The Canadian Animal Germplasm Technical Experts Board has prepared discussion papers to provide a rational approach for decision-making on major issues vis-à-vis farm animal genetic resources in Canada. These discussion papers are expected to evolve as technical advances emerge and as changes occur in such areas as intellectual property and disease control. Additional discussion papers will be prepared as the Canadian Animal Germplasm Technical Experts Board or the Steering Committee decides. These discussion papers and improved knowledge of farm animal genetic resources in Canada should greatly assist decision-making to support activities on conservation and use of animal genetic resources.
The scope of efforts for conservation of farm animal genetic resources is broad. It includes all farm animal species used for production of meat, milk, eggs, traction and fibre. Research and its application to advanced technologies for conservation, evaluation and use of genetic resources are part of the mandate as is the support of conservation of live animals and genetic resources in gene banks. It is beyond the scope of the Canadian Animal Germplasm Technical Experts Board to consider direct action on wild species. However, it will consult with other agencies on efforts for conservation of wild species that are "farmed" to assure there is adequate effort to support a sustainable genetic base.
Canada is one of many countries that is undertaking the conservation of farm animal genetic resources. Some will be working closely with the Food and Agriculture Organization (FAO) of the United Nations or their efforts may be sponsored by other countries through the FAO.
Canada is not the original home of any of the major farm animal species with the exception of turkeys. Many of the breeds important in Canada had their origin in other countries, mainly the United Kingdom, France, The Netherlands and other European countries.
The breeds that originated elsewhere have undergone profound changes. This applies in particular to those that have been in Canada for many generations. Canadian strains of common breeds are not necessarily identical to the same breeds represented in other countries. The closeness of relationships and the conservancy efforts of other countries are important considerations for setting priorities for Canadian efforts.
There are several unique populations of animals in Canada. These have arisen by natural selection, as in the case of the Sable Island horses, and by carefully controlled research protocols for specialized strains or lines. This breadth of diversity and its utility to meet breeding and research needs must be considered in setting priorities.
The Canadian Animal Germplasm Technical Experts Board puts forward the following twelve points as criteria to consider for establishing general priorities:
In conservation of genetic resources, a choice is invariably necessary because of the large number of identifiable populations and the limited resources available. Therefore criteria need to be applied to help in assessing the risk of losing specific populations. Two sources can be cited to help in establishing these criteria. As part of a paper presented to the FAO Expert Consultation, Maijala (1992) lists some factors that may be useful in determining which populations are most at risk. The other source is the Recommendations of the FAO Expert Consultation (1992). These publications developed sets of guidelines based on population size, effective population size and special considerations. The criteria are based on measurable data and as such can provide a defensible basis for assessing risk of loss of a population or of significant samples of the population genetic diversity.
1. The size of the population is of primordial importance in determining if a population is at risk because it determines the rate of inbreeding (defined as F) which is a measure of the loss of genetic variability. However, the actual population size (N, for the number of individuals) is less valuable than is the effective population size (Ne, for the effective number of individuals) because the rate of inbreeding is affected by the sex ratio, by changes in population size between generations and by the family size. Inbreeding is calculated as F= 1/2Ne per generation, which suggests that with Ne=50, genetic variability will be lost at the rate of one percent per generation.
With random matings, equal numbers of males and females, and a population that maintains a constant size between generations, the effective population size is approximately numerically equal to the actual size: N=Ne .
When sex ratios are skewed (in our domestic animals there are normally more females than males) Ne is reduced and can be calculated approximately as follows:
where Nm is the number of males and Nf is the number of females. A large discrepancy between the number of males and females results in a large reduction in effective population size. The sex with the least number of individuals is of greater relative importance in reducing Ne.
Variation in population size between generations can be adjusted for by calculating the harmonic mean of the population size per generation using the formula:
where t is the number of generations, and N1 to Nt are the population sizes in generations 1 to t. The smallest generations are most important in decreasing the effective population size.
Based on these mathematical formulae, it can be seen that it is important in a small population to attempt to equalize the number of females and males and to avoid generations with very few individuals.
This discussion assumes that the number of individuals in each family is random. If this is true and the population size is constant, family size follows a Poisson distribution with a mean and a variance of two. Effective population size can be increased if family size is controlled. At the extreme, if all families are composed of one male and one female, the variance for family size is zero and the effective population size is approximately doubled: Ne=2N .
