Papers presented at the Expert Consultation on FAO Programmes for the Preservation of Animal Genetic Resources (continue)

PROGRAMME AND FINANCE ASPECTS OF FAO WORKPLAN FOR ANIMAL GENETIC RESOURCES

Animal Production Service

Animal Production and Health Division ¹

¹ FAO, Rome

The document on the Preservation of Animal Genetic Resources presented to the Committee on Agriculture (COAG), which forms the background paper for this Consultation, summarized the situation regarding the rationale, methodology and strategies for preservation. The Committee recognized the importance of this area of work and the need for documentation of these resources. It emphasized the importance of training and of technical and financial support and urged FAO to develop further its training programmes on the subject. The FAO Council later accepted the COAG views and called for the programme of preservation of Animal Genetic Resources to be expanded and further developed. It recommended that “lack of funding should not be the cause of delay in implementation nor lessen the impetus already achieved”. An action plan has therefore been developed, which relies primarily on FAO's own resources and, as proposed in the COAG paper, it is hoped that this seed funding will lead into development programmes financed on a more substantial and long-term basis.

The responsibility of implementing a programme for the preservation of animal genetic resources rests primarily with the national governments and national institutions. The required activities (data collection and establishment of the required information system, germplasm collection, freezing and storage and the establishment of breeding units for live animal preservation) would need to be based at appropriate national institutions. Regional and global facilities have been initiated by FAO to facilitate this process. The FAO/EAAP Global Animal Genetic Data Bank would need inputs from national institutions in the same way as the FAO Regional Animal Gene Banks would receive frozen semen or embryos from national institutions. FAO's programme is designed to assist the national institutions with training, technology development and provision of equipment. The activities involved are consultancies, meetings, publications, surveys and training courses. A list of these activities is given in the Annex.

Having recognized that preservation may well be of breeds which are not, at present, highly successful economically, FAO can hardly be expected to divert a large proportion of its animal production regular programme funds to this at the expense of programmes designed to increase food production and increase the income of rural populations. The present staffing of AGR Group in FAO is two professional officers and two support staff. The Group is responsible for all FAO breeding development work including AI, Semen Donation Scheme, ET, animal biotechnology, etc. The present staff has also been assigned the responsibility of implementing the programme for preservation including regional gene banks and global data bank activities. FAO inputs available for the purpose are limited and consist of expertise and seed funds to initiate essential activities. There is, therefore, a need for increased investment in order to support the required activities at national, regional and global levels.

The Committee on Agriculture recommended increased activity in documentation and preservation concurrently - particularly with regard to indigenous breeds in developing countries. These countries require most inputs and technical support in order to achieve the objectives. Many of these countries often lack the basic infrastructures required for data collection and for developing a long-term preservation programme. Through regional cooperation some of these activities may be concentrated at one national institution which may be designated as a regional centre.

The regional gene banks at present provide the storage facility. There is a need now to identify endangered breeds in each participating country, to collect and freeze semen and embryos and to pass them on to the regional banks for storage. These activities require considerable inputs in terms of expertise, training and equipment. The joint activity with EAAP in establishing the global data bank owes much to the support of the host country and, more particularly, the host university and staff. The increased work load placed upon this facility will clearly need increased external support in the future.

Both COAG and Council recognized the need for urgent funding and also the necessity of seeking funds on an international basis. The funding could be based on commitments by donor governments, industry and non-governmental organizations by setting up of Trust Funds. The determination of the amount of funding depends on the level of activity envisaged. There is a clear opinion that activity should not be restricted by funds and yet it must always be so - the debate is about the level. The answer lies partly in the time scale involved. Serious implementation across all species in all areas of the world within two to three years is a different proposition from a few breeds in limited areas over 5 to 10 years. The investment costs in these two cases would differ markedly. But the question that needs to be addressed is of cost in terms of lost genetic resource. One cannot argue that genetic resources must be saved at any cost and then a question arises: what is an acceptable cost or an acceptable loss of diversity?

FAO has the expertise within its ranks and has access to expertise on a global basis. It has the capacity to identify and to mobilize international expertise as well as to provide support services. The various needs of the programme in the immediate future (surveys, censuses, documentation, training, storage techniques etc.) could be identified and provided to the country or institution in which it is needed. However, this requires funds both for technical expertise and for support services.

In terms of actual costs for different preservation strategies, several estimates have been produced; one such source summarizing several authors was provided in COAG paper. The relative costs differ markedly - as do the efficiencies of preservation of genetic resource. Again, a balance has to be achieved. Similarly, not all breeds (not even indigenous breeds) at low population numbers can necessarily be preserved “in pure form”. Priorities have to be assigned within any particular time period and within available funds.

Once these questions are resolved and a well-considered practicable programme for the preservation of animal genetic resources has been recommended, funds need to be located to implement the programme. FAO's Regular Programme funds serve the purpose of initiating the required activities. Donor funds from the governments of developed countries, international organizations, or the industry, are usually made available to FAO to implement Trust Fund projects in a manner agreed both by FAO and the donors. Trust Fund projects are now being prepared to support the various activities at national institutions as well as at regional and global facilities.

It is hoped that with the cooperation of the international community, FAO's programme for the preservation of animal genetic resources will be effectively strengthened in the near future.

Annex: FAO activities on animal genetic resources

A. Selected Publications
1948 Breeding livestock adapted to unfavorable environments
1950 Improving livestock under tropical and subtropical conditions
1950 Report of the inter-American Meeting on Livestock Production
1953 Zebu cattle of India and Pakistan
1957 Types and breeds of African cattle
1958 Pig breeding, recording and progeny testing in European countries
1966 European breeds of cattle
1970 Observations on the goat
1976 The buffaloes of China
1977 The water buffalo
1977 Animal breeding: selected articles from World Animal Review
1977 Bibliography of the criollo cattle of the Americas
1977 Mediterranean cattle and sheep in crossbreeding
1978 Declining breeds of Mediterranean sheep
1979 The sheep breeds of Afghanistan , Iran and Turkey
1980 Prolific tropical sheep
1980 Trypanotolerant livestock in West and Central Africa , vol. 1
1980 Trypanotolerant livestock in West and Central Africa , vol. 2
1980 Le bétail trypanotolérant en Afrique occidentale et centrale, vol. 3
1981 Recursos genéticos animates en América Latina
1981 Animal genetic resources - conservation and management
1982 Sheep and goat breeds of India
1982 Breeding plans for ruminant livestock in the tropics
1984 Animal genetic resources: conservation by management, data banks and training
1984 Animal genetic resources: cryogenic storage of germplasm and molecular engineering
1985 Livestock breeds of China
1985 Sheep and goats in Pakistan
1985 Awassi sheep
1986 Animal genetic resources data banks, vol. 1
1986 Animal genetic resources data banks, vol. 2
1986 Animal genetic resources data banks, vol. 3
1986 Sheep and goats in Turkey
1986 The Przewalski horse and restoration to its natural habitat in Mongolia
1987 Animal genetic resources - strategies for improved use and conservation
1987 Trypanotolerant cattle and livestock development in West and Central Africa , vol. 1
1987 Trypanotolerant cattle and livestock development in West and Central Africa , vol. 2
1987 Crossbreeding bos indicus and bos taurus for milk production in the tropics 
1989 Animal genetic resources of the USSR
B. Periodicals 
1972 World Animal Review: articles on breeds
1984 AGRI Newsletter: 2 issues per year 
C. Recent meetings
1980 FAO/UNEP Technical Consultation 
1983 OAU/FAO/UNEP Animal Genetic Resources in Africa - Indigenous Livestock in Africa 
1983 First FAO/UNEP Expert Panel in Rome 
1985 FAO/UNEP Expert Consultation in Rome
1985 FAO/UNEP Expert Consultation on the Przewalski Horse in Moscow
1986 Second FAO/UNEP Expert Panel in Poland 
1989 Workshop on Open Nucleus Breeding Schemes in Poland 
D. Training courses
1983 FAO/UNEP Training Course on Animal Genetic Resources - Conservation and Management - in Budapest
1988 ET Training Course in Czechoslovakia
1989 ET Training Course in Cuba
1989 ET Training Course in China 
1989 Meeting of the Coordinators of the FAO/EAAP Global Animal Gene Bank in Hannover
E. Important surveys which have not been subject of official publications
1984 Report on the conservation of Kenana and Butana in Sudan
1984 Cattle genetic resources in Iraq
1985 Deep-freezing of Gobra semen in Senegal and storage at the University of Milan . A preliminary trial.
1986 Cattle genetic resources in Iran
1987 Genetic analysis of Sahiwal cattle in Pakistan

TECHNICAL PROBLEMS ASSOCIATED WITH THE PRESERVATION OF ANIMAL GENETIC RESOURCES

REVIEW OF REGIONAL ANIMAL GENE BANKS AND RECOMMENDATIONS FROM HANNOVER WORKSHOP ON ASSOCIATED TOPICS RAISED BY THE TENTH COMMITTEE ON AGRICULTURE

John Hodges ¹

¹ Senior Officer, Animal Breeding & Genetic Resources, Food & Agriculture Organization of the United Nations, Rome, Italy

1. Introduction

1. The basis for the Regional Animal Gene Banks is given in the paper considered by the Tenth Committee on Agriculture in April 1989, entitled Preservation of Animal Genetic Resources, which is available as a background document. The Committee on Agriculture raised some technical questions on the topic which were subsequently addressed as part of the Workshop held in Hannover FRG in June 1989 attended by all the Coordinators of the Regional Animal Gene Bank Centres from Africa, Asia and Latin America, together with staff of the Global Animal Genetic Data Bank in Hannover and other resource experts.

2. This paper consists of two parts:

1. Operational, management and organizational aspects of the Regional Animal Gene Banks and linkages with the Global Animal Genetic Data Bank.

2. Recommendations from the Hannover Workshop on the issues raised by the Tenth Committee On Agriculture.

2. Operational, management and organisational aspects of regional animal gene banks

3. There are two centres in Africa (Ethiopia and Senegal) and Asia (China and India) and three centres in Latin America and the Caribbean (Argentina, Brasil and Mexico). The centre in Mexico is to provide storage facilities for germplasm from the Disease Free Zone of Central America since animals and animal tissues may not pass into that Zone from other parts of the Region.

4. In each centre, a Regional Animal Gene Bank Coordinator has been appointed by the host country. These are the 7 Coordinators who took part in the Workshop in Hannover in June 1989. The Coordinators are either animal genetics or specialists in reproduction or both.

5. The governments of each country in the regions have been asked if they wish to participate in the programme for the preservation of germplasm from their endangered breeds. Where there is interest, National Coordinators have been appointed by each government for liaison with the Regional Animal Gene Bank Centres.

6. When fully established, the centres will contain facilities for the receipt, processing and long term cryogenic storage of germplasm (semen and embryos and later possibly oocytes). Provision is also to be made for the storage of blood serum to be available later for further disease diagnosis tests not carried out on the donor animal at the time of collection. The possibilities of storing DNA independently of the germplasm (probably from the blood since it is the most accessible body tissue) are also being explored, so that the Regional Animal Gene Banks may in the future be able to participate and contribute to the growing international interest in mapping the genome and in transfer DNA.

7. The Regional Animal Gene Banks are also to be equipped with the documentation facilities which will contain all relevant genetic information on the breed and donor animals. These data will be in two parts, passport data and populations statistics. In addition, there will be documentation on the samples of germplasm including details of health control tests carried out on donor animals and on the processing of the samples themselves. The documentation system of the Regional Animal Gene Banks are being developed to be compatible with the Regional Global Animal Genetic Data Bank in Hannover. It is planned that the Regional Animal Gene Banks will become Regional Focal Points for the collection and flow of animal genetic data. The precise details of which data will pass between them and the Global Animal Genetic Data Bank is currently being studied by the EAAP/FAO Working Group on the latter.

8. In principle it is anticipated that samples from donor animals will be split and sent to the two centres in the Region for security of long term storage.

9. Initial studies for the establishment of the Regional Animal Gene Banks were carried out by the experts in each country with modest financial support from FAO. The output from these studies and from work carried out by FAO staff have been incorporated into an Operating Manual for Regional Animal Gene Banks.

10. The basis of operation of the Regional Animal Gene banks is TCDC (Technical Cooperation between Developing Countries). Each country will be responsible for contributing the needed resources within their own country. Additional external funding for the regional needs beyond those national resources will be provided. It is anticipated that TCP (FAO Technical Cooperation Programme) will be available for the initial training and establishment of the system. The host countries are currently in the process of making TCP applications.

11. The most important component for operational establishment now is the provision of regional training courses for nationals from the participating countries and supply of minor equipment where this is not available from national resources. The training courses will take place at one of the Regional Centres and will focus upon three aspects:

1. The technique for identification of endangered breeds, sampling methods for donor animals, genetic characterisations of the breed and evaluation of the donor animals.

2. The collection, processing, evaluation and freezing of semen and embryos together with animal health tests.

3. The documentation procedures, record system and management controls including inventory operation.

12. The coordinators of the Regional Animal Gene Banks and the national coordinators will become key experts in the organization of animal genetic resources activities, including the collection of census data, preparation of breed characterisations, identification of breeds which are endangered, matching of breeds in adjacent countries which have different names, but which may be genetically identical, etc. In this way they will provide the routing for the flow of information from countries via the Regional Animal Gene Banks to the Global Animal Genetic Data Bank and subsequent flow of output from the latter of processed data of value and interest to participating countries.

13. The collection of survey/census type data on indigenous breeds is beginning in a few large developing countries which already have their own national animal genetic data banks. Two of these are China and India. It will be important that the national software programmes and breed descriptors used in national data banks should match the Global Animal Genetic Data Bank system. Therefore in May 1989 FAO arranged for two software experts from the Chinese and Indian animal data banks to spend several weeks at the Hannover Centre working with the experts there on comparison and adjustment of systems. It is planned that where national data banks exist they will be routinely linked with the Hannover Centre. In most developing countries however with less resources and little possibility of creating a national data bank, the Regional Animal Gene Bank Centres will become depositories of national data, which, as mentioned earlier, will then be passed in appropriate form to the Hannover Centre.

14. One of the early tasks awaiting attention now is the design of suitable software programmes for the storage of this information in the Regional Animal Gene Bank Centres.

15. The organization of surveys and census programmes at the national level will reveal which breeds are threatened. It will often not be possible to carry out genetic evaluations of these breeds to ascertain their unique traits before they disappear completely. Therefore the plan is to take samples of semen and embryos from breeds whose numbers fall below 10000 animals. It will not be policy to preserve everything indiscriminately, nor to insist upon genetic characterisation before cryogenic preservation. The choice of 10000 animals, while pragmatic and in excess of the estimates used for in European conditions (200–1500), is felt to be realistic for developing country conditions. Often climatic stresses such as drought, lack of feed, disease epidemics, market and human needs can affect livestock numbers suddenly and drastically; also the transhumance and nomadic systems make accurate census data difficult to obtain and reproductive rates are usually less than in temperate conditions. All these reasons lead FAO to recommend the precautionary level of 10000 minimum population size.

16. The practical activities of semen and embryo collection in some developing country conditions can be difficult. Semen is often easier to collect, although with untrained animals in remote locations this too may be uncertain. Electro-ejaculation is a possibility. The collection of embryos is also uncertain due to the unknown hormonal responses of different breeds to MOET treatment. There are also the realities that in some species it is still difficult or impossible to collect and successfully freeze semen and embryos.

17. The comparative costs of collecting and preserving semen versus embryos have been estimated in temperate country conditions showing the much higher costs of the latter. Ideally, cryogenic stores should be accompanied by the preservation of live animals. However, this cannot be assured in many developing country conditions. If the last live female of a breed dies and there are cryogenic stores of semen and embryos, then live animals can be regained through the use of recipient mothers of another breed of the species. This process clearly requires the storage of representative samples of embryos. As already indicated, they are difficult and expensive to obtain.

18. A different approach is therefore being considered, namely the possible use of the developing technique with cattle, of cloning embryos at the 16 or 32 cell stage. By this method already carried out in research conditions, the nucleus from each cell is removed and inserted into an enucleated oocyte taken from the ovary of a cow at the abattoir. The combined new cell develops into a new embryo. Thus there is the potential for 16–32 new clones from one embryo. The possibilities of repeating this multiplication are also reported and, if successful, will open the way to extensive cloning.

19. In the context of the preservation of genetic variation from an endangered breed in developing countries the technique will, when fully developed, offer an alternative approach. Since all the genetic variation of a breed is contained by semen from a properly constituted, representative and random sample of males, the storage of large numbers of both embryos and semen is no longer essential to preserve the full range of genetic variation. The special value of storing embryos with semen has so far been foreseen as a means of overcoming the practical problem of regaining live animals of the breed in pure form. By currently available techniques, each stored embryo can be expected to provide only one live animal. Therefore it has been visualised that many embryos should be stored together with semen. In this way, it has been expected that a population of purebred live animals could be regained. However, under currently available techniques, in the absence of stored embryos, the samples of semen would have to be used in a long process of crossbreeding over several generations starting with females of other breeds. Only in this way with semen alone would it be possible to achieve ultimately a high level of original breed purity.

20. The advantage of the new approach now being researched and briefly described on para. 18 above, is that the full genetic variation in diploid form could be regained by preserving only a few embryos together with semen from a representative sample of males. In an extreme case, the breed could in fact be regained by embryos from only one female, albeit with a population of half-sib families. This situation would need a careful mating programme for use of the semen in subsequent generations of animals deriving from the half-sib families.

21. The procedure under the new method, when fully operational would be as follows: At the time of regaining the live animals, the few embryos would be multiplied as described and then many of them having identical diploid variation could be inserted in recipient females of another breed. The mature females resulting from these cloned embryos will then be inseminated with semen from the gene bank, thus regenerating the full genetic variation in the diploid form.

22. Although the idea of using few embryos may be considered less than ideal, it should be weighed against the present alternative, which is to collect larger numbers of embryos from many females in order to guarantee that the ultimate population to be regained will possess the full diploid genetic variation of the original breed. The costs and practical difficulties in collecting such numbers of embryos from endangered breeds are substantial at present, especially in many developing country situations. The new cloning technique is currently being developed with cattle, but clearly it will become a very attractive research target in the near future and may later be suitable for other species. It can therefore be expected to make a significant contribution towards reducing currently perceived difficulties and costs of cryogenic storage of embryos from developing countries in Regional Animal Gene Banks.

23. The creation of a World Watch List providing an early warning system is expected to be an output from the Animal Gene Banks and the Hannover Centre.

3. Recommendations from the Hannover workshop on items raised by the Tenth COAG

A. Surveys, genetic classification, risk assessment, census data and World Watch List

The Workshop recommended that the methods for surveying indigenous populations should be developed first by the Global Animal Genetic Data Bank thus providing a standard format for populations data and the simplified animal descriptors for the genetic characterisations; the drafts should then be studied by the Regional Animal Gene Bank Coordinators, who will consult the national coordinators. The final agreed system will then be tested for suitability for use in the Hannover Centre and suitable software developed in Hannover and in the PC's to be used in the Regional Animal Gene Banks.

B. World Watch List

The Workshop recommended that the World Watch List should be developed in the Hannover Centre and felt that the data flow from cooperating countries will be sufficient to make it meaningful in the first instance. Modifications can be made on the basis of experience. The frequency of publication is left open at this stage.

C. Risk Assessment

The Workshop recommended that standard levels of risk assessement should be developed before the World Watch List is published and that these standards should be worked out after having taken account of the classes of risk assessment used by specialists in other species.

D. Regional consultative groups

The Workshop recommended that for many technical aspects of assessing national information on endangered breeds and the needed techniques for preserving them, it would be appropriate to establish informal consultative groups in each region, consisting of the Regional Animal Gene Bank coordinator, the national coordinators and any other specialists able to contribute expertise.

E. Animal health, disease assessment, controls, tests and records; movement of germplasm within the region

In view of national differences in disease control regulations, the Workshop recommended that the Regional Consultative Group should be responsible for establishing any special procedures needed. It was noted with satisfaction, that, with the exception of the Disease Free Zone of Central America, no host country has felt that there are insurmountable problems in the movement and storage of germplasm within the region to and from the designated centres.

F. Use of mobile teams of experts to serve countries in assessing endangered breed status and in the collection and handling of semen and embryos

The Workshop recognised the apparent attractions of using external specialists for these tasks, but felt that in practice it will be more effective and cheaper to train nationals to undertake the work. The Workshop therefore recommended that priority be given to the organization of regional training courses in English, French and Spanish as appropriate. The Workshop also recommended that once a national team has been successfully trained and has gained experience in their own country, then they should be made available on TCDC principles to another country in the region to work alongside and train nationals as they start their programme. This approach will provide continuing education and on-the-job training in the home country for nationals already having been through the training course. The Workshop noted that both Brasil and China have developed simple field freezing techniques for embryos and recommended that these should be included in the Operating Manual.

G. Standards for genetic selection and evaluation of donor animals

The Workshop feld that this point is important and recommended that special attention be given to it in the training programmes. It is felt to be particularly important to have full documentation of the relatives (especially parents) of donor animals.

H. Wildlife

The Workshop recommended that close liaison should he developed between the Regional Animal Gene Banks and those organizations wishing to store semen and embryos from wild species. It recommended that such organizations should also be encouraged to collect germplasm not only from truly wild animals, but also of species related to domestic animals, since their DNA may be of value in domestic animal programmes in decades to come. The Workshop emphasized that special attention will be needed for the animal health controls of wild animal germplasm stored in Regional Animal Gene Banks. The Workshop also recommended that wild animal species now being farmed or ranched (such as deer), should be included in the programme.

I.Biotechnology

The Workshop recommended that FAO maintain very close contact with research in the biotechnology of animal breeding, genetics and animal reproduction in view of the great impact that new discoveries may have upon the operation of Regional Animal Gene Banks. The Workshop also recommended that the Hannover Centre system should be designed to include future data on transgenic domestic animals so that there may be one global reference centre for the genetic descriptors of these new genomes.

J.Live Animal Preservation

The Workshop recommended that wherever possible live animals should also be preserved as a supplement to the cryogenic storage of germplasm. It was recognised that the problems of organizing such programmes are great and not well understood in developing countries and recommended that FAO continue with its plan to provide a Guidance Manual on live animal preservation as soon as possible.

K. Documentation

The Workshop placed great emphasis finally upon the importance of comprehensive documentation as part of the combined programme of Regional Animal Gene Banks and the Hannover Global Animal Genetic Data Bank. The Workshop recommended that strenuous efforts be made and that sufficient funding be provided at the start of this programme to ensure that compatible, flexible and open-ended systems be developed so that regions and countries do not become isolated from each others' information systems.

The Workshop recommended that the Dictionary of Breed Types of Domestic Livestock by Mason, be provided to each Regional Centre and also to participating countries since it will permit uniform nomenclature. The Workshop also recommended that FAO should issue specifications on the types of PC to be used in the Regional Animal Gene Banks to most advantage. The problems arising from the use of different languages were discussed, including the experiences arising with the software experts from China whose data base is in Chinese characters. Recommendations on languages for input, storage and output options should be made by the EAAP/FAO Working Group after appropriate study. The Workshop also recognised the need already experienced at the Hannover Centre of having a competent geneticist working with data to ensure its plausibility as well as checks on its validity and integrity. The Workshop supported the continued study by the Hannover Centre of the use of Data Base Management Software.

FUTURE BIOTECHNOLOGICAL POSSIBILITIES IN PRESERVING ANIMAL GERMPLASM

G. Brem ¹

1 Lehrstuhl für Molekulare Tierzucht, Ludwig-Maximilians-Universität, Veterinärstr. 13, FRG-8000 München 22.

1. Introduction

Cryopreservation of sperms and embryos has been developed over several decades and is used widely now in practical animal breeding programmes. Methods have been available for over ten years for manipulating embryos for production of identical twins, chimaeras and recently also cloned mammals. Gene transfer techniques for livestock have been established since 1985.

In addition to possibilities of manipulation and cryoconservation of embryos a number of methods have been developed for isolation and cloning of genes by molecularbiological methods. Using these techniques chromosomes or genes can be isolated and stored in the form of genomic DNA or cDNA libraries. It must be pointed out, however, that these techniques usually do not allow preservation of genomes in a form which can be reactivated in total at a later stage. Nevertheless they permit individual genes, or a multitude of undocumented and unknown genes to be stored.

Both cryoconservation of sperms and embryos and storage of genes can and should be used for the ex situ preservation of animal germplasm of breeds. In Figure la summary of the various components of genetic information and the techniques required for their conservation is shown. This paper will focus on the most important ways employed for storing genetic resources in the light of technical feasibility. Aspects of application and costs for usage of ex situ cryoconservation of genomes and genes of endangered cattle breeds by means of modern biotechnological methods in developing countries are available in a FAO paper published recently (Brem et al., 1989a).

2. Cryoconservation of genomes

2.1 Semen

Cryoconservation of semen was described for the first time by Polge, Smith and Parkes (1949). Storage of semen preparations at a temperature of -196°C more or less arrests cellular metabolism. The ability of semen stored for a long time in liquid nitrogen to fertilize oocytes is not reduced if at least 50% of the spermatozoa are motile after thawing at the time of insemination. However, the necessary motility required for insemination must be tested in the AI station with at least one dose from a frozen ejaculate before storage and also before use. It has been shown that the motility of high-quality semen is decreased only slightly by storage. A lot of experience in cryoconservation of semen is available all over the world. However in developing countries it may be difficult occasionally for some logistic reasons like, for example, the availability of liquid nitrogen for freezing, transport, and storage of semen.

Genetic information	Techniques used for preparation and collection
	nucleotide	biochemical
	codon	biochemical
	exon	biochemical, genetic engineering
	gene-cluster	biochemical (in the future), genetic engineering
	chromosome	genetic engineering, cryoconservation, microinjection
	karyotypegenome	micromanipulation
	semen (haploid)	cryconservation, artificial insemination
	oocyte (haploid)	in vitro fertilization
	pronuclei (haploid)	micromanipulation
	nucleus	cloning, micromanipulation
	embryo	cryoconservation, embryotransfer
	cells	cloning, gene library
	adult animals	small populations, zoos

Figure 1: Components of genetic information and techniques available for preserving genetic resources at different levels.

As shown by some experiences it is also possible to collect spermatozoa from the epididymis by dissecting the ducti deferentes with adjacent distal candae epididymis. These spermatozoa can also be used for IVF procedures even after freezing and thawing. Thus spermatozoa can be collected even from slaughtered male animals if there is no other possibility available.

2.2 Embryos

In the last decade techniques allowing collection, storage and transfer of bovine embryos have been simplified considerably by the development of non surgical techniques and methods for cryoconservation of embryos. Nevertheless embryo transfer projects are still hampered by pronounced differences in the reactions of individual donor animals towards superovulation. Embryos can be obtained by flushing the oviducts or the uterus using surgical or non surgical methods. The techniques necessary for embryo collection have been extensively described and are already available in many developing countries.

Before the embryos are stored in an embryo bank it is important to grade them qualitatively to guarantee good results after revitalisation. Grading of embryos usually takes into account a description of the state of development and morphology and only embryos graded as “excellent” or “good” will allow good results of cryoconservation.

To prevent embryos from being damaged upon freezing and thawing it is necessary to add anti-freeze compounds to avoid the formation of ice crystals. Cryoprotectants minimize mechanical damage to cells by crystal formation and they also strengthen cellular membranes. Most of the methods for freezing embryos use glycerol (10%) as cryoprotectant. Glycerol can either be added in several concentration steps or in one step. Embryos are then stored in plastic straws and cooled down from ambient temperature to -7°C. At this temperature crystallization of the freezing medium is initiated by seeding. The embryos are then allowed to equilibrate and after 5–10min. they are cooled at a rate of 0,3 – 0,5°C per minute until the final temperature of -28°C to -35°C is reached and they are transferred to liquid nitrogen. The embryos may be stored in liquid nitrogen for decades and as far as we know neither the genome nor extrachromosomal genetic material will be altered in any way during this time.

Recently a new technique for cryoconservation has been developed. The so-called embryo vitrification has yielded a viable alternative to conventional techniques. Embryo vitrification has been successfully used for cryopreservation of bovine, mouse, rat, rabbit and sheep embryos.

Vitrification is the solidification of a liquid brought about by an extreme elevation in viscosity during cooling. Highly concentrated aqueous solutions of cryoprotectants are able to supercool to very low temperatures and became so viscous that they solidify without the formation of ice. Successful vitrification requires the use of a highly concentrated yet effectively non-toxic solution of cryoprotectants and high cooling and warming rates.

Rall and Fahy (1985) have used a vitrifiation solution consisting 20,5 % w/v dimethyl sulphoxide (DMSO), 15,5 % w/v acetamide, 10 % w/v propylene glycol and 6 % w/v polyethylenglycol (PEG) in a modified Dulbecco's saline at pH 8 for vitrification of mouse embryos. Cooling rates between 20 and 2500°C/min. and warming rates between 300 and 2500°C/min. have lead to 80 % or higher survival rates of eight-cell mouse embryos cooled to -196°C. However, embryos warmed at 10°C/min. were always killed (Rall and Fahy, 1985).

Scheffen et al. (1986) have developed a simple and efficient procedure for preservation of mouse embryos by vitrification consisting of the successive use of two solutions:

1. 10% glycerol - 20% 1–2 propanediol in PBS as equilibration and intracellular cryoprotecting medium;

2. 25% glycerol - 25% 1–2propanediol in PBS as extracellular vitrification medium.

Dilution of the cryoprotectant was achieved by using a 1 M sucrose solution. This procedure was also used for bovine embryos (Massip et al., 1987). Late morulae and blastocysts were exposed for 10 min. at room temperature (20°C) to solution (1). Then they were loaded in straws by aspirating them into a drop of solution (2). Immediately after loading, the straw was plunged directly in liquid nitrogen.

After storage, warming was done by putting the straw in a water bath at 20°C. Transfer of these embryos resulted in pregnancy rates of more than 40% in good recipients. Massip et al. (1987) think that with good management conditions and a rigorous selection of embryos and recipients it is possible to improve greatly the pregnancy rates. Van der Zwalmen et al. (1989) have developed a vitrification procedure for bovine blastocysts. They first exposed the blastocysts to a solution of 3.4 M glycerol in PBS (25% V/V) for 13 min and then to a solution of 3.4 M glycerol - 0,25 M sucrose in PBS for 7 min. Afterwards the blastocysts were transferred to a drop of 3.4 M glycerol - 3.4 M 1–2 propanediol/PBS precooled at 4°C within a 0,25 ml transparent French straw. Of 45 blastocysts stored in liquid nitrogen, 35 were cultured after thawing and 20 reexpanded. 14 were transferred to recipients resulting in 7 pregnancies.

Robertson et al. (1989) have developed an ultrarapid freezing procedure of mouse embryos using DMSO and trehalose. The survival rate of two-cell embryos frozen in trehalose vitrification -freezing medium was 86% and 71% developed into blastocysts. Lopes et al. (1989) have shown that the in vivo survival of mouse embryos frozen by direct plunging into liquid nitrogen was lower than of embryos after vitrification.

2.3 Oocytes

The recovery of mature bovine oocytes requires surgery, laparatomy or slaughtering of donor animals and is a rather laborious process. These procedures are not suitable for animals of endangered breeds. An alternative to the recovery of mature oocytes has been suggested by experiments designed to recover immature oocytes from ovaries of slaughtered animals. The technique must therefore be considered as a complement to other methods of conservation. Recovery of immature oocytes