of resistance by modifications in the way antibiotics are used. Therapeutic use, just as much as, or more than, feed additive use, selects for resistance and we now almost certainly have combined plasmids, determined for resistance and also for good gut adhesive and colonising ability, and modifications in the way antibiotics are used are unlikely to cause their disappearance. However, the veterinary profession in this country has a notable reputation for the control of disease by methods other than medication. E. coli bacillosis of neonates should be approached by modifying production methods appropriately and by oral rehydration techniques. Salmonellosis in calves should be dealt with similarly, together with radical changes in the calf trade and rigorous isolation procedures. We have the knowledge, the skill and the resources to do this if society demands it. This article is based on a paper read at the Conference on Infection in Britain Today held at the Royal College of Physicians in November 1980. | v3-fos |
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} | s2 | Twenty-Five Years of Progress in Understanding Major Infectious Diseases of Dairy Cattle
Disease, whether it occurs in cats, cattle, caribou, or other species, is the manifestation of insult to the structural or functional integrity of the living being. Infection may occur with or without evidence of disease. Disease may represent only the “tip of the iceberg” of infection. Prevalence of disease is the proportion of a defined population that meets specific criteria at a point in time, and incidence refers to the proportion of a defined population in which the onset occurs during a specifiable interval. Risk is an instantaneous rate of incidence. Prevalence, incidence, and risk in infectious diseases are quantitative inferences derived from assessment of implications — caveats derived from variables that often are elusive, such as antibiotic resistance to plasmid mediated determinants transferable between species of enteric bacteria, or such as increases in population density among insect vectors influenced by climatic conditions favorable to the insects. Carefully orchestrated manipulation of the etiologic agent or the host response is made possible by ever-expanding understanding of the molecular biology of pathogenicity on the one hand and cellular and humoral immunity on the other. Such understanding often leads to new caveats or destroys old ones based on incomplete information. This paper deals with a selected limited number of infectious diseases of dairy cattle. Knowledge developed around the cited parameters in 13 infectious diseases during the past quarter century is brought into focus. Ten geographically dispersed specialists in infectious diseases of cattle were asked to identify priorities among the array of transmissible diseases important to North America. Except for the mastitis syndrome, which will be given special attention elsewhere in these anniversary papers, the diseases discussed are those wherein major advances in knowledge or major needs for advanced knowledge were of special importance.
INTRODUCTION
In the past 25 yr, there have been major contributions to knowledge about infectious processes and immune responses in dairy cattle, the most troublesome of which (exclusive of mastitis, which is given special attention elsewhere in this series of anniversary manuscripts) appear to be those afflicting mucous membranes of the respiratory, alimentary, and genital tracts. Many of them manifest similar signs of illness, so that differential diagnosis is difficult. Many are caused by viruses against which therapeutic agents are ineffective and against which immunoglobulin is not always available at the site of attack. Despite these deficiencies, unfolding knowledge about molecular characteristics of viruses and immune systems has clairified much of the mystery about infectious diseases and body responses to invading microorganisms. This paper deals with selected infectious diseases that have tested the cutting edge of scientific inquiry during the past quarter century. They are arranged alphabetically to avoid illusion that one is more important than another; related diseases are introduced within the discussion of the major title. Omissions may concern readers who may feel that certain diseases have been overlooked. Those included were compiled after querying colleagues in the United States and requesting that they assign priority ratings to diseases they judged of major importance. Represented here, then, are those regarded as being contemporary problems with advanced, but incomplete, knowledge.
Bluetongue
In the past quarter century, bluetongue virus has emerged as a ubiquitous infectious agent of major importance in international commerce of the cattle industry and in commercial distribution of bovine semen. Bluetongue is a noncontagious, infectious viral disease of ruminants, especially sheep, but to less extent goats and cattle. The virus is transmitted by insect vectors, particularly gnats of the Culicoides genus.
In sheep, the disease is characterized by fever, emaciation, oral lesions, lameness, and a substantial death rate with heaviest losses in lambs.
In cattle, the infection usually is nonclinical, resulting in a persistent, silent infection and a virus-shedding carrier state. Occasionally cattle sicken, however, and develop erosions in the mouth, encrustations and excoriations on the muzzle, nasal discharge, laminitis, and acute dermatitis of a patchy nature involving the flanks, groin, perineum, udder, and teats. Necrosis of the skin in the interdigital spaces has been observed. These signs occur in a number of other diseases (the so-called mucosal diseases), and differential diagnosis may be difficult.
In utero transmission occurs in cattle and can result in abortion, hydranencephaly, congenital malformations, and immunotolerant calves. Further, the virus can produce pathologic changes in the reproductive tracts of bulls. Such changes include focal degeneration of the seminiferous tubules with inflammation and hemorrhage in the colliculus seminalis.
While of relatively minor importance to dairy cattle per se, fear of bluetongue infection causes restrictions in international commerce. It has been suspected that cattle may be the reservoir of virus, serving as the source of infection for sheep,, especially in areas where Culicoides gnats abound (24).
Twenty serotypes of bluetongue virus are known. In addition, cattle can be infected with several closely related viruses including the viruses of epizootic hemorrhagic disease of deer and Ibaraki disease, agents which cause similar disease in wild ruminants, resulting in diagnostic difficulties. Ibaraki virus was isolated first in the Ibaraki Prefecture, Japan, from the blood of a 10-yr-old cow. It is a bluetongue-like virus producing lesions in cattle that resemble bluetongue in sheep, but the virus has little pathogenicity for sheep.
Antibody against Ibaraki virus commonly is found in cattle in all regions of the United States except the northwestern states (23). There appears to be no antigenic relationship between bluetongue virus and Ibaraki virus, but Ibaraki virus and two types of epizootic hemorrhagic disease virus (EHDV) in deer are related serologicalty. A common complement-fixing antigen is shared by the two EHDV serotypes and bluetongue virus serotypes (42). The significance of Ibaraki virus disease of cattle in the United States is unknown, but judging from its ulcerative effects on Japanese cattle and the wide distribution of antibody against the virus in cattle in the United States, concern must be expressed.
Bovine Herpes Mammillitis
In the last quarter century, a new chapter has been added to the study of virus-induced lesions on the teats of dairy cattle. In 1966, Martin et al. (35) reported that bovine ulcerative mammillitis is caused by a herpesvirus. In 1970, Yedloutschnig et al. (59) identified the disease in the United States. It has been established recently that the disease has global distribution.
The disease is known as bovine ulcerative mammillitis as well as bovine herpes mammillitis. It can be manifested in two forms in a milking herd. In one form, from which the name of the disease was derived, ulcerative lesions appear on the teats and less frequently on the udders of affected milking cows. The virus usually causes gross swelling of the teat wall. Within 48 h, the skin over the affected areas becomes soft and sloughs, revealing an irregularly shaped, painful, deeply ulcerated area which heals slowly, healing accompanied by the formation of brown scabs. Healing usually occurs within a week after skin lesions appear. The scabs begin to shed after about 2 wk. Lymph gland swelling occurs, and mastitis follows in approximately 22% of the cases. The causative herpesvirus probably is transmitted mechanically by milkers and milking machines or by biting flies (24). This form of the disease must be differentiated from paravaccinia or pseudocowpox, another affliction of the teats, and sometimes mouths of calves, which is a slow-healing nonulcerative virus disease, manifested by vesicle formation.
The second manifestation of this disease is the appearance of multiple nodules in the skin, and this is known as dermopathic bovine herpesvirus disease. The nodules appear as pronounced, circular, raised areas in the skin over the entire body. The nodules are deep and undergo tissue destruction with the formation of superficial scabs that ultimately fall off. Tufts of hair are cast off in the desquamation, leaving ulcerated lesions that resemble lumpy skin disease, a severe poxvirus infection indigenous to parts of Africa but currently exotic to the United States.
Bovine Viral Diarrhea and the Mucosal Disease Complex
In the past 25 yr a group of viral infections affecting the mucous membranes of the bovine alimentary tract has been studied extensively. These have gained a prominent place among the dairy cattle diseases because of their debilitating effects.
When necrosis, ulcers, or erosions are observed in the oral mucosa of cattle, the differential diagnosis should include bovine viral diarrhea-mucosal disease (BVD-MD), malignant catarrhal fever, papular stomatitis, rinderpest, bluetongue, the vesicular diseases, and ingestion of caustic substances.
Bovine Viral Diarrbea-Mucosal Disease. Bovine viral diarrhea-mucosal disease was described initially in 1946 (49) as an epizootic diarrhea of cattle. Today it is known that BVD-MD virus is a togavirus that causes multiple clinical and pathologic manifestations (29). The detection of serum antibody in much of the world's cattle population suggests that inapparent infection is common. After natural infection, serum antibody frequently persists for the lifetime of the animal.
The classic clinical syndrome occurs sporadically and is characterized by elevated temperature, leukopenia, diarrhea, lacrimation, nasal discharge, and erosions of the oral mucous membranes. Most cases occur in cattle aged 6 to 24 mo, and many of these die. Survivors may develop a chronic debilitating disease characterized by weight loss, intermittent diarrhea, inefficient feed utilization, and erosions of the oral mucosa. Occasionally lameness occurs due to laminitis, necrotic interdigital dermatitis, or ulcerative coronitis. In some epizootics BVD-MD is regarded as a respiratory disease because nasal discharge, cough, and rapid respirations are prominent clinical signs (28).
Cattle infected during pregnancy can have normal pregnancies; however, abortions or malformed calves may be seen, even if the inciting infection is mild or inapparent.
Bovine malignant catarrhal fever (MCF) resembles BVD-MD in that crusting of the muzzle and oral erosion and necrosis occur. It usually can be differentiated by a severe panophthalmia with corneal opacities, prominent enlarged superficial lymph nodes, stertorous labored breathing and nervous behavioral changes, all of which are characteristic of MCF. When these signs are observed, they usually are associated with fatal encephalitis. The disease seems to occur when cattle contact sheep or wild ruminants. The nature of the virus causing MCF in Africa is known, but studies are still underway to determine the cause of the disease in North America.
Papular stomatitis, a mild poxvirus infection usually occurring in calves, causes raised or flat lesions in the oral mucosa. These lesions frequently contain characteristic streaks of various shades of brown and have irregular edges, which helps to distinguish them from BVD-MD or MCF erosions. Only rarely are papular stomatitis lesions manifested as punched-out depressions in the mucosa. Papular stomatitis infections usually are inapparent. The lesions are noticed only if the oral mucosa is examined. Occasionally, however, overwhelming infection is manifested by clinical signs.
Rinderpest, enzootic in Africa and in parts of Asia, is exotic to the United States. In this acute, devastating disease of cattle and other ruminants, ulcerative lesions identical to BVD-MD are found, except that they usually are more severe. Introduction of rinderpest virus into the highly susceptible bovine populations in heretofore rinderpest-free areas presumably will result in high clinical attack rates, high case fatality rates, rapid transmissibility between animals and herds, and a disease problem likely to be devastatingly more drastic than that of enzootic BVD-MD. Rinderp~t always must be considered when diagnosing BVD-MD because variations in virulence of rinderpest strains are known. A mild rinderpest strain easily can be misdiagnosed as BVD-MD, permitting considerable dissemination of the virus before the disease is diagnosed.
Vesicular stomatitis virus causes vesicles on the oral mucous membranes, teats, and the coronary bands in the feet of cattle. These are indistinguishable from the lesions of foot-andmouth disease. The vesicles rupture rapidly, and as healing or secondary bacterial invasion occurs, the lesions may resemble the erosions of BVD-MD, especially if the epithelial flaps, the sole remnants of the vesicles, have been completely desquamated. Vesicular stomatitis virus is probably insect-borne. The disease is enzootic in the southern United States. Occasionally, however, it appears in the northern United States or Canada during summer months. Continued studies are needed to understand the epidemiological nuances of this disease, especially since it is caused by a rhabdovirus. Rhabdoviruses frequently have wide host spectra.
Bovine bluetongue infection occasionally results in a clinical syndrome similar to BVD-MD, with necrosis of the oral mucosa, crusting of the muzzle, and lameness. In clinical bovine bluetongue, diffuse necrosis of the oral mucosa, particularly the dental pad, can serve as a differentiating feature when contrasted with the usually discrete erosions of BVD-MD. Bluetongue occasionally is manifested also as drying, cracking, and exfoliation of the skin (pityriasis), suggestive of photosensitization -a finding rarely associated with BVD-MD (29). When skin lesions occur in BVD-MD, it is said that they resemble moist eczema.
Diarrhea, frequently a feature of BVD-MD, MCF, and rinderpest, is not always a consistent sign. Profuse intractable diarrhea in cattle also is seen in salmonellosis and Johne's disease.
Salmonellosis can be differentiated from mucosal disease by its lack of the latter's characteristic oral lesions and by isolation of Salmonella organisms from feces or tissues of infected animals. Acute arsenic poisoning and poisoning with organophosphate insecticides also cause diarrhea, and this must be noted in differential diagnosis.
When epizootics of BVD-MD sometimes are misdiagnosed as one of the more common respiratory diseases, such as shipping fever or infectious bovine rhinotracheitis (IBR), the diagnosis usually is based on elevated rectal temperature, nasal discharge, increased respiratory rate, and cough. The so-called respiratory form of BVD-MD usually is not accompanied by pneumonia, however, unless there is superimposed infection. When BVD-MD is manifested as a respiratory disorder, careful examination of the oral mucosa, gums, tongue, and hard palate of ill animals and apparendy healthy herdmates sometimes reveals oral erosions. If oral erosions are found in catde, and if there is a leukopenia, a diagnosis of BVD-MD is appropriate. In differential diagnosis, the nasal mucosa must be examined closely for evidence of white fibrinous plaques found in IBR.
There is a strong immunologic relationship between the hog cholera virus and the agent causing bovine virus diarrhea. Because of that, studies were instituted to determine if BVD-MD virus could be used to immunize swine against hog cholera. Such immunity could be induced if unattenuated live BVD-MD virus were used. However, it was learned subsequently that BVD-MD virus occurs naturally in some swine herds, and great concern has been expressed that such infected swine, if virus-shedders, would serve as sources of infection to susceptible cattle. That method of vaccinating hogs against cholera no longer is advised. A modified-live virus vaccine has been developed, however, and now is available from commercial firms for the vaccination of cattle against BVD-MD (23).
While it is recognized that BVD-MD modified living vaccines are quite effective and relatively safe for most cattle under field conditions, there have been reports indicating that in some cattle this vaccine may be a predisposing factor, or possibly the primary cause, of severe reactions in some animals. Such adverse reactions, which usually involve low morbidity and high mortality, are not clearly understood. The use of vaccine in pregnant cows is ill-advised because the virus does cause abortions and is responsible for congenital abnormalities (23). In consideration of these reactions, BVD-MD vaccine is recommended only where previous or anticipated disease problems are of sufficient magnitude to warrant the risk.
Brucellosis
During the past 25 yr, it became painfully obvious that with the national trend toward larger dairy herds, with increases in cattle density, the probabilities of introducing brucellosis and the persistence of brucellosis were becoming greater. The trend also led to higher prevalence in some areas and, indeed, greater difficulty in eliminating the disease. Further, the trend has led to recognition of the probability that the disease might not be eliminated by conventional test and slaughter methods. This new awareness has reactivated an interest in brucellosis research. Renewed enthusiasm has been engendered for scientific explorations in a disease that has serious public health and economic importance to the cattle industries.
Bovine brucellosis is primarily a disease of sexually mature cattle in which the inciting organism, usually Brucella abortus, circulates systemically for variable periods of time after invading the body. It is believed that invasion is most commonly by ingestion and that the source of infection usually is in discharges of an infected cow (47). However, other methods of transmission have been reported, including inhalation. The organism localizes and multiplies in the reticuloendothelial tissues, such as the spleen and lymph nodes. Further, it has a predilection for reproductive organs including the udder, uterus, and testicle, epididymis and seminal vesicles and, less frequently, other structures, such as bones and joints. The most dramatic clinical manifestation of brucellosis and usually the only sign of infection is abortion resulting from placentitis (14).
Brucella abortus is an intracellular parasite.
It multiplies especially abundantly in the placenta where there is a high concentration of erythritol. Placental lesions result in fetal anoxia and death, usually in the last trimester of pregnancy in initial infections. In the female, localization in the udder and supramammary lymph nodes is most common and more permanent than other sites. After parturition or abortion, vaginal excretion of the organism continued for the first 15 days, then diminished and became intermittent (47).
The morphological features of the genus Brucella are remarkably consistent, but the organism shows limited activity in the conventional biochemical tests used for identification. It is recognized, however, that organisms identified as members of the genus Brucelta possess a range of properties that can be clustered into species or biotypes, but clearly defined boundaries between species do not exist in all cases. For convenience of identification, it is recommended that strains should be classified into species by the preferred natural host, sensitivity to brucella phages; and oxidative metabolic profiles (44). Nearly all brucellosis in cattle is caused by ingestion of, and subsequent infection by, one of the biotypes of Brucella abortus.
Brucella endotoxins possess many structural and biological properties common to the lipopolysaccharide endotoxins of enterobacterial pathogens (33). The initial reaction to endotoxin shock is sensitized vascular damage. There is fever, release of histamine and catecholamines, and intravascular clotting. Repeated insults by endotoxin may result in tolerance to further vascular damage (32). However, exposure stimulates cell-mediated immunity in 'addition to humoral antibody production. Histiocytic granulomas appear to be manifestations of cell-mediated immunity in chronic brucellosis. Brucella agglutinins developed in response to endotoxin antigen will crossreact with some Salmonella, Pasteurella, Yersinia, and Leptospira genera (47), complicating diagnosis.
In an evaluation of brucellosis research by a committee serving the National Research Council, National Academy of Sciences, Sanford S. Elberg, Chairman, and six other preeminent committee members stated that there are few aspects of brucellosis so unexplored as its epidemiology: When one views the volumes of literature which have been amassed on vaccination procedures, serologic tests, shedding of the organism and pathogenesis, it is appalling to see the limited number of studies relating to routes of transmission, relative importance of various sources, survival of the organism, means of transmission within and between herds, the characteristics of high-risk herds, management practices to reduce transmission and the dynamics of herd immunity. The danger of accepting field observations as truths is well illustrated in brucellosis by the history of the questions concerning the role of the bull in the transmission of the etiologic agent... (18). Nicoletti (47) has commented that it is accepted widely that sexually immature cattle are quite resistant to exposure to B. abortus and that susceptibility increases with sexual development and pregnancy. Calves may acquire infection in utero or by ingestion of contaminated milk. A small but important percentage of heifer calves infected in early life are negative to serologic tests and abort or have an infected calf during the first pregnancy. These are referred to as latent infections. Latent infections are difficult to diagnose early in the course of the disease and may present serious problems in the elimination of brucellosis from cattle herds (47).
In brucellosis, the term "incubation period" usually implies that period between exposure to infection and the time at which clinical or serological evidence indicates that infection has occurred. That period varies considerably and is affected by several factors such as gestation, exposure dose, age, vaccination, and other unknown host-resistant influences. The variable incubation period and the difficulties of diagnosing infection until after transmission are among the most serious technical problems in unraveling the epidemiologic patterns of brucellosis (47).
For many years, diagnosis was based upon isolating the organism from infected tissues or secretions or by employing agglutination tests on serums from cattle under surveillance. Serious limitations were found in the agglutination test. It was often ineffective in detecting chronic infections and could be influenced seriously by vaccines or other antigens. Subsequently, a battery of other serologic methods was developed including the ring test, card test, Rose Bengal test, complement-fixation, rivanol, and mercaptoethanol tests for humoraI antibody. And recently the lymphocyte stimulation test has been evaluated for evidence of cellmediated immunity (47). Varied sensitivity and specificity among these have made it obvious that no single test is entirely reliable in the diagnosis of brucellosis, but in wisely selected combination, they are highly effective.
Strain 19 vaccine remains the most widely used immunogenic agent for the control of bovine brucellosis. In more than five decades of research on this strain, there has been no proved change in virulence or immunogenicity (47). It rarely causes permanent infection in vaccinated cattle, especially when administered in calfhood. There is no apparent pathogenicity for human beings except when it is injected. A reduced dose, proposed by Nicoletti (47) and administered subcutaneously, has enhanced the practicability of the use of the vaccine regardless of age of cattle.
Calf Diarrhea Caused by Viruses
Major progress in the understanding of calf diarrhea has resulted from the isolation and characterization in the late 1960's of a reo-like virus (now called rotavirus) and of a coronavirus in the early 1970's. These accomplishments resulted largely from the efforts of Mebus and coworkers at the Nebraska Agricultural Experiment station.
Rotavirus. In recent years, the importance of rotaviruses in the ubiquitous and troublesome diarrheal diseases in calves has become well established. And, as for some bacterial diarrheal diseases, rotaviruses have the propensity to infect more than one species of animal so that human, bovine, porcine, and equine strains possibly may produce diarrhea in neonatal calves (24). Although rotaviral infection may occur at all ages, including adult cattle, it is in neonates that the disease is often most severe and frequently fatal. The disease in calves usually becomes apparent between 3 days and 15 wk after birth.
It is likely that the disease heretofore known as pneumoenteritis in calves and piglets is primarily a rotaviral disease. In pneumoenteritis, diarrhea usually precedes pneumonia, occurring between 1 and 10 days of age.
In calves on a total milk diet, feces in rotavirus infection usually are brilliant yellow to white in color, not always putrid, and resemble those of classic milk scours. In other calves, however, feces may be watery, brown, gray, or light green and tinged with fresh blood and mucus. The color appears to be dietary dependent. If the diarrhea is prolonged, dehydration becomes apparent, and the calf may die within 4 to 7 days after onset, showing a significant loss of body weight. Severely ill calves have recovered after administration of glucose and saline mixtures substituted for milk. Continued feeding of milk is harmful and probably accounts for the severity of epizootics in calves maintained in cow/calf operations. Inclement weather may complicate an outbreak. Many calves have developed severe pneumonia after the onset of diarrhea, especially when they were exposed to inclement weather. Severely affected animals may die 2 or 3 wk later (24).
The pathogenesis of rotavirus infection in calves is similar to that for transmissible gastroenteritis in pigs. The virus infects villous epithelium in the intestine. Immature enterocytes, which migrate from the crypts to the tip of the villus, fail to differentiate fully in their migration. This failure appears to be the major factor, which produces an ultimate electrolyte transport disorientation for sodium ions, resulting in fluid and electrolyte loss manifested by diarrhea (24).
Resistance to rotavirus disease appears to be mediated by local immunity at the epithelial surface of the small intestine (55). Unfortunately, the passive protection afforded by colostrum has limited value. Calves are protected when they are fed colostrum with rotavirus antibody of high content, but that colostrum must be fed continuously in the likelihood of constant exposure to virus (24).
The duration of immunity is not known, and the period of persistence of virus in the feces after illness also is unknown. The effectiveness of cell-mediated immunity apparently has an influence on the persistence of virus which is shed in the feces. It is likely that immunity is only partial and not long-lasting. Therefore, adults can become infected from infected neonates. Since the incidence of the disease is extremely high, and since reinfection may occur, it is likely that the disease is perpetuated through persistence of virus within an animal species. Also, there is a possibility that interspecies infection may be important in transmission of the infectious agent.
In control of this disease, despite the limitations cited, the feeding of colostrum to newborn calves is extremely important. The colostrum must contain reasonably high antibodies to the rotavirus, and it must be fed every day during the crucial neonatal age period in which rotavirus infection is a threat if it is to be effective.
An attenuated bovine-derived strain of rotavirus has been used for production of a vaccine. The vaccine currently is given to dayold calves. Although there is some question about the efficacy of this new vaccine, it seems to show effectiveness when used in herds with serious rotavirus diarrheal problems (24).
Coronavirus. A second major virus, a corona-
virus, also has been isolated from dairy calves with neonatal.diarrhea (39). The coronavirus and the rotavirus both affect the absorptive epithelium of the gut. Both cause similar clinical syndromes and contribute significantly to calf morbidity and mortality. Both viruses are studied with similar technologic methods, and both are combined in a commercially available modified live virus vaccine. They are, therefore, frequently discussed together, even though they have slightly different pathogenic mechanisms and different virologic characteristics.
Coronavirus infection cannot be distinguished clinically from other causes of diarrhea in newborn calves. A laboratory diagnosis can be made by finding the virus in feces by immunofluorescent microscopy or electron microscopy.
Prevention of coronavirus-induced diarrhea is complex. It requires hygienic calving conditions, assurance that each calf receives colostrum, and sanitary rearing practices. Where a specific diagnosis has been established, use of the modified live virus in pregnant cattle and newborn calves may be of value.
Colibacillosis
The two most common bacterial enteric diseases of young cattle, especially calves, marked by severe diarrhea, dehydration, and high death loss, are colibacillosis and salmonellosis.
Colibacillosis is associated with infection by Escbericbia coli, a ubiquitous organism found normally in the lower bowel of all warm-blooded animals. It is found abundantly in carnivora and omnivora but sparsely in horses and cattle and may or may not be pathogenic. Its pathogenic propensity is regulated by plasmid-mediated determinants that, in addition to providing filamentous surface antigens on the organism that facilitate adhesion to the epithelium of the intestine and thence colonization and proliferation, cause the organism to produce a path opotentiating diarrhea-inducing enterotoxin or sometimes a neurotoxin, which also is vasotoxic and produces enterotoxemic systemic disease (13). A third toxin, which is produced by all coliform bacteria, rarely causing diarrhea, is endotoxin. E. coli endotoxin is responsible for systemic colibacillosis in immunoglobulin-deficient calves. This occurs even in calves that ingest but do not absorb protective quantities of colostral antibody (19,41). Although the alimentary tract is usually the portal of entry, signs of nonenterotoxemic systemic colibacillosis in calves may or may not be accompanied by diarrhea or alimentary tract injury. It is important to emphasize that ordinarily endotoxin does not cause fluid loss from the intestine. Usually, manifestations of endotoxin injury are associated with hyperthermia, lethargy, complement activation, neutrophil degranulation, and various toxic effects on the vascular system that may culminate in irreversible, fatal shock. Signs include fever, depression, vomiting, trembling, edema, increased respiration, and some hemorrhage (41).
The vasotoxic neurotoxin elaborated by some varieties of Escbericbia coIi produces enterotoxemic colibacillosis, as in edema disease of swine. Brain hemorrhages in calves are believed to be induced by the same bloodvessel-damaging neurotoxin.
The most common and most troublesome of the toxins of E. coli is enterotoxin, and the disease it induces is enteropathic enterotoxic colibacillosis, so-called "white scours" or "calf scours". Enterotoxic colibacillosis is the only form of colibacillosis which is truly an enteric disease. It occurs in the young; adults are spared (41). Calves exposed to enteropathic enterotoxic E. coli during the 1st day of life most likely will develop disease whereas those exposed to the same organism 2 days later may not show signs of illness.
There is a gradient with the anterior small intestine being highly sensitive to enterotoxin and the posterior small intestine being relatively resistant. As with cholera in man, enterotoxininduced secretion in the small intestine of calves takes place across an intact, unaltered mucosa except for the discharge of goblet-cell mucus. Enterotoxic colibacillosis produces a profuse, watery, dehydrating diarrhea, not by hyperperistalsis (whip-lashing) of the gut but by biophysical directional alternation of water and electrolyte flow in the intestinal membranes.
It has been baffling that in enterotoxic colibacillosis the organism adheres to villus, not crypt, epithelium. Yet, it is the crypt epithelium that contains numerous secretory granules responsible for the diarrhea. It is believed that the enterotoxin mimics or stimulates the release of villus hormones, including prostaglandins, which regulate adenylcyclase and the intestinal secretory process and secretion of water and electrolytes (41).
When such secretion by the small intestine exceeds colonic absorption, diarrhea occurs. Losses of water, sodium, and carbonate result in dehydration, metabolic acidosis, and hyperkalemia. Acidosis leads to decreased intracellular potassium and then increased extracellular potassium. This effects cardiac arrhythmias and then myocardial failure, the immediate cause of death in most cases. Death frequently occurs in less than 24 h.
In the most acute form of white scours, the calf shows weakness, appears sleepy, and soon dies without other signs. In the usual form, however, the calf shows a severe diarrhea, the fecal material often being full of gas bubbles and whitish in color because of lack of bile. Some animals may die after a few days. In more protracted cases, lameness may appear because of acute inflammation of one or more joints. In such joints, the capsule is distended with a cloudy fluid in which there are myriads of colon bacilli. These animals almost always eventually die (24).
In controlling calf scours, it is essential to emphasize that colostrum and good management to prevent environmental stress are equally important. Colostrum, containing immunoglobulin, will interfere with the adherence and colonization of the enterotoxic coliforms on the intestinal villi.
The success of vaccination of cows to prevent diarrheal disease in calves appears to depend upon preparation of an antigen (vaccine) to stimulate formation of appropriate multivalent anticapsular immunoglobulins, not antitoxin, because the common enterotoxin is not immunogenic (24).
Morgan et al. (43) vaccinated brood sows with purified somatic pili from clones of piliation phase enterotoxigenic Escbericbia coli and protected neonatal suckling pigs against diarrheal disease caused by enterotoxigenic E. coli strains that possessed the same pili. The practicality of such a vaccine is diminished by the heterogeneity of pathogenic strains unless ways are devised to produce polyvalent immunoglobulin by genetic or hapten linkage of pilus immunogenic determinants. If that can be accomplished for pigs, it most likely will have benefit for calves.
Foot Rot or Infectious Pododermatitis
Foot rot remains a troublesome and enigmatic disease in cattle, although careful and difficult research continues to chip away at its elusive nature.
The anaerobic Fusobacteria, which produce copious amounts of butyric acid, and Bacteroides species, which produce copious volumes of isovaleric and isobutyric acids (24), have been isolated from cases of foot rot in cattle and studied extensively by Berg (4) and others, including Thorley et al. in Great Britain (56).
The disease in cattle frequently has been associated with Fusobacterium necropborurn but also associated with Bacteroides melaninogenicus (4) and Bacteroides nodosus (56).
These organisms have little or no capacity to invade normal epithelium, but under anaerobic conditions they readily enter and multiply in tissues damaged by injury or infection caused by other organisms (24). In all species of animals suffering from foot rot (or liver abscesses associated with F. necropborurn and pyogenic bacteria), the typical lesion is necrosis, and it has a putrid odor.
The genesis of the lesion in cattle is not understood completely; it is more thoroughly known in sheep. The early lesions of foot rot in cattle are similar to those of sheep; that is, swelling, and moistness in the interdigital cleft. In sheep, but not in cattle, inflammation of the sensitive laminae of the hoof occurs, often underrunning the sole.
The most important bacterium in foot rot of sheep is Bacteroides nodosus. The disease is initiated by the penetration of B. nodusus into the epidermis damaged by injury (54). This is accompanied or followed by invasion of the same site by F. necropborum, which extends the tissue damage, creating new invasion sites for B. nodosus (5). Perhaps the genesis of the foot lesion in cattle parallels that of foot rot in sheep. Treponema penortba may be a pathopotentiator. Pus-producing bacteria, like Corynebacterium pyogenes, are secondary invaders, but they operate in synergy with the primary invaders to produce tissue destruction and sometimes abscess formation. F. necropborurn facilitates the establishment and growth of C. pyogenes in the tissues by the phagocyteinhibiting and leucocyte-destroying action of its exotoxin (48). C. pyogenes produces a macromolecular substance that stimulates the growth and invasiveness of F. necropborurn, particularly by removing oxygen from the tissue site under microbial attack. Whether a liberation of exotoxin is activated by phage (as for some species of Corynebacteria) is currently unknown (48).
Attempts to vaccinate animals with formolized bacterial cell preparations and with culture filtrates of F. necropborum have not been successful, although it appears that most animals develop antibody as they age and are exposed to the organism (24). It is not clear that this antibody is protective against the disease, however.
Some prophylactic agents, like broadspectrum antibiotics, are effective in preventing F. necropborum-associated liver abscesses in cattle. Such agents may have potential in preventing or treating bovine infectious pododermatitis.
Infectious Bovine Rhinotracheitis
Twenty five years ago this disease had been newly described (8,29) and during the intervening period, elaboration of the many f~cets of infectious bovine rhinotracheitis has resolved many perplexing questions.
Infectious bovine rhinotracheitis (IBR) is the name given to the respiratory form of a herpesvirus infection that also sometimes is manifested by a genital syndrome called infectious pustular vulvovaginitis (IPV) or coital exanthema. Close confinement of feedlot cattle and cattle in large dairy herds provides favorable conditions for the rapid transmission of the virus (51).
Sometimes the disease appears simply as a granular conjunctivitis, occasionally by meningoencephalitis, especially in calves under 6 mo of age. Frequently, the infection is manifested solely by abortions. Abortion can be a sequel either to respiratory infection by IBR virus or to injection of modified live IBR vaccine. Abortion usually is not a sequel to IPV.
The IBR virus, like other herpesviruses, may be latent, therefore being shed intermittently from infected animals for long periods following recovery from clinical disease. Latent infection was activated by the use of corticosteroids in studies reported by Sheffy and Rodman in 1973 (52).
The respiratory form of IBR infection probably is the most significant and economically important form of the disease in feedlots, but for dairy cattle abortion may be more costly. An outbreak may be mild with numerous subclinical cases, or it may be fulminating. It is not unusual to find cattle in an infected herd that appear bright and yet have markedly elevated body temperature (20). There may be noticeable decrease in feed consumption and signs of upper respiratory infection. The affected animals exhibit rapid rhythmic and sometimes dyspneic breathing, inappetence, elevated temperature, coughing, nasal discharge, foamy salivation, catarrhal conjunctivitis, and loss of weight. The course of disease usually runs between 7 and 10 days.
The muzzle and nasal mucosa become inflammed, giving rise to the term "red nose". Occasionally respiratory signs are not evident. Conjunctivitis, with ocular discharges which initially are clear but later infrequently become mucopurulent and sometimes with centripetally originating corneal opacities, may appear as principal manifestations of infection. Such infection may be misdiagnosed as pinkeye. A high percentage of cattle with classic pinkeye, caused not by a virus but by Moraxella boris bacterium, have corneal opacities originating in the center of the cornea and spreading centrifugally. The diagnosis of IBR-associated conjunctivitis should be considered if opacities in a herd are few and appear to originate at the corneoscleral junction (29). Another disease wherein corneal opacities occur frequently and originate at the corneoscleral junction is malignant catarrhal fever (MCF). That disease, too, is associated with dyspnea due to profuse accumulation of tenacious mucopurulent exudate in the nasal passages and, to some extent, the trachea and bronchi. Partial blockage of these airways may result in the forced open-mouthed breathing seen in IBR and MCF.
White pustules frequently appear and coalesce in the respiratory, ocular, and reproductive tract mucous membranes. When these lesions appear in all these loci, they are sufficiently distinctive for IBR to suggest that the disease in question is, in fact, IBR. In the nasal passages, however, similar lesions may occur in MCF.
Following natural IBR virus infection or vaccination with modified live virus 1BR vaccines, cell-mediated and humoral components of the immune system are activated. The humoral response, usually measured by serum neutralization tests, has served traditionally as an indicator of prior infection and as an indirect measure of resistance. Evidence now suggests that a crucial role in resistance and recovery from early IBR involves locally deployed elements of the cell-mediated immune system. Thus, detection of neutralizing antibody in serum probably is not an accurate indicator of immunity but may be used until methods of measuring cell-mediated immunity to IBR come into common use (29).
The IBR can be controlled by maintaining protective antibody in a herd and by reducing the number of susceptible animals to a minimum. This can be accomplished by helping cattle acquire both passive and active immunity at an early age.
The calf receives IBR antibody from cotostrum within the first 24 h after birth. The quantity that a calf receives depends upon the dam's serum antibody and the amount of colostrum ingested. Because the dam transfers IBR antibody to her offspring comparable to that in her own serum, some workers suggest maintaining a protective antibody titer in the brood stock. This can be accomplished by booster vaccinations 3 to 4 wk prior to breeding or by vaccinating in the last trimester of pregnancy with vaccines approved for pregnant cattle.
Colostral antibody may persist as long as 4 mo and may prevent an antibody response to vaccine given during that period. Therefore, calves vaccinated before 6 mo of age should be revaccinated after that age. Vaccination may provide immunity for longer than 2 yr (51). As an additional protective measure, all additions to a herd should be isolated and observed carefully for signs of illness every day for 30 days.
Today live modified combined vaccines against IBR, bovine virus diarrhea-mucosal disease, and parainfluenza-3 are available and are compatibly effective when administered at 5 to 7 mo of age to calves that have had colostrum immediately after birth and earlier to calves deprived of colostrum.
The first IBR vaccines were modified live virus products developed to be used intramuscularly. These had the disadvantage of causing abortion. In 1969, intranasal-administered, modified live-virus IBR vaccine was introduced and has gained widespread acceptance. Unlike vaccines for intramuscular injection, the intranasal vaccines usually are safe for use in pregnant cattle. Among the advantages ascribed to the intranasal vaccines are rapid protection attributed to interferon production and rapid induction of secretory antibody at mucosal surfaces (29).
Intranasal vaccines are believed to induce production of humoral antibody at titers comparable with those of intramuscular vaccines. The intranasal vaccine can be administered to cattle of the same ages as applied to use of the intramuscular vaccine. It has been suggested that intranasal-administered vaccine may override low colostrum-acquired maternal immunity, resulting in successful vaccination of some calves that do have colostrum-acquired antibody. If calves less than 6 mo old are vaccinated, booster doses are recommended for animals to be retained for long-term purposes, especially as breeding animals.
Inactivated IBR vaccine has been available from time to time in combination with bacterin containing organisms of the Pasteurella genus and inactivated myxovirus parainfluenza-3. The latter two agents have been associated with the shipping fever complex. There is controversy regarding the efficacy of inactivated vaccine for these respiratory illnesses, however. As in the case with many other inactivated vaccines, the initial vaccination procedure should be repeated, and annual booster doses are recommended. Advantages of inactivated vaccine are that it eliminates the risk of postvaccination abortion associated with live-virus vaccine and avoids unfavorable postvaccination reactions except anaphylaxis. Also, with inactivated vaccine, there is no shedding of vaccine virus and seeding of populations otherwise not exposed. In addition, it eliminates concern that the vaccine virus itself may establish a latent infection that could be reactivated at a later date.
Although research on IBR continues, the past quarter century has seen it emerge from an undescribed disease to a well understood and well controlled infection.
Johne's Disease (Paratuberculosis)
Johne's disease is a chronic granulomatous enteritis of ruminants caused by an acid-fast bacterium, Mycobacteriurn paratuberculosis. Johne and Frothingham first described this disease in 1895 in Germany (23). They cited an unusual case of tuberculosis in a cow wherein they found acid-fast bacteria, which they thought to be either avian or bovine tubercle bacilli (9). In 1906, Bang in Denmark recognized it as an infection distinct from tuberculosis and suggested that it be named bovine pseudotuberculosis or paratuberculosis (9).
Paratuberculosis has been recognized in the United States for about 75 yr, and concern has been expressed that it is increasing in incidence. The disease is characterized by progressive emaciation and, in cattle, by a recurrent diarrhea. There is thickening, corrugation, and convolution of the mucosa of the distal end of the small intestine. The granulomatous enteritis that occurs is expressed by infection-damaged, malfunctioning short, club-shaped intestinal villi that assume wart-like configurations.
Diagnosis has been extremely difficult for a variety of reasons. Clinical disease usually occurs only in a small number of infected animals. Infected animals that shed the organisms can be inapparent (silent) carriers showing no evidence of disease. Fecal culture isolation techniques to recover the organism require 2 to 3 mo for completion, because the organism grows extremely slowly in laboratory media. Further, these techniques detect only a small number of infected animals. Heretofore, immunologic tests, including the complementfixation test and the agar gel immunodiffusion test, used prior to the development of the lymphocyte transformation test that employs phytohemagglutinin as mitogen and purified protein derivative from the organism as antigen, have lacked specificity or sensitivity (9). The difficuh T in diagnosis has been compounded by the fact that humoral and cell-mediated immunologic status of infected animals varies with the stage of the disease. However, the availability and assessment of the lymphocyte transformation test, as described by Buergelt (9), offers encouraging potential for the diagnosis and ultimate eradication of this troublesome disease. The test provides an in vitro evaluation of cell-mediated immunity and offers a fairly sensitive method to assess the extent of disease in populations of cattle under surveillance.
To complicate the diagnosis, there is a close antigenic crossreactivity between Mycobacteriurn aviurn, the cause of avian tuberculosis, and Mycobacteriurn paratuberculosis. However, these two infections may be differentiated by the lymphocyte transformation test, provided appropriate purified protein-derivative antigens from each of these agents are used in parallel on a given serum specimen (9). Paradoxically, even with the hopefully effective lymphocyte transformation test, a humoral mitogen-suppressor substance has been found in serum fractions containing immunoglobulins lgGl and IgG2. Notwithstanding the difficulties in serologic interpretations, the lymphocyte transformation test shows promise of being useful in the diagnosis of subclinical and clinical cases of Johne's disease and may provide an overall impression of the extent of exposure to M.
paratuberculosis within a herd. Nonruminant hosts may serve as reservoirs from which the organism may be shed to infect cattle (9). Although Johne's disease has been historically considered a disease that involves the lower end of the small intestine, the ileocaecal valve, and the adjacent lymph nodes, the organism has been isolated also from the liver, blood, udder, uterus, ovaries, milk, urine, feces, and fetus. Therefore, there is evidence that the disease is transmitted not only by fecal contamination but also by milk and by transplacental infection of the fetus (9).
A typical clinical case of Johne's disease is characterized by intermittent or continuous chronic diarrhea in young animals, especially in 2 or 3-yr-old lactating females. The disease most often occurs in the early part of the first or second lactation. Feces are Soft and watery and are passed without straining. Diarrhea may decrease in severity during late pregnancy, only to reappear more severely after parturition. Valuable high-producing animals may be affected suddenly. Milk production decreases, and the haircoat may become rough or loss of hair occurs. The appetite remains good. There is no fever. Usually only one or two animals show evidence of disease in a herd at the time of initial observation. After a course of weeks to months, with unresponsiveness to treatment, the disease terminates in death of those animals showing severe dehydration, emaciation, and weakness. Decreased absorption of amino acids associated with increased protein leakage results in a negative nitrogen balance with the clinical manifestations of a wasting disease.
Although M. paratuberculosis has been cultured from pharyngeal lymph nodes, tonsils, bronchial lymph nodes, spleen, lungs, kidney, reproductive tract, mammary gland, and placenta, gross and microscopic lesions usually are absent in these organs. Lymphatic and hematogenic spread through the thoracic and portal venous circulation, respectively, begins early in the disease process. However, the organisms do not seem to multiply to any extent or give rise to distinct lesions except in the intestine and its adjacent lymph nodes. The spread is assumed to occur passively by way of circulating macrophages. In the liver, these bacteria-laden macrophages may become trapped in sinusoids and, in conjunction with lymphocytes and possibly antibody, they may give rise to multiple small granulomas (9).
Merkal (40) introduced recent immunologic concepts to the understanding of the pathogenesis of Johne's disease. He postulated that an antigen-antibody reaction in the infected intestine causes the release of histamine in an immediate-type hypersensitivity reaction, resulting in mediation of diarrhea. Delayed hypersensitivity, also a phenomenon in this disease, involving Specific antigens from the organisms and specifically sensitized lympbocytes, causes the release of cytotoxins and pyrogens that mediate emaciation and anemia.
Attention has been called in early literature to the similarity between leprosy and paratuberculosis (9). Both diseases are caused by acid-fast organisms that are difficult to culture and are characterized by long and slow growth periods of organisms in culture. The granulomatous inflammatory response in both diseases is similar. Despite involvement of different organ systems, the microscopic lesions are dominated by epithelioid and Langhans' giant cells. In advanced leprosy the typical inflammatory cells have been termed "leprosy cells", enlarged maerophages with vacuolated, frothy cytoplasma resulting from the accumulation of lipid substances and bacillary debris. Although such cells are not identified clearly in Johne's disease, vacuolation of cytoplasm and lipid degeneration of epithelioid cells has been noticed (9), with clumping, distortion, and dissolution of bacilli within these cells. In the adjacent draining lymph nodes, large areas of lymphoid cells within the paracortical zone have been replaced by granulomatous inflammatory infiltrates in both diseases (9). The similarity in the histopathologic findings of leprosy and of paratuberculosis have attracted pathologists studying paratuberculosis to the adaptation of Ridley and Jopling's morphologic classification of leprosy for paratuberculosis, hence, the inclination among some pathologists to refer to Johne's disease on cellular morphologic grounds as bovine leprosy (9).
Vaccination of cattle with heat-killed or live, attenuated organisms has been used with some claims of success in France and England. Where vaccination is practiced, one dose for a 1-wk-old calf is recommended (9). But it generally is agreed that the vaccine does not confer absolute immunity, and some animals vaccinated with the live, attenuated cultures have excreted the organisms. A major problem in using vaccines of this nature against Jobne's disease, in addition to their questionable reliability, is that vaccinated animals become sensitized and react to mammalian tuberculin when tested for tuberculosis. Further, the immunologic significance of antibody in Johne's disease and its relationship to the infectious agent remain unsolved.
Doubts have been raised about the protective nature of antibody in chronic, progressive, infectious diseases. In such diseases, it is thought that antibody may protect the etiologic agent rather than the host (9). There is evidence that such antibody abrogates effective cellular immunity and actually enhances dissemination of lesions in some chronic infectious diseases. The mechanism of enhancement of disease by antibody should be suspect when a disease normally controlled by cellular immunity suddenly undergoes rapid dissemination of lesions, such as is seen classically in miliary tuberculosis (9). One must examine this question critically in Johne's disease. Does this occur in those clinical cases of Johne's disease resulting in diarrhea, emaciation, and death, as distinguished from the infected animals that show inapparent infection? If so, the value of vaccination needs critical reevaluation. In that evaluation, however, one must be sensitive to experience with other vaccines in similar modes. For example, the appearance of miliary tuberculosis in young children is rare after BCG vaccination (15). The BCG is a strain of originally virulent M. boris attenuated by Calmette and Guerin by serial passage on inhibitory media. It is assumed that the effectiveness of the vaccine is dependent upon development of humoral antibody.
Due to the lack of adequate or effective vaccines or effective drugs for treatment, control of Johne's disease in infected herds is achieved best by strongly enforced management procedures, which include prompt isolation and culling of infected animals and their offspring as determined by serologic (9) or culturing (45) methods, segregating calf-rearing quarters, and good hygienic procedures.
Malignant Lymphoma of Cattle (Enzootic Bovine Leukemia)
The past quarter-century has brought remarkable progress in an understanding of bovine malignant lymphoma, particular!y the identification of a virus as the cause of some such tumors in cattle. This progressive and ultimately fatal disease of cattle also is known as bovine lymphosarcoma, bovine lymphomatosis, or bovine leukosis. In clinically apparent cases, it is manifested by enlargement of lymph glands. The glandular enlargements in malignant lymphoma of cattle may be preceded by hematologic change, usually a persistent lymphocytosis. Many European workers believe that cattle may develop these hematologic changes without tumor formation (24).
Some cattle in herds afflicted with this disease show enlarged and firm superficial lymph nodes. Generally, the disease in these animals progresses rapidly. Emaciation develops, and death usually occurs in animals that show signs of illness. Sporadic cases appear in calves, or endemics appear in adults, the incidence being highest in animals from 5 to 8 yr of age. Calves under 6 mo of age showing the sporadic form usually present generalized enlargement of lymph nodes and nodular or diffuse lesions of the spleen, liver, kidneys, or bone marrow. This is called the neonatal multicentric form.
Animals between 6 mo and 2 yr of age may show enlargement of limited groups of lymph nodes, usually with involvement of the thymus gland. Also, in that age group, the skin may show multiple tumors. These are referred to as the thymic and skin forms, respectively, and these conditions appear to be unrelated to bovine leukemia virus (24).
In animals over 2 yr of age, the multicentric form of malignant lymphoma usually is observed. Sometimes an eyeball may protrude because of tumor formation in the orbit. Chronic bloating occurs in some because of enlargement of thoracic nodes that interfere with normal eructation. Abomasal lesions may lead to digestive disturbances or abomasal ulceration. Lameness and paralysis may occur because of pressure on parts of the spinal cord or peripheral nerves from tumors. Lymphocytic infiltration of some of the internal organs may be seen in some cases with pronounced increase in the number of circulating lymphocytes, hence, the term lymphocytosis or leukemia (24).
There is evidence that B-lymphocytes are the target cells for bovine leukemia virus (BLV) infection. The B-cells are lymphocytes from which immunoglobulin-producing plasma ceils are derived. Also, it is now clear that the agent is a C-type virus, an oncogenic RNA virus in the subfamily Oncornavirus. The C-type particles are budding forms at cell membranes, also observed as free particles in intercellular spaces. Particles with central nucleoids are designated C-type. The B-type particles, by way of contrast, have acentric nucleoids.
In laboratory diagnosis, phytomitogen is used in buffy coat cultures of leucocytes from cattle with persistent lymphocytosis or lymphosarcoma to enhance the appearance of C-type particles in the cell cultures. By this technique, C-type particles, heretofore demonstrated in cattle with persistent lymphocytosis and lymphosarcoma, also have been found in "normal" cattle (24). Perhaps this indicates inapparent, silent infection which, in the absence of antibody, will skew the epidemiologic evidence of distribution of the infection in the national cattle population. Nonetheless, in the United States there is evidence that the disease is becoming more prevalent, with high and low incidence areas (23). Evidence is emerging that BLV is responsible for producing several forms of natural disease (24), but not all bovine lymphosarcomas have been associated with BLV.
Sero-epidemi61ogic evidence indicates that the virus in cattle is transmitted horizontally by contact as well as vertically, and it also is transmitted similarly in experiments with sheep and goats (24,25). Further, BLV replicates in monolayers of bovine embryonic spleen cells, fetal lamb spleen, and human diploid embryonic cells with the production of syncytia (23). This observation, and the fact that BLV will produce lymphomatosis when inoculated into a variety of species, i.e., sheep, goats, and subhuman primates (25,37), and the finding of BLV in cows' milk (17) have prompted careful studies on the human risk from exposure to BLV.
A seroepidemiologic study was conducted by Donham et al. (16) in an attempt to identify antibody against the bovine leukemia virus in people exposed to cattle with lymphosarcoma. Farm families, farm employees, and veterinarians in contact with dairy herds having documented cases of lymphosarcoma were tested for precipitating antibody to the BLV, using the agar-gel immunodiffusion test. The cattle also were tested by serologic procedures. Further, information was collected from the farm families regarding consumption of unpasteurized milk from their dairy herds. Twenty-one dairy herds with documented cases of lymphosarcoma were identified. A total of 846 cows from these herds were bled and 33% showed positive serologic tests.
No positive serums were found in the 45 dairy farmers, family members, and farm employees associated with the herds with lymphosarcoma. Consumption of raw milk was reported by 77% of the farm group.
In addition, 83 veterinarians, 30 leukemia patients, and 200 control human serums were tested and found negative for antibody to the BLV (16).
Although 77% of the farm people in this group consumed raw milk and showed no seropositive evidence of infection, it is still advisable for people to drink pasteurized milk inasmuch as it has been demonstrated that flash pasteurization inactivates the virus in milk (23).
Special attention is called to the studies of 12 neonatal chimpanzees removed from their mothers at birth, nursed separately, and fed either unpasteurized BLV-containing milk or a prepared infant formula. Hematologic changes were noted at days 119 and 200 in two animals fed BLV-containing milk. The animals died at 33 and 46 wk, respectively. The bone marrow, peripheral blood, and other autopsy findings for both animals were indistinguishable from leukemia and were consistent with the Di-Guglielmo syndrome, erythroleukemia (37). However, neither the bovine lymphosarcoma virus nor BLV antibody was in these animals. Whether the disease developed as a direct result of BLV ingestion remains a moot question since none of the other chimpanzees developed illness nor antibody (1). In a symposium on bovine leukosis sponsored by the United States Department of Agriculture in May 1979, Glyn G. Caldwell reported the results of Public Health serologic studies and concluded that "adequate serologic tests which are sensitive and specific have failed to demonstrate infection in humans despite close contact or raw-milk ingestion from BLV-infected herds. Consequently, BLV does not appear to represent a public health hazard for humans" (10).
Further studies are needed before we understand fully the epidemiologic patterns of BLV and the details of its association with the variety of lymphoid tumors affecting dairy cattle.
Salmonella typbimurium and S. dublin.
Unlike the emerging enteric viral diseases, satmonellosis has plagued dairymen for years. The past 25 yr, however, have brought considerable understanding to this ubiquitous enterobacterial disease.
Diarrhea, severe dehydration, prostration, and death characterize salmonellosis in young cattle, especially calves. Nevertheless, once established in young animals in a herd, adult animals tend to become infected. This appears more commonly today than 10 yr ago (20). At that time it was restricted primarily to young animals (20).
Salmonellosis is usually an enteric disease. Sometimes, though, it becomes systemic, involving organs and tissues other than the intestine, especially the joints. Most clinical cases in calves are due to Salmonella typbimuriurn. West of the Rocky Mountains, however, some cases in calves and adults are due to Salmonella dublin, (8) Cattle that contract S. dublin infection continue, after recovery, to excrete some of the bacteria in their feces for many years; about 30 to 40% remain shedders during their lifetimes because the organism colonizes the gall bladder and liver. In contrast, in the more ubiquitous S. typbimurium infection of cattle, which is indistinguishable clinically from S. dublin disease, recovered cattle remain carriers and shedders for only a month or two because S. typbimurium does not colonize the gall bladder or liver in mammalian species, except perhaps rodents.
Notwithstanding these facts, it is possible to eliminate one of these infections from a herd while virtually impossible to eliminate the other. Elimination of host-adapted S. dublin can be accomplished by identifying and discarding reservoir carrier animals through culturing fecal specimens. This cannot be done in nonhost-adapted S. typbimurium infection because of the ubiquitous distribution of that bacterium among many animal species which can contaminate the farm environment. Although outbreaks of S. typbimurium infection in cattle may originate from other cattle, there are usually opportunities for crossinfection from other species of livestock (especially swine), wild rodents, or poultry. However, in Great Britain there are specific phage types and clones peculiar to cattle alone (58). These can be eliminated from a herd. This emphasizes the importance of phage-typing all isolates of salmonellae from dairy herds. ]458
POPPENSIEK AND KAHRS
The detection of salmonellae in a calf shipped from one locality to another does not necessarily indicate infection at the origin. Calves showing clinical evidence of salmonellosis are often those which have come from a clean source hut which apparently have encountered the organism for the first time while in transit.
Acute salmonellosis in adult cattle, while by no means as common as in calves, occasionally can be very troublesome (30). Abortion is a common feature of adult infection (30). Diarrhea and dehydration in the adult usually are severe when they do occur.
Colonization of the latter part of the small intestine and colon is a necessary first step in pathogenesis of enteric salmonellosis. The normal intestinal flora may block access to attachment sites on the mucosal cells needed by the salmonellas. Also, indigenous fusiform bacteria that lie in the mucous layer coating the epithelium of the large intestine normally inhibit growth of salmonellas by producing volatile organic acids which are toxic for the salmonella organisms (24).
Factors such as antibiotic therapy and diet and water deprivation that disrupt the normal colonic flora, or otherwise stress cattle, greatly increase the host's susceptibility to enteric and septicemic salmonellosis (24). Clinical salmonellosis must be regarded as having a multifactorial cause.
After invading intestinal epithelial cells, salmonellas cause net secretion of water and electrolytes, possibly by means of lipopolysaccharide in the bacterial endotoxin, causing inflammatory release of prostaglandins that in turn activate adenylcyclase (24). This enzyme acts upon secretory granules in the crypt cells to stimulate water and electrolyte secretion.
Bacterial endotoxin also effects elevated body temperature, damage to capillary endothelium resulting in hemorrhage, thrombocytopenia, depletion of liver glycogen with prolonged hypoglycemia, and shock. Shock often induces severe circulatory collapse resulting in sudden death.
Relapses are certainly an important practical problem in salmonellosis, but a number of these are believed to be from physical tissue damage from treatment rather than from first-order resurgence of infection.
There is widespread agreement that celtmediated immunity is more important than humoral immunity in resistance to salmonellosis. Humoral antibody contributes primarily to bacterial clearance (24).
Chemically inactivated vaccines (bacterins) have been prepared for immunoprophylaxis against Salmonella species with varying and ofttimes questionable effectiveness. In assessing effectiveness, intracellularly-located organisms are not. affected by humoral antibody. The body must depend upon effectiveness of cell-mediated immunity, the effectiveness of macrophages which are activated by sensitized lymphocytes, to phagocytize and kill the tissue-invading salmonellas. Live vaccines prepared from avirulent cultures ofS. typbimurium and S. dublin are used widely in Europe. They promote cell-mediated immunity (58).
Macrophages also release a cloning-inhibiting factor, a lymphokine that like the secretory immunoglobulin IgA, blocks adherence and thus prevents colonization, multiplication, and cloning of the organisms on the intestinal epithelium.
Salmonella anatum. During the past decade, there has been a noticeable appearance and increase in incidence of Salmonella anatum infection in cattle (6,7), vying only with S. dublin for second place in order of frequency (24). Next to S. typbimurium, it is the most widely distributed Salmonella type (24). The source of infection appears to be mostly wild birds that contaminate pastures and hayfields with droppings containing the organisms (37). However, the organism has a wide distribution among a great variety of birds and mammals, so feedstuff and water, in addition to pasture or hayfield, may be contaminated.
In infected lactating cows, there is a marked reduction in milk production associated with protracted illness (20,37).
Salmonellosis and Antimicrobial Resistance. Another problem relating to enteric bacteria has appeared during the past decade. Increasing resistance among members of the Enterobacteriaceae to antimicrobial agents has been noted throughout the world (46). Particular interest has focused on Salmonella species (46). The antimicrobial resistance patterns and presence of plasmid-bearing resistance factors in isolates from human and animal sources have been studied.
Considerable anxiety has been expressed about the use of antibiotics in animal feeds as Journal of Dairy Science Vol. 64, No. 6,1981 growth promoters and prophylactics, as this practice has been suspected to be a major factor in the increased antimicrobial resistance. No effective method for assessing the magnitude of risk to the welfare of man has been devised. Opinions about the degree of risk are highly polarized.
The increase in resistance has not been sustained in at least one instance. An abrupt decline in ampicillin resistance of Salmonella typbimurium in humans was reported in New York in the late 1970's, in contrast to an increasing frequency of resistant isolates from upstate New York calves over a similar time (12). The sudden, unanticipated drop in antibiotic resistance, reported in this singular but noteworthy urban experience, introduces new and unanswered questions as to why a population of S. typhimurium suddenly should lose its ampicillin resistance (12). It is possible that plasmid coded protein on the Salmonella cell surface is antigenic to the host animal, thus resulting in immunoglobulin against the ampicillin-resistant strain and an emerging population immunity (58).
Genetic control mechanisms will continue to be studied. But the role of the gut flora in support of or antagonism toward survival of R-factor-bearing strains versus antibiotic-sensitive strains and clone-preference effectors needs review.
The life-span benefits of incorporating antibiotics in feeds as growth stimulants are not understood fully, because it is not known how the antibiotics act at the molecular level as growth promoters.
Considerable practical evidence of the prophylactic benefits of antibiotics in feed or water does exist. Nonetheless, it is not always certain that diseases against which protection is claimed actually exist in all areas where antibiotics are being used as prophylactic feed additives. Improved surveillance and monitoring methods are needed.
Further the need for better understanding of the ecologic impact of different R-factor incompatibility groups in enteric genera such as Escbericbia and Salmonella (58) is of fundamental importance.
Sideropbores. Since serum and tissue extracts contain all nutrients essential to bacterial growth, it is assumed that all materials required by the pathogen are available for its utilization. This is not so. The potentiality of various pathogens to survive in an animal depends upon their ability to obtain sufficient iron for active metabolism (31). All microorganisms, with the possible exception of lactic acid bacteria, require iron. Iron is used in the cytochrome system for energy production. Lactic acid bacilli do not use that system. They derive energy from the glycolytic system. The same is true for anaerobes, although anaerobes may utilize both pathways.
Although iron is one of the most prevalent of the earth's elements in aerobic environments the amount of free iron available for assimilation by microorganisms is restricted due to the proclivity of ferric iron to form large, insoluble aggregates at neutral or alkaline pH. To acquire necessary iron from these aggregates, most aerobic microorganisms have evolved specialized iron-solubilizing and transporting ligands, chelating agents, which have been termed siderophores.
A pathogen does not have to contend with the insolubility of ferric iron but must obtain iron from its host. Although the amount of iron in host fluids is more than adequate for microbial growth, the iron-binding proteins, transferrin and ferritin in the serum and lactoferrin in secretions, sequester essentially all the iron in these environments.
The most likely mechanism whereby a bacterial pathogen successfully competes with these iron-binding proteins is via siderophore production.
Enterochelin or enterobactin is a siderophore produced by Salmonella typbimurium and possibly other enteric bacteria. It antagonizes the iron-restricting mechanisms of the host and, therefore, is a virulence factor for S.
typbimurium (60).
One of the most effective defense mechanisms a host might develop against iron-dependent parasites would be the production of antibody to siderophores. Perhaps anti-siderophore vaccines for animals might reduce the risk to man of antibiotic-resistant enterobacterial pathogens if the vaccinal antigens were rendered siderophore-specific for predetermined genera, not the entire aerobic microflora.
The ubiquity of salmonellas suggests that they will be a problem for dairymen for some time, in fact, as long as dairy cows and calves are subjected to stressful environments.
Shipping Fever, *'Hemorrhagic Septicemia", Pasteurellosis
Hemorrage is not a prominent feature of the bovine respiratory disease, which has borne the name "hemorrhagic septicemia" or shipping fever for many years. The name evolved from habit rather than from a description of major lesions. All septicemias show some evidence of hemorrhagic lesions, many of which, however, are far Iess than those which characterize the extensive hemorrhagic diatheses.
In 1880, Louis Pasteur described the organisn~ that caused cholera in fowls, a disease characterized by enteritis with submucous and subserous hemorrhages and vascular congestionsurely a hemorrhagic disease. Later, it was learned that Pasteur's fowl cholera bacillus was indistinguishable from the organism of rabbit septicemia and of swine plague, two other hemorrhagic diseases wherein isolations of this common generic organism had been made.
In 1886, Hueppa, in Germany, grouped the organism under one name, Bacterium septicemiae hemorrhagicae; the "hemorrhagic septicemia" bacillus. Even in that neophytic era of microbiology, it was reported that Bacterium septicemiae hemorrbagicae was isolated from cases of pneumonia (and sometimes septicemia) of cattle. While hemorrhage is a common lesion of septicemia, only occasionally has that lesion been identified with bovine pneumonia. In view of the primitive status of microbiology of that day, no assurance can be given that the hemorrhagic lesions were, in fact, attributable to the organism isolated.
Bacterium septicemiae hemorrhagicae subsequently was renamed. The genus Pasteurella was proposed, and the species name of the animal from which the organism was isolated was assigned according to a classification scheme proposed also in 1886 by another German scientist, Flugge.
Pasteurellosis was a name proposed shortly thereafter for a group of diseases in which Pasteurella species were associated. The fact that pasteurellosis in cattle is identified as a respiratory syndrome, particularly in groups of confined young cattle exposed to crowded stressful conditions associated with shipping and not a hemorrhagic diathesis like fowl cholera, was not sufficient cause to disassociate the firmly established synonym "hemorrhagic septicemia" from the disease, and so it has remained, even to this day.
Pasteurellosis (shipping fever) in cattle attributed to Pasteurella rnultocida is characterized by a bronchopneumonia with thickened pulmonic septae and moderate amounts of fibrin on the lung surface. This is in contrast to the pneumonia attributed to Pasteurella hemolytica, where the amount of fibrin is much greater and the lesions represent a true fibrinous pleuropneumonia (24). Affected animals show elevated temperatures, difficulty in breathing, pulmonary rRIes, diarrhea, pulmonary and subcutaneous edema, and in advanced cases, cyanosis of mucous membranes due to oxygen deficit brought about by the diminished area of functional lung parenchyma. It is likely that primary infections by viruses like parainfluenza-3 virus, IBR, respiratory syncytial viruses, BVD-MD virus, or others, or by mycoplasmas, can predispose to secondary invasion by pasteurellas through damaging the protective mucociliary clearance mechanisms in the trachea and bronchi and by impairing alveolar macrophage function. There seems to be a marked tendency toward seasonal incidence of pasteurellosis that corresponds to activity of respiratory virus and mycoplasma infections, particularly in the fall and winter.
The local inflammatory effect produced by viruses such as parainfluenza-3 or IBR leads to increased fluidity of the mucus coating with consequent sneezing and coughing. These inevitably form endogenous aerosols in the respiratory passages that can result in the downward vortical carriage of bacteria from upper parts of the tract during inspiration (24). Researchers The riddles of pathogenesis and immunoprophylaxis remain elusive and, therefore, speculative. However, in many areas of the country practicing veterinarians are becoming increasingly alarmed at the frequency of intractable and often fatal "shipping fever" cases from which antibiotic-resistant pasteurellas are being recovered (20,23). The pathopotentiators are unknown. A search for virulent clones among nonpathogenic oropharyngeal commensals continues.
Perhaps the behavior of another oropharyngeal commensal may shed some light on the mechanism of pathogenesis. Corynebacterium dipbtberiae, a normal inhabitant of the oropharynx, is harmless until it is attacked by phage. Then it releases a deadly exotoxin which produces serious disease in its host.
If phage is not a likely incriminating pathopotentiator, perhaps the role of plasmids, extrachromasomal genetic elements bearing pathogenic potentiators, should be explored.
There appears to be some evidence that the number of Pasteurellae resistant to one or more antibiotics is increasing (48). Therefore, the search for an effective immunogen becomes increasingly important. Live-culture vaccines for cattle are under study following earlier experience with live Pasteurella multocida for use in poultry (24), the results of which have been encouraging but for relatively short duration. Studies of serological relationship between strains of Pasteurella multocida by Shigidi and Mustafa in Kartoum, Sudan (53), confirmed observations that there is considerable serologic heterogeneity of strains of P. multocida. Heterogeneity of somatic antigen may be responsible for irregular results in the current system of vaccination against pasteurellosis, even though indirect evidence indicates that immunogenicity of P. multocida might be primarily, but not completely, dependent upon the capsule antigen.
As long as cattle are exposed to stress, shipping fever or pulmonic pasteurellosis will be a problem. Knowledge obtained in the last quarter century has brought us partially closer to an understanding of the disease.
Winter Dysentery
Winter dysentery is an acute, highly contagious catarrhal hemorrhagic enteritis of cattle characterized by a brief explosive attack of diarrhea. Occurrence of abdominal pain with the profuse, watery, sometimes bloody diarrhea identifies the disease as dysentery. In the northern United States it occurs usually in housed cattle during winter and results in a moderate to marked drop in milk production. In infected herds, the attack rate may reach 100%, but fatalities are rare. The causative agent is unknown but is suspected to be a virus (11,34).
In 1931, Jones and Little (26) described the disease and attributed its cause to a new species of bacterium, Vibrio jejuni, subsequently renamed Campylobacter jejuni. In 1957, MacPherson in Canada challenged the vibrionic cause when he reproduced the disease with filtered fecal material and suggested that it is caused by a virus (34).
There followed a period of some diagnostic confusion in differentiating winter dysentery, infectious bovine rhinotracheitis, and bovine viral diarrhea-mucosal disease in the 1950% until Roberts (50) characterized winter dysentery after a 3-yr study of 25 outbreaks, and Kahrs (27), employing serologic methods, compared winter dysentery, BVD-MD, and IBR. He found winter dysentery was unrelated to these other infections.
Roberts (50) reported that the incidence of the disease peaks every 10 yr, suggesting that immunity develops in herds in a given area, holding the disease in abeyance until susceptible herd replacements ultimately make up the milking herd, permitting the disease to cycle again.
Winter dysentery seems to affect young dairy cows most frequently. Those that are lactating, pregnant, or have recently calved usually are affected most severely. Milking cows often are first affected, followed several days later by younger stock (11).
Changes in feed and weather often are suspected as predisposing factors. Other stress factors that might increase the incidence or severity of the disease include poor nutrition, parturition, stressful stabling, and respiratory disease in confined animals. The disease is not seen in cattle on pasture in the United States (11), but apparently it has been observed in cattle on pasture in Australia (23).
Winter dysentery is not manifested by a noteworthy change in body temperature. When elevated temperature does occur, it usually precedes the onset of diarrhea by 24 to 48 h (11) and is accompanied by cough. By the time diarrhea is apparent, body temperature usually is normal.
The onset of diarrhea may be preceded by dullness, depression, decreased milk production, slight to marked inappetance, extreme thirst, cough, nasolacrimal discharge, and excessive salivation with drooling. Abdominal pain is manifested by kicking at the abdomen and lying down and getting up at frequent intervals. Rumen atony is observed sometimes. Also there is loss of condition, dehydration, weakness, tottering gait, and occasionally recumbency with reluctance or inability to get up. Probably the two most common signs preceding the onset of diarrhea are a nasolacrimal discharge and a dry, harsh, hacking, or moist cough (11).
Diarrhea strikes suddenly, swiftly, and copiously but is of short duration. It often is characterized as explosive or projectile because of the considerable velocity with which intestinal contents are released. Another characteristic feature of this disease is a moderate to severe decrease in milk production. Animals that recently have calved seem to be most affected (11). In individual animals, winter dysentery may last for a few hours but more often it lasts for a few days but less than a week. In an infected herd the duration ranges between 3 and 4 wk, with 2 wk most commonly cited (11).
Whether substantial immunity develops to winter dysentery is not known. MacPherson hypothesized that young calves are not susceptible because of maternal antibody acquired in colostrum or perhaps freedom from stress (34). He felt susceptibility was greater in 1 to 3-yr-old animals when passive maternal antibody protection has disappeared and physiologic stresses are mounting.
The sudden onset of diarrhea in adult cattle merits consideration of several diseases in the differential diagnosis. Among these are BVD-MD, dietetic gastroenteritis, coccidiosis, parasitosis, salmonellosis, Johne's disease, and rinderpest.
No effective vaccine is available for this disease, and it is likely that such will not be available until the precise cause is determined. It may be a distinct disease caused by a single infectious agent or a syndrome.
Many treatments have been advocated, but their merits have been questioned (50). Abundant drinking water should be available for affected animals. Fox has suggested intravenous blood tranfusions for gaunt recumbent animals supported by gruels of high nutritional value given by mouth (21).
Winter dysentery has been known for 50 yr, but because its cause has remained elusive, it is still an enigma needing intensive study.
EPILOGUE
Opinions may vary about the relative economic importance of these diseases. Arguments point to the need for an effective, urgent system of reporting and analyzing morbidity and mortality data in dairy herds so that predictors and estimators can be provided on an immediate feedback, continuous-flow basis. In that way incidence and prevalence can be assessed in the population at risk, predisposing factors hopefully will be made visible, patterns of disease movement can be foretold and monitored, and management more wisely attained.
The technology of microbiology and immunology, if it advances as rapidly in the next quarter century as in the one past, undoubtedly will answer many questions raised in this chronicle. | v3-fos |
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} | s2 | Organization of the Core Lipids of High Density Lipoproteins in the Lactating Bovine *
During lactation, cows develop greatly increased levels of plasma high density lipoproteins (HDL), including a population of particles of larger size than normal human plasma HDL. To study the relationship between lipoprotein size and organization of their core lipids, HDL from lactating Jersey and Holstein cows were fractionated by isopycnic density gradient ultracentrifugation, and individual fractions between densities 1.06 and 1.13 g/ml were examined by differential scanning calorimetry and negative stain electron microscopy. With increasing density, particles decreased in mean diameter from 16.0 to 9.0 nm. Scanning calorimetry of HDL of diameters 12.0 to 16.0 nm showed a reversible thermal transition between 20 and 35”C, resembling the liquid crystalline transitions of cholesterol esters in the lipid extract of bovine HDL. The enthalpy of this transition was markedly reduced compared to pure cholesterol esters or to the cholesterol ester transitions described previously in larger cholesterol ester-rich lipoproteins. Scanning calorimetry of bovine HDL of diameter less than 12.0 nm showed no thermal transitions between 0 and 60°C. However, the disappearance of the low temperature cholesterol ester transition was associated with the appearance at high temperatures (94 to 113°C) of a distinct second component of the lipoprotein denaturation endotherm. This component represented a constant entropy change of cholesterol ester (0.0032 cal/g/”C) which was similar to that of the smectic-liquid transition of pure cholesterol esters. Thus, as HDL particles decrease in diameter from 16.0 to 12.0 nm, their core probably becomes too small to allow melting of liquid crystalline cholesterol esters. The high temperature component of the thermal denaturation of small HDL particles may be due to disordering of cholesterol esters that becomes possible secondary to lipoprotein disruption.
During lactation, cows develop greatly increased levels of plasma high density lipoproteins (HDL), including a population of particles of larger size than normal human plasma HDL. To study the relationship between lipoprotein size and organization of their core lipids, HDL from lactating Jersey and Holstein cows were fractionated by isopycnic density gradient ultracentrifugation, and individual fractions between densities 1.06 and 1.13 g/ml were examined by differential scanning calorimetry and negative stain electron microscopy. With increasing density, particles decreased in mean diameter from 16.0 to 9.0 nm. Scanning calorimetry of HDL of diameters 12.0 to 16.0 nm showed a reversible thermal transition between 20 and 35"C, resembling the liquid crystalline transitions of cholesterol esters in the lipid extract of bovine HDL. The enthalpy of this transition was markedly reduced compared to pure cholesterol esters or to the cholesterol ester transitions described previously in larger cholesterol ester-rich lipoproteins. Scanning calorimetry of bovine HDL of diameter less than 12.0 nm showed no thermal transitions between 0 and 60°C. However, the disappearance of the low temperature cholesterol ester transition was associated with the appearance at high temperatures (94 to 113°C) of a distinct second component of the lipoprotein denaturation endotherm. This component represented a constant entropy change of cholesterol ester (0.0032 cal/g/"C) which was similar to that of the smectic-liquid transition of pure cholesterol esters. Thus, as HDL particles decrease in diameter from 16.0 to 12.0 nm, their core probably becomes too small to allow melting of liquid crystalline cholesterol esters. The high temperature component of the thermal denaturation of small HDL particles may be due to disordering of cholesterol esters that becomes possible secondary to lipoprotein disruption. X-ray scattering and electron microscope studies have shown that plasma lipoproteins are quasispherical particles in which a core of apolar lipid is surrounded by a more polar surface of lipids and apoproteins (1). The plasma low density lipoproteins (density 1.019 to 1.063), the major cholesterol transporting lipoproteins of human plasma, display reversible order-disorder transition of their core cholesterol esters in the * This research was supported by National Institutes of Health Grants HL 22682, HL 18673, HL 07291, HL 07286, and GM 13914 and by a grant-in-aid from the American Heart Association (316-3070-2286, NY Affiliate). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vicinity of body temperature (2)(3)(4)(5)(6)(7)(8). A similar transition of cholesterol esters was observed in HDL,' (15 to 19 nm) isolated from swine fed an atherogenic diet (6, 9). By contrast, intact human HDLz (diameter 10 to 12 nm) do not display any transition of cholesterol esters between 0 and 60°C (10). These observations suggest that between 15 and 12 nm in diameter there is a critical size change that dictates an alteration in the organization of the lipoprotein core lipids.
During lactation, cows develop hypercholesterolemia due to a marked increase in plasma high density lipoproteins (11)(12)(13). The increment in high density lipoproteins occurs mainly in fractions of lesser density (density 1.06 to 1.12 g/ml (11)(12)(13). Such large HDL particles are normally absent from human plasma or present in low concentration (13). Bovine HDL of density 1.06 to 1.12 g/ml have diameters from 12 to 16 n m , intermediate between human HDLz and swine HDL,. They therefore offer the opportunity to test the hypothesis that there is a critical change in the thermal behavior of the lipoprotein core in this size region. We have isolated HDL from lactating cows by density gradient ultracentrifugation and examined the individual fractions by differential scanning calorimetry and x-ray scattering. The results show a markedly perturbed order-disorder transition of cholesterol esters present only in the larger bovine HDL particles. MATERIALS AND METHODS Four nonpregnant lactating cows (1 Jersey and 3 Holsteins) were used for this study. Blood was drawn from the jugular vein into bags containing acid/citrate/dextrose and the plasma was recovered by centrifugation at 4'C. The plasma and all salt solutions used for centrifugation contained Naa.EDTA (0.04%), NaN3 (0.05%), and gentamicin (0.005%). Solution densities were determined using a Mettler/ Paar Densitometer (Graz, Austria).
Lipoproteins were prepared by sequential preparative ultracentrifugation using a Beckman 40.3 rotor a t 39,000 rpm and 16'C. Cellulose nitrate tubes were fded with 6 ml of plasma. The d < 1.006 and the 1.006 to 1.050 g/ml fractions were pipetted from the top 2 ml following separate 18-h periods of centrifugation. The 1.050 to 1.21 g/ml fractions were recovered in the top 1 ml in a similar manner after 24 h of centrifugation. In each case, the infranatants were adjusted to the higher density by the addition of NaCI/NaBr solution according to the methods of Lindgren (14).
For preparation of density gradients, separate 2-ml aliquots of a pool of the 1.050 to 1.21 g/ml fraction were transferred to cellulose nitrate tubes (1.4 X 8.9 cm). These were overlaid with a mixture of 4 ml of the 1.050 to 1.21 g/ml fraction pool and 2 ml of 0.195 M NaCl solution. The tubes were filled with 4.5 ml of a NaCVNaBr overlaying solution ( d = 1.050). Following 40 h of centrifugation at 37,000 rpm and 16'C in a Beckman SW 41 rotor, ten 1.2-ml fractions were I The abbreviations used are: HDL, high density lipoproteins; SDS, sodium dodecyl sulfate; DSC, differential scanning calorimetry; LDL, low density lipoproteins; apo, apolipoprotein. collected from the top of each tube using an ISCO density gradient fractionator (Lincoln, Neb.).
In each fraction, concentrations of total and free cholesterol (151, triglycerides (16) and lipid phosphorus (17) were determined. Protein concentrations were determined by a modified Lowry procedure (18) employing 1% SDS in a Na2CO:, buffer and using bovine albumin as a standard. Polyacrylamide gel electrophoresis was performed in 0.1%
SDS-15% acrylamide by a modification (19) of the procedure of Laemmli (20).
Differential scanning calorimetry was performed on HDL samples using a Perkin-Elmer DSC-2 (21,22). Samples were concentrated by vacuum dialysis prior to DSC experiments, using 0.15 M NaCI, pH 8.0. Seventy-five-microliter aliquots containing 50 to 150 mg of lipoprotein/ml were sealed hermetically in stainless steel pans and examined at heating rates of 5"C/min and cooling rates of 10"C/min. Baseline curvature in these experiments largely reflects differences in radiative heat loss between the sample and reference pans. Polarized light microscopy, x-ray scattering, and negative stain electron microscopy were performed as described previously (4,231.
Plasma HDL obtained from lactating cows (1 Jersey and 3
Holsteins) was fractionated by isopycnic density gradient ultracentrifugation between 1.05 to 1.20 g/ml. Representative compositional data are shown in Table I, for a Jersey and a Holstein. The greatest concentration of HDL was recovered in fractions of 1.069 to 1.083 g/ml. Increasing density of the isolated fractions was accompanied by increased relative content of protein and a decreased content of cholesterol and cholesterol ester (note that percentage compositions are shown in brackets in Table I). Triglyceride represented less than 1 weight % of the lipoprotein mass, whether determined by fluorometric assay (16) or by quantitative thin layer chromatography (24). The bottom fractions of the gradient contained small amounts of phospholipid and proteins. For all of the HDL fractions, the major apoprotein consisted of a single band with electrophoretic mobility in 15% polyacrylamide SDS gels similar to human apoA-I (Fig. l), as reported by Jonas for nonlactating bovine HDL (25). The fast migrating bands seen in the gels presumably represent small amounts of apoC-like peptides or reduced apoA-I1 and were more prominent in the less dense fractions.
Differential scanning calorimetry was performed on each of density fractions 2 to 6 obtained from all four animals. Fraction 1 was not examined because it contained pas well as amigrating lipoproteins and fractions 7 to 10 contained insuf-of Bovine HDL 171 ficient material for calorimetric analysis. The DSC curves obtained from one set of fractions from a lactating Holstein cow are shown in Fig. 2. Lipoproteins from gradient fractions 2 and 3 displayed reversible transitions between 20 and 35°C (Fig. 2, a and 6 ) . There was no similar transition in the intact lipoproteins of greater density (Fig. 2, c to e). On heating to higher temperatures, all fractions showed an irreversible denaturation endotherm. Following thermal denaturation, all lipoproteins displayed reversible liquid crystalline transition of cholesterol esters between about 30 to 45OC as exemplified in Fig. 26 The identity of the 30 to 45OC transition was confmed by examination of the lipid extract of the bovine HDL by DSC and by polarized light microscopy of heatdenatured HDL as described previously (10). These results indicate that the transition between 20 and 35OC observed in the intact HDL samples was probably due to an order-disorder transition of cholesterol esters. However, the enthalpy of this transition (0.1 to 0.6 cal/g of cholesterol ester) was reduced compared to pure cholesterol esters (1 cal/g) (23), low density lipoproteins (0.7 cal/g of cholesterol esters (4)), and HDL, (0.7 to 0.8 cal/g of cholesterol esters (6)).
In Fig. 3 is shown the relationship between lipoprotein diameter and the enthalpy of the order-disorder transition of cholesterol esters. The individual data for the bovine HDL samples and the mean enthalpies for LDL (4) and HDL, ( 6 ) are shown. The data for LDL and HDL, suggest no change in ' CE values were obtained by subtracting UC from TC and dividing the difference by 0.6.
Thermal Studies o f Bovine HDL There is a marked decrease in enthalpy of the order-disorder transition of bovine HDL between about 16 and 12 nm in diameter, and no transition in smaller particles. The sizes of the HDL shown in Fig. 3 are based on negative stain electron micrographs. Diameters of the same fractions determined from quasielastic light scattering measurements were in agreement to within 1.0 nm.* On heating the bovine HDL fractions to higher temperature, there was an irreversible lipoprotein denaturation endotherm (Fig. 2). For fractions which displayed a cholesterol ester transition between 20 and 35"C, the denaturation endotherm consisted of a single peak with a small secondary peak at a slightly higher temperature (Fig. 2, a and b ) . With increasing density of the lipoproteins, the high temperature component of the denaturation endotherm became more clearly separated and increased in size. The fist broad component had a constant peak temperature of 90 zk l.O"C, while the second component had a peak temperature that increased up to 113°C. The smaller HDL also showed a greater enthalpy of denaturation. Expressed in calories/g of lipoprotein, the total denaturation endotherm increased in enthalpy from 0.67 cal/g (fraction 2) to 1.72 cal/g (fraction 6). The enthalpy and entropy changes associated with the two components of denaturation were calculated in terms of each of the lipoprotein S. T. Kunitake, unpublished observations. To investigate the identity of the two peaks of the denaturation endotherm of bovine HDL, paired samples were subjected to incremental heating studies in the calorimeter. One sample was heated directly to 130°C while the other was heated to a series of progressively higher temperatures (Figs. 4 and 5). The release of apoA-I was monitored by the appearance of the endotherm of peak temperature 60°C. The latter transition was present in bovine apoHDL and resembles the reversible unfolding endotherm of human apoA-I, except that it is at a slightly higher temperature (22). The release of cholesterol esters was monitored by the appearance of the cholesterol ester liquid crystalline transition between about 20 and 45°C. In Fig. 4, when fraction 6 was heated directly to 130°C it showed two peaks at 90 and 113°C ( a ) . When a second sample was heated to just below the onset of the 90°C peak ( b ) , it showed no release of cholesterol esters or apoA-I. However, further heating through the 90" peak ( c ) released apoA-I as shown by the peak at 60" ( 4 . No cholesterol esters were released, however, until the sample was heated through the 113°C peak ( d ) . Reheating then clearly showed the cholesterol esters as a double peak at 20 to 45°C ( e ) . The results suggest that the fist component of the denaturation endotherm was associated with release of apoA-I from the HDL particle, while the second component was associated with release of cholesterol esters and lipoprotein disruption. In the larger HDLs, these components are not well separated and some cholesterol ester is released simultaneously with apoA-I (Fig. 5). However, in the smaller HDLs, it appears that 70 to 80% of apoA-I is released with the first component of the denaturation endotherm without any release of cholesterol esters (Fig. 4).
X-ray scattering was performed on the larger HDL (frac- the calorimetrically defined transition (Fig. 2, a and b ) . At 1O"C, the scattering profile showed three well defined fringes resembling the pattern of HDL (1). It was notable that no fringe was present at 1/3.6 nm" as observed in HDL, and LDL (4, 6). On heating bovine HDL to 35"C, there were no major changes in the positions or intensities of the scattering fringes.
DISCUSSION
The present investigation is an extension of previous studies of the structure of the core lipids of plasma lipoproteins (2-8). Low density lipoproteins from a variety of species (2-8) and HDL, from swine (6, 9) display order-disorder transition of their core cholesterol esters in the vicinity of body temperature. This transition has been attributed to a change of cholesterol esters from a smectic-like or layered state to a more disordered state. The evidence is that, 1) the transition is identical in temperature and enthalpy to the smectic-cholesteric transition of cholesterol esters isolated from the same sample, and 2) below the transition there is a maximum at I/ 3.6 nm" in the x-ray scattering profile which disappears above the transition (4). An identical fringe is displayed by smectic liquid crystals of pure cholesterol esters (4). Moreover, Fourier transform analysis of the x-ray scattering profiles of both LDL and HDL, shows an interior electron density maximum -4.5 nm from the surface of the particles which arises from a region of overlapping steroid ring moieties (5-7). Assuming the particles have spherical symmetry, these findings can be most readily explained by a model of LDL and HDL, containing radially oriented layers of cholesterol ester (5.6). Luzzati and co-workers (26) have shown that the x-ray scattering profile of LDL below the transition can be attributed to micellar, rather than layered structures of the core cholesterol esters, provided initial assumptions of nonspherical particle symmetry are made.
In the present study, HDL from the gradient fractions of density 1.06 to 1.09 g/ml displayed reversible transitions between 20 and 35°C (Fig. 2) which probably represent orderdisorder transitions of cholesterol esters. This transition occurred at a similar temperature to the liquid crystalline transitions of the isolated bovine HDL cholesterol esters. Also, in the incremental heating studies, the 20 to 35°C transition in the intact lipoprotein gradually merged with the 30 to 45°C cholesterol ester transition in the heat denatured lipoproteins (Fig. 5). However, in intact HDL the peak temperature of this transition was depressed by about 12°C relative to the smectic-cholesteric transition of released cholesterol esters, and the enthalpy of the transition was reduced compared to pure cholesterol esters, LDL or HDL. In addition, the x-ray scattering profie of these particles did not show the thermotropic changes previously attributed to smectic-disordered transitions of cholesterol esters in LDL and HDL,. Thus, our results suggest that the endotherm occurring at 20 to 35°C in intact HDL was due to a transition of cholesterol esters between states of similar enthalpy, probably representing a minor structural rearrangement.
The thermal denaturation of the smaller bovine HDL particles was accompanied by a characteristic double-peaked endotherm resembling that of human and swine HDL (10,6). Previous studies of human HDL have shown that the fist broad component of the denaturation endotherm is associated with release of apoA-I and the second sharper component with disruption of the particle and release of apoA-I1 and cholesterol esters (10). Since the bovine and the swine HDL (6) lack significant amounts of an apoA-11-like protein, the present results show that the second component of the denaturation does not necessarily reflect a conformational change of apoA-11. In both bovine and human HDL, this component was associated with release of cholesterol esters. The entropy change associated with this transition was constant when expressed in terms of cholesterol esters (0.0032 cal/g/"C). It is notable that the second peak of the denaturation endotherm of human HDL is also associated with an entropy change of -0.003 cal/g/"C (10). Thus, the second endotherm may reflect disordering of cholesterol esters associated with particle disruption. When particles of increasing density are compared, this endotherm appears as the low temperature transition of cholesterol esters disappears (Fig. 2, a to c ) . Thus, in smaller HDL particles there may be an ordered structure involving cholesterol esters which cannot melt at low temperatures because of constraints placed on the core by particle size. Following release of most of the apoA-I, these particles are no longer stable and cholesterol esters display order-disorder transitions in association with particle disruption. Although the investigations of HDL by nuclear magnetic resonance spectroscopy have not shown ordered structure of cholesterol esters (27), it may be that such structures cannot be observed on the NMR timescale, as suggested for the boundary lipid of intrinsic membrane proteins (28).
The lactating cow may represent an exaggerated model of the structure and metabolism of HDL in other species. During lipolysis of triglyceride-transporting lipoproteins, there is transfer of surface material (phospholipids, apoA-I, and apoC) into the HDL fraction in man and the rat (29-33). Studies on the uptake of phospholipids and apoproteins by HDL in oitro show that the incorporation of chylomicron or VLDL surface material into HDL may be associated with a decrease in density of the HDL fraction (34, 35). The massive lipolysis of triglyceride-transporting lipoproteins that occurs during lactation in the cow may cause the marked increase in HDL, particularly in the less dense subfractions. Studies of human HDL subfractions isolated by density gradient ultracentrifugation show that the thermal denaturation pattern changes as a function of density in a similar way to bovine HDL, ie. there is greater thermal stability in the more dense HDL fractions (35). Incorporation of phospholipids into the more dense human HDL subfractions leads to formation of HDL with lower hydrated density and decreased thermal stability (35). The differences in thermal stability of subclasses of bovine HDL may reflect structuural alterations associated with acquisition of surface components of lipolyzed triglyceride-transporting lipoproteins. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Fading with light and greying with age in the fleece of black Australian Polwarth sheep
Summary In order to measure the variation of colour related to some external and internal factors, 136 samples of wools were collected in 1979 and 1980 in a flock of black Polwarth sheep kept in the South of Victoria State, in Australia, 38" latitude South in open air all year long. The genetic formula of these animals at Agouti, Brown and Extension loci was probably aa, B+B+, E+E+. The animals were female or wethers, they were rugged for control or non rugged with a coat opaque to visible and U.V. radiations and cut off at 2.5, 3.5 months or one year of wool growth. The colour measurements were made with a colour atlas based on the D.I.N. System. Except for very rare cases, the growing wool (at the base of the staple) was in the grey scale (which goes from pure white to jet black, without a hue component). With age one could see a greying or silvering of the coloured fleece. This is due to an increase in the number of white fibers and seems independant from light exposure. Due to exposure to solar radiation there was a fading of the staple to the range ot browns. This means the addition of a spectral colour component : the hue, orange in this case. The fading first affects the tip on the staple, about 2 cm after one year of exposure, and is less intense on the average with lighter greys. In a second step the middle staple is affected, after at least 3.5 months. The middle staple fading is less intense but affects a longer length : about 4 cm after one year of exposure. The brown shades after fading overlap the shades genetically induced by mutants at the Brown locus B. There were some rare cases of spontaneous browning from the base of the staple not due to the light of sun but in these animals the hairy parts remained jet black or dark grey. Greying and fading were mainly restricted to the fleece and result probably from physical properties of the wool fibers deriving from their greater fineness compared to the coarse hair of hairy parts.
-Introduction
Although it comprises only a small fraction of the Australian sheep industry the breeding of coloured sheep in Australia is not negligible ; about 50 000 head are bred by specialized producers (CuRTis, 1979 ;L AUVERGNE , 1980). The coloured wool is marketed primarily to those interested in handicrafts (spinning, knitting and weaving) and is desirable because it produces naturally coloured yarns which need no dye treatment and because the colours obtained with the natural wool provide a wide variation of shades which can hardly be matched by artificial means.
The wide range of shades among the black pigmented animals which constitue the majority of the coloured population is given primarily by two phenomena : greying and fading.
Greying is an intermixing of white fibers among the coloured ones. Also called silvering the greying may be seen in many breeds where it is congenital or developping with age resulting sometimes in a nearly white fleece in the adult, for example in Karakul breed, see SERRA (1948). This is due in particular by senescence of the melanocyte system, a widely spread behaviour of mammalian pigmentary systems well studied in Man (see F ITZPATRICK et al., 1964).
Fading is the discolouration of coloured fibers, from black towards brown, the shades progressively becoming lighter with time. This phenomenon is well known among producers of naturally coloured wool (cf. SERRA, 1965 ;N APIER , 1976;M ELDRUM , 1979;C URTIS , 1979). According biochemical experiments the melanin is decolourized by oxydizing agent (remember for example the peroxide blond obtained after H 2 0' 2 applications, NicoLAus, 1968) and one knows that U.V. of solar radiation are oxydizing agents (see also S PEARMAN , 1977).
The papers by SERRA (1965) in Portugal and B ROOKER and DO LLING (1965) in Australia were the first attempts to measure these color variations with a color scale. Since, in Australia, the widespread use of rugging the sheep during the last few years provides new experimental facilities to measure fading and greying as the sheepcoat, originally developed to reduce fleece damage due to weathering and contamination from burrs, sand, etc. (L IPSON et al., 1970;A BBOTT , 1978), does seem to prevent bleaching of pigmented wool (Anonymous, 1979 ;D ENNIS , 1979) hence furnishing control data. The present article is an attempt to use this type of data for a measurement of fading and greying in naturally coloured wool using a color chart.
II. -Material and methods
A. -The data 1. The flock and the breed The variations in coloured wool were measured in three trials on black coloured Polwarth sheep of the Tarndwarncoort flock belonging to one of us (Wendy D ENN I S at Warncoort, Victoria).
The white Polwarth breed was established one hundred years ago in Victoria on three properties (among them Tarndwarncoort) by crossing the Lincoln with the fine Saxon Merino and then backcrossing to the Merino and interbreeding the resultant progeny. The wool quality number is 23 E!, on the average, the fleeces weigh an average of 5 kg (greasy), the staple length ranges from 10 to 14 cm, with an average of 12 cm (J EFFRIES et al., 1979 ; see also Polwarth Centenary Souvenir Publication, 1980).
The trials
In three trials a portion of the sheep were « rugged with a sheepcoat (G OLLIN and Company, Ltd. Austral. pat. No. 428078) made of woven pigmented polyethylene which is an adequate filter of both visible and ultra-violet radiation, according to A BBOTT (1980) (personal communication) ; see also the leaflet by G OLLIN and co (1978).
The coloured animals were black wethers or ewes. Their genetic formula in resp. Agouti, Brown, and Extension loci was probably aa, B+B+, E+E+ as all of them had a jet black coat (9-1 or 10-0 in our grey scale, see fig. 1) in the hairy parts. Some with the HST white pattern (head, stockings, tail) did certainly carry the gene s (A D p LSTE irrssorr's, 1974 terminology) in the Spotting locus S.
In the trials, rugged and non-rugged animals were co-grazed on pasture having some shelter and shade.
The farm of Tarndwarncoort is approximately 100 km South West (40 km due South) or Laveston, the closest weather station in Victoria, which lies at approximately 38° latitude South. Sunshine data obtained from the Laveston Station (B.F. DANIEL, Bureau of Meteorology, Melbourne ; personal communication) indicate that the long term monthly averages in hours of sunshine in the test area range from a low of 133.3 in May to a high of 258.5 in January. The actual hours recorded for the test period were very close to the long term averages.
Other areas which lie at a similar latitude (North) are Sicily ; Tunisia ; Richmond, Virginia; Saint-Louis, Missouri; Pueblo, Colorado and Sacramento, California.
Trial. I : Twenty purebred coloured Polwarth sheep (ewes and wethers) ranging in age from one to eight years were « rugged » on 8 January 1979. All animals had been shorn previously on October 20, 1978. Fifteen animals, shorn at the same time, were left unrugged as controls. Wool samples were taken from all animals from the midback at the next shearing on 22 October 1979 : groups 1 and 2 of table 1.
Trial II : Thirty-two purebred, coloured Polwarth sheep (ewes and wethers) ranging in age from one to eight years were rugged immediately « off-shear p on 22 October 1979. Six similar animals remained unrugged as controls. Wool samples were taken from the midback and midneck of each animal on 6 February 1980 : 3,5 months later. They gave the groups 3, 4 and 4 bis of table 1.
Trial III : At the shearing of october 22, 1979, fourteen wether Polwarths were left unrugged and 25 others were rugged. Wool samples were taken from the midback of each animal on October 28, 1980, after one year of growth giving the groups 5 and 6 of table 1.
B. -The treatment of the wool samples Wool samples from the three trials were gently washed with mild wool detergent and luke warm water then rinsed and air dried. When completely dry, they were evaluated for colour by the same person (J.J. L AUVERGNE ) between 10 am and 2 pm near a shaded window in natural light at the latitude of Paris (approximatively 49°N orth) in December 1979 and November 1980 for resp. the samples from first and third trial or under similar light conditions in Warncoort, Victoria, in February 1980, for samples from the second trial.
C. -The scale of colour Colour measurements were made using a MÜ LLER color Atlas (see L AUVER - GNE , 1966). Basically with this systemwhich is derived from the german DIN systemeach colour is defined in a three dimensional scheme using variables defined in Ntcxettsorr (1958) (see also D!RIB!R!, 1964) : 1) Hue (« teinte » in French) which is that attribute of colours which derive from the spectra and are classed as reds yellows blues, etc. There are 60 hue classifications in the Mu LLER Atlas. Wool generally falls into the range of 7 through 12 which is the yellow/red range.
2) Value or lightness (« valeur » in French) which gives more and more dark colour : from 0 to 10 in the MÜ LLER ' S Scale.
3) Saturation of chroma (« saturation » in French) which adds more and more brightness : from 0 to 9 in the MÜ LLER ' S Scale.
In the MÜ LLER Atlas a shade is quoted by a triple number (hue, from 1 to 60, value from 0 to 10 and saturation from 0 to 9) unless if it is a colour without a hue which is simply in the grey scale (between 0-10 : full white and 10-0 : jet black) and quoted only with two numbers, see in fig This plotting of value against saturation is similar to the quality/intensity plotting already used by WRIGHT (see for example WRIGHT, 1974) in order to measure the colour of various genotypes in the Guinea Pig. Staples with multiple banding from region to region across the animal which do exist in Australian flocks and even sometimes in the Tarndwardcoort flock have not been found among our samples.
The staples of our trials were monocoloured, sometimes bicoloured, more rarely tricoloured, with usually a clearcut partition of colour.
The frequency of various types of pattern : no fading or greying, fading of the tip with or without fading of the middle, fading of the superior half of the staple, greying at the basis of the staple is presented in table 2.
One must note that (lines 5 and 7) greying may occur in a group in the same time as fading.
B. -Details on fading
The scale of browns is tridimentional but in our experiment we had practically only one hue, 8 (an orange). An approximative linear scale has been then set up from the darkest to the lightest shades and is used in table 3 and 4 in order to describe the degree of fading according the shade of the wool growing which is always in the linear grey scale, as seen above.
In table 3 which presents data of tip fading 3 types of treatments are distinguished : 2.5 months, 3.5 months and one year exposure : groups 1, 2, 3, 4 bis and 5.
In table 4 one has pooled middle staple fading in groups 1 and 5 which both were exposed during one full year.
-The fading process in these groups is illustrated in figure 2.
C. -Details on greying The evolution of greying with age is presented in two tables : no. 5 and 6.
In table 5 are pooled groups 1 and 2 : females and wethers cut in 79 and whose ages are given by the year of birth.
In table 6 are pooled groups 3 and 4 of wethers and females cut in Feb. 1980 and whose age is measured in years.
The greying process is illustrated by fig. 3 where the linear scale of grey has been considered from 10 for 10-0 (jet black) to 0 for 0-10 (full white).
IV. -Discussion
A. -The fading process If fading is due to exposure to solar radiation then the coated fleeces must not. be faded at all as we know the rugs are opaque to visible light and to U.V. This is the case for samples 4 and 6 [see table 2 (1)].
(1) With the exception of a small percentage in sample 6, see further on for an explanation.
Moreover this discoloration must be proportional with the time of exposure. This is also the case for sample 3 and 4 bis (3.5 months of exposure) which are less faded than sample 4 and 5 (1 year of exposure) (see figure 2). The fading process involves first the tip of the staple as seen above. It seems proportional in intensity with the degree of greyness of the growing wool (see table 3) but, in light grey wool sometimes, this is not the case.
After the tip, the middle of the staple is reached by the process but in a less intense way (table 4) and after a duration which excesses 3.5 months, as we do not see any fading midstaples in our 3.5 months samples (table 2 lines 2 and 3). As we have not sampled between 3.5 months and one year the commencement of this middle staple fading cannot be determined precisely.
The other characteristic of this middle staple fading is to affect a greater length than the tip fading : about twice the length, see table 2, lines 4 and 5.
There is no precise description of this two step of fading in the earlier literature. In the paper by SERRA (1965), this author has unfortunately mixed measurement of fading and greying and proposed a scale which includes full staples. This, after the various association of shades at the base, middle and tip of the staple makes its use quite difficult. Moreover no reference to a physical measurement of colour is given. Even with these defects the S ERRA 's article must nevertheless be considered as the pioneer work in the field as the pointed out the necessity of using colour charts.
In the article by B ROOKER & D OLLING (1965), the Munsell soil color chart was used and the authors said : « To date no adult sheep has been observed with brown wool growing from the skin. Brown wool found at the distal end of a pigmented staple has invariably been associated with light grey to black wool at the proximal end. » This fits our present observations well. Nevertheless, these authors said further on that brown fibres have been observed in the birth-coat of black lambs, a rare feature which has been confirmed in checking lambs at Tarndwarncoort and which could explain the « fading p of the 3 cases of table 2 line 7 among one year wool, coated animals which, according our views, must not fade : the brown upper zone could be a congenital brown which has disappeared at with age. When there is a light grey to black wool at the proximal end of the staple there is no possible confusion but there still is no explanations for the brown lamb coat described above or the brown adult fleeces of jet black headed animals which have also been seen and measured by some authors of this article (D OLLING & L AUVE RGNE, unpublished data). The question is still open but already it appears that the checking of colour of hairy parts of the body must be done carefully, in order to distinguish genetically true brown sheep from genetically black sheep whose fleece has faded to brown.
As one can see in table 2 (lines 4 and 5 in particular) there are variations in the fading ability of fleeces since about 30 p. 100 of the animals had no intermediary fading zone in their back staple. This indicates there is probably a genetic component for fading.
In addition, one of us (M.J. B URRILL ) in U.S.A. (but it is of common observation in France also) has had communications with breeders of black animals whose jet back fleeces do not fade, a feature which, in our data, is observed only among grey fleeces.
It was also of common observation in our samples that pratically no fading was seen among hairy zones such as the head, even when fleece was much faded. This may be due to the fineness of the wool fiber which, having a thiner screen of keratin offers less protection against oxydising solar radiation.
Even if fading is apparently rather rare in coarse hairy coat of mammals (in fact we have not found any article devoted to that point) some unpublished observations on the goat (specially in the long hairy strains such as the French Poitevine or Corsican Goat) show that some tip fading may be observed.
B. -The greying process
The greying in Polwarth is obviously an endogenous developing process with age as one can seen in fig. 3.
The process seems to be a progressive one but, in some cases, it is quite variable as one can see some staples with a dark grey tip and a light grey basc : see table 2, lines 5 and 7.
The already quoted Portuguese author SERRA (1948) summarized the knowledge on greying known up to that time. In some breeds such as the Karakul the depigmentation with age is quite constant but with variable expressivity, according the strains. This author suggests the possible influence of fineness of wool. A list of possible mendelian factors is given but these must be considered in light of the knowledge of that time.
Since S ERRA 's review, A DALSTEINSSON (1970) has shown that at least one mendelian factor, the A! (grey) allele at the Agouti locus, may play a major role in greying with age. This factor or a close allele may be responsible for the special colour of Gotland sheep pelt (A DALSTEINSSON et al., 1978).
In our case it is difficult to give a conclusion for the Polivarth. The greying tendency with age is noticeable (see fig. 3) but there is great individual variation : see table 5 line 8 or table 6 line 8, for example. A polygenic inheritance could explain these variations in a coloured strain where no selection for grey has yet been done. The genes could combine their action to the general process of greying with age linked with the senescence and known in many species, specially Man (SrEnRNtnN, 1977). The wool greying quicker than the hair as generally observed in our data where the hairy faces remained black or dark grey may be due to the fineness of the wool fiber where the inclusion of melanosomes were more difficult than in much coarser hair, a point already faced by S EKRA (see above).
C. -Some practical considerations The tip and middle staple fading is responsible for the final shade of yarn spun with uncoated black wool which is never jet black but « frieze » (« bure » or « burel » in French). As a matter of fact, the darkest shade of coloured yarn produced in spinning mills in France (La Noisette, Auzances, Creuse) as well as in Australia (SACSOS) are in the brown scale (12-8-0 and 8-8-0) and not in the grey one (hueless).
By using sheep rugs the coloured wool producers are able to furnish customers with fleeces in the black-grey range of colour.
Coating, a practice already utilized by some coloured wool producers, after the CSRIO trials (see A BBOTT , 1978) is an advantage for colour control. Our data confirm another already recognized advantage by A BBOTT : that of the length of the staple (see in table 2 One must keep in mind that these data come from a commercial flock with the simplest sampling procedures. This explains why some facets have been only briefly studied.
A comprehensive study of greying with age, following animals year after year and checking the variation in different strains need to be established.
For fading, more data needs to be collected between 3.5 months of exposure and the time when the middle staple fading starts.
For greying, as well as for fading, one needs extended topographical studies, the mid dorsal sampling (with some neck samples) being only a partial description of the phenomenon.
More data also is needed for a comparison between males and females with detailed information obtained in the first period of the life of the animal.
In addition some further research on heritability of fading in all black flocks could be done in order to precise the part of genetical control on fading of melanins.
Of course, the present experiment, which is restricted to the Polwarth breed. needs to be extended to the other coloured breeds.
V. -Conclusions
The study presents several weaknesses but describes already the way fading occurs in black pigmented fleeces submitted to natural light : first the tip, then, after at least 3.5 months, the middle staple. For greying the age tendency is clearly demonstrated.
Received for publication in may 1981.
Acknowledgements
Thanks are due to Dr A BBOTT (C.S.I.R.O., Division of Textile Industry, Geelong, Vic.) and Dr DANIEL (Bureau of Meteorology, Melbourne) for having provided indispensible details upon rugging and meteorology. | v3-fos |
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