This is clearly an efficient way of reducing the rate of inbreeding with limited resources.
2. In addition to the effective population size, other factors must be considered when assessing risk.
Recent trends in the population size. A breed or population group which is popular and increasing in size is in much less danger than one which is decreasing in size. This point could also include the danger to a given population posed by cross breeding programs with a different breed. A population is classified as endangered in Germany, according to Maijala (1992), if more than 10 percent of the matings are performed with males of another breed.
Number of herds. When individuals of a population are kept in only a few herds or flocks, the population is in danger from disease, fire, accident, or dispersal. Again, according to Maijala (1992), in Germany the existence of a breed in fewer than 10 herds is considered to place a population at risk. This point needs to be assessed in each specific situation according to local conditions.
The existence of cryopreserved material. If genetic material from a population is in storage, the risk to the population is alleviated to a certain extent according to the number of individuals stored and the security of the storage facilities. Stored individuals can be considered in the calculation of Ne. Ideally, material should be stored in more than one location to reduce risk of loss.
Different species. The reproductive rate should be considered in determining risk since species or breeding groups with a low reproductive rate will be more difficult to rescue. There are also species or population differences in risk of disease, in longevity and in the production system which is used (intensive, extensive or nomadic). All of these can be associated with the risk of loss.
Different global regions. Geographic isolation of the population or its concentration in regions that are politically, economically, or environmentally unstable may place a population at greater risk.
Outstanding traits or special environments. A population that carries a known outstanding trait should be given some priority. These may be specific genes with potential value, significant social value or adaptation to a specific environment.
3. Based on criteria cited, Maijala (1992) and the FAO Expert Consultation (1992) have established classifications for populations at risk.
RISK Ne Nf Nf, Special STATUS (Maijala,) (FAO, 1992) Consideration CATEGORY 1992) (FAO, 1992) ------------------------------------------------------------- CRITICAL <100 <1000 ------------------------------------------------------------- ENDANGERED <50 100-1000 1000-5000 ------------------------------------------------------------- VULNERABLE 50-100 1000-5000 5000-10000 ------------------------------------------------------------- RARE 100-200 5000-10000 10000 ------------------------------------------------------------- MONITOR 10000Maijala (1992) based his classification on the effective population size (Ne) and the FAO Expert Consultation used the number of breeding females (Nf). Although Maijala's estimates appear to be much smaller than the FAO's, Ne and Nf are very different units of measure and the differences are much smaller than it would appear. As well, the FAO's "critical" category may be considered to be Maijala's "endangered" category. Ne gives a relatively precise estimate while Nf gives an estimate that is very easy to understand and apply. Maijala (1992) mentions the importance of an assessment of the degree of threat and the priority for conservation by local experts.
Cryopreservation is a system of conservation that can provide opportunity to sample the broad genetic base of a population and retain that genetic material indefinitely. Present technology limits the utility of cryopreservation of semen from some species. Embryos of some species can be readily cryopreserved, but other species, e.g, swine, are not, at present, prime candidates for successful embryonic cryopreservation. DNA can be readily frozen, but its usefulness is currently limited to research studies on gene mapping and location of quantitative trait loci. A relatively new area of great potential is the cryopreservation of embryonic stem cells or germ cells which can be grown in cell culture in large numbers and potentially recovered as the whole organism. Biological materials of domestic animal species and their suitability for cryopreservation are tabulated on the following page.
Animal health considerations are extremely important for cryopreservation. Most commercial cryopreservation units must maintain extreme care of animal health and exposure to disease organisms if they are to protect, and compete in, domestic and international markets. The Animal Health Division, Agriculture and Agri-Food Canada administers regulations that govern the safe collection and storage of semen and embryos.
Collection and storage of gametes and embryos can be expensive because of the need to assure the donors are free from specific diseases and their pathogens. On-farm collection of semen is not permitted because of lack of control of disease causing organisms. On-farm collection of embryos, which can then be processed to rid the surface of potential pathogens, is probably ultimately the least expensive process for collection of genetic resources for cryopreservation of rare and expensive animals without putting them at significantly greater risk.
Sources of material for cryopreservation depend on the specific aims of the gene bank owners. Commercial artificial breeding organizations may wish to set aside a portion of their storage for long term cryopreservation of semen or embryos that are no longer of commercial value. The surplus genetic material occurs frequently as organizations review the demand for existing sires, review the proofs of young sires, and determine which will be marketable and which will be discarded. There is an opportunity here for establishment of a background broad genetic base as cryopreserved semen from these sires.
Research establishments may have specimens of specially selected lines of breeding stock which would be costly to reproduce. They could be cryopreserved and form an additional source of specific genetic material for future use.
Conservancy groups may wish to establish a back-up of cryopreserved material in case there is loss of live animals due to disease or insufficient resources to maintain the entire population.
BIOLOGICAL SPECIES MATERIALS --------------------------------------------------------------------------------------------------------------------------------------------- CATTLE SHEEP SWINE GOATS HORSES POULTRY -------------------------------------------------------------------------------------------------------------------------------------------------------- SEMEN Well developed Technology Technology cf. sheep Technology Cryopreserved technology. Some developed but is approaching usable for poultry semen sire's semen may less efficient commercial conservation has low fertility not survive than cattle viability but lower (Lake, 1979) but (Coulter, 1992). (Fiser et al., 1987) (Fiser et al., 1993) fertility than could be useful as cattle. a backup for conservation (Fiser, personal communication). OVA Ova are large cells cf. cattle cf. cattle cf. cattle cf. cattle cf. cattle and as such, are extremely difficult to cryopreserve (Fiser, personal communication). EMBRYOS Well developed Commercially No suitable Embryos Embryos can Embryo technology in viable technology can be be cryopreservation commercial use. technology (Niemann, successfully successfully is not yet used in 1991). frozen frozen applicable conservation (Niemann, (Niemann, to poultry. (Robic et al., 1992) 1991). 1991). TISSUES Stem cells can be - cf. cattle - - Culture of cryopreserved and blastodermal cells provide 90 % and their viability on whole organisms thawing is now feasible (Teale et al., (Etches and 1994). Gibbins, 1994). Cryopreservation of cells is now possible (Reedy et al., 1994). DNA DNA freezes cf. cattle cf. cattle cf. cattle cf. cattle cf. cattle readily and retains its stucture. Methods to use it need long term research.
Cryopreserved material should be maintained in more than one location. If the risk of loss of cryopreserved material is 1:100 in any one facility, then having two independent facilities changes the risk of loss of all that genetic material to 1:10 000. Similarly, having samples duplicated in separate storage devices at each location decreases the risk for each location.
In summary, the following are principles that should form a sound basis for a gene bank strategy:
Canada has an outstanding record of animal health and disease control. This situation has resulted from many years of strenuous efforts by Animal Health officials with the cooperation from the animal industries to prevent entry and eradicate in Canada many diseases common in other countries. The future for Canada's export of genetic material, as breeding stock or as embryos or gametes, depends upon the maintenance of its animal health status which currently allows exports from Canada to almost every country in the world.
Canada has the ability to act as a reservoir and source of important genetic material, acceptable from the standpoint of animal health to other countries, with resultant economic benefit to Canadian animal producers. A strong conservation program for farm animal genetic resources represents an economic advantage to the country.
Disease concerns, on the other hand, potentially limit the ease of importing animal genetic resources to broaden the genetic base of Canada's farm animal populations. Except from a very limited number of countries, importation of genetic resources is either extremely complex and costly or not permitted. It is thus essential that the existing diversity be retained since the cost and health risk of replacing existing genetic resources is excessive.
Ongoing research and advances in technology will undoubtedly influence disease control strategies applied to animal semen and embryos. Specific inquires concerning animal health issues may be directed to the Animal Health Division, Agriculture and Agri-Food Canada.
Some of the principles for consideration in disease control for conservation include:
1. Importation of all animal genetic resources, whether live animals, gametes or embryos must be approved by the Animal Health Division, Agriculture and Agri-Food Canada. Imports from some countries are prohibited because of the risk of disease importation which would be disastrous for the Canadian animal industry.
In all instances, live animals must undergo prescribed tests and quarantine either before or after arrival in Canada, or both. Movement of animals between Canada and United States is easiest and regulations of the two countries tend to be compatible because of frequent review and discussion amongst animal health officials in the two countries.
Intact embryos, subjected to appropriate treatment to remove disease organisms, are considered the safest means of importing genetic material. New guidelines for handling micro-manipulated (sexed or split but excluding embryos derived by nuclear transfer) embryos and embryos derived by in vitro fertilization techniques have also been developed for international movement.
Semen is much more of a problem from the disease transmission standpoint because the sperm lack a protective coating and thus cannot undergo the rather stringent treatments applied to embryos.
2. Export of animals must be cleared by the Animal Health Division which certifies that the animals meet the health standards of the importing country. Exceptions apply for animals shipped to the United States for immediate slaughter.
Some countries, concerned over the health of their farm animals may not allow imports from Canada for a specific disease situation. Australia has recently limited some imports on animal health basis. Some countries may try to use animal health concerns as non-tariff trade barriers although this may be mitigated by the provisions under the General Agreement on Tariffs and Trade.
3. Semen collection, testing, storage and import/export is under the federal Health of Animals Act. Semen from certain species can be collected only on Animal Health "approved" premises. Such artificial insemination (AI) units are concerned to assure that the health status of their stud is not impaired by the collection of semen from animals from other sources. AI units are very strict in their controls of disease and are extremely adverse to the risk of introducing a disease to their premises and animals. Health of Animals Regulations require 30 days isolation on-farm for sires destined for semen collection, followed by testing and 30 days quarantine at the stud prior to collection of semen. This poses problems for collection and storage of semen in gene banks. The costs involved to meet health regulations must be borne and there is no guarantee that the sire will meet health or semen quality standards. Owners of approved establishments have to be reassured that the collection will not cause undue risk to their premises and animals.
Similarly, owners of rare genetic resources are concerned over the risk of moving animals to a centre for collection. Consideration is being given by Agriculture and Agri-Food Canada to on-farm collection of semen for restricted or defined use. This could have application to conservation of animal genetic resources but needs to be reviewed carefully.
Conservation of semen in non-approved premises could result in risks of disease transmission with potential litigation if, for example, a valuable genetic resource was destroyed by disease transmitted through improperly collected and tested semen. The possibility of setting up a system of testing and isolation of semen from on-farm collections in gene banks may be feasible.
A separate conservancy gene bank could be established for semen from those rare specimens that cannot be readily collected in existing approved premises. An example of an appropriate location is Agriculture and Agri-Food Canada's Greenbelt Research Farm. With its proximity to Animal Health officials for advice and direction on testing and quarantine, this site could serve an essential function. The conservancy gene bank would handle only rare breeds and special lines of animals bred at research establishments and use the latest technology of cryopreservation.
4. Embryos are considered the most effective way of conserving animal genetic resources free of pathogenic organisms. There is little regulation except that the procedures for treatment of embryos to remove pathogens are to be followed. It may be relatively less costly and risky to super-ovulate animals, collect embryos and cryopr5. eserve them than to concentrate on traditional semen stores. This technology would apply mainly to cattle, goats and sheep at present but could also apply to other species as the technology is developed.
5.Ova have similar qualities to embryos. There is little international trade but research on collecting ova and using in vitro fertilization is underway in a number of locations in Canada. The potential to collect the ovaries at slaughter from rare breed females that have finished their useful life could be a potential means of expanding the gene bank. The application of appropriate laboratory procedures should reduce animal health risk to a minimum.
6. DNA cryopreservation is feasible but at present is of limited value as a means of long term storage. Laboratory procedures can produce DNA free of external viral or bacterial DNA. The ability to use such cryopreserved material will depend on the research currently underway to understand the genome and identify genes and their location on chromosomes (gene mapping). Ultimately, identified genes may be obtained from cryopreserved animals and incorporated into an embryo or ovum and the important traits recovered.
7. The identification of heritable genetic defects from potential donors to a gene bank poses an additional consideration, requiring a transparent process be developed. Technical advances in the diagnostic capability to detect genetic diseases also provides the potential that new disorders will be identified in the future.
Ownership of conserved genetic resources is an issue that has caused severe problems for international conservation efforts on plants. In particular, less developed countries feel short-changed by past events. They perceive that their resources, indigenous plants, are taken by developing countries and incorporated into new cultivars which then cost the country of genetic origin dearly if they wish to use the technology to increase their food supply.
The movement of live animals between countries is carefully controlled for animal health reasons and the surreptitious movement of animals from less developed to highly developed countries is unlikely. The costs and health risks of importing animals from many parts of the world are prohibitive. There seems little reason to anticipate problems from other countries over ownership stemming from importation of live animals.
In the future, it will be possible to identify, locate and transfer specific genes, for example, a gene that codes for resistance to a specific disease. A source of appropriate DNA, such as a blood or semen sample will be the only requirement. If the source of the gene was obtained for other reasons, such as a blood sample for testing health status, the originating country may well have legitimate claims for compensation, not unlike the situation in plant species.
For the present, the concerns around conservation need to relate to domestic conservation and ownership. New technologies will develop opportunities for use of conserved animal genetic resources that will have implications for domestic and international trade. It is preferable to confront potential problems now and start a dialogue on ownership, access and use to avoid future conflicts.
1. Live animal conservation.
Joywind Farms Rare Breeds Conservancy Inc. has a system stipulating its ownership of the original animals placed on an individual farmer's premises. Contracts stipulate that the increase in numbers are jointly owned by the Rare Breeds Conservancy Inc. and the individual farmer (Chiperzak, personal communication). Other, independent breeders, own breeds that are important to be conserved and have complete control over their own operations. They may be part of a breed organization and cooperate with others. Many try to have surplus animals for sale as a source of income to defray their costs. These systems need various supports such as tracking genetic parentage, managing resources and reducing risk of disease losses.
The Joywind Farm Rare Breeds Conservancy Inc.'s system seems ideal from the standpoint of protecting rare breeds since any animal placed with farmers who are no longer able to care for them can be recovered and placed with another farmer.
It appears well beyond the capability of governments to take ownership or part ownership of animals or to set up a continuing program to provide funds for routine maintenance of animals except under some very specific circumstances and for a very short period of time. Live animal conservancy with concomitant ownership is preferably left in the hands of the private sector.
2. Cryopreserved genetic resources.
This area is potentially more contentious. Costs of recovering genetic material, testing it for viability and freedom from disease, and long term storage and inventory control may not be recoverable through sale of semen, ova or embryos. Some extremely valuable and irreplaceable genetic material will be held in storage. A clear, readily understood contract needs to be developed so that rights and obligations are clear and non-controversial.
Usually, once a "sale" of semen, embryos or ova is complete, the new owner can use the resulting progeny for breeding and have no further obligations back to the original owner. The type of contractual arrangement to resolve ownership and use of such genetic resources is not likely to be contentious and will reflect present industry practices.
The ownership of DNA, either a complete genome or specific gene sequences will need to be resolved. Created, "artificial" genes and "discovered" genes may carry an intellectual property control. This may apply, not only on the original use, but on any subsequent use. This is an additional complication that could also apply to transgenic animals which may also carry an intellectual property control.
The ownership of existing banks of genetic material, such as a blood testing laboratory's samples, is also a subject for resolution. Samples provided for confirmation of parentage are a valuable gene bank, but the original owners presumably have not provided authorization for use of their genetic resources by third parties.
It is anticipated that other forms of genetic resources storage, such as cell cultures may be important for future conservation efforts, particularly for poultry. Poultry semen has limited ability to withstand current cryopreservation techniques and poultry ova are considered completely impossible to cryopreserve. Germ-cell lines of poultry could be a means of setting up a cryopreserved gene bank. The technology in this area is advancing and the potential exists to have such stored germ-cell lines transferred to developing embryos to produce living poultry. This new area would appear to have the same ownership questions as surround other single- and multiple-cell stored genetic resources except if the process itself is also under some form of intellectual property protection.
An associated area of concern is the right to protection of information. Commercial organizations may wish for protection of their future research and resulting business to limit access to information on what they have stored in gene banks. As well such organizations may not provide information to a Canadian inventory on farm animal genetic resources unless their data is kept commercially confidential.
It is important to have a complete inventory of Canadian farm animal genetic resources so that the appropriate steps can be taken to assure diversity is protected that will provide the base for sustainable development. It is essential that agreements be developed with commercial organizations that will give them control over the use of their data.
4. Some potential guidelines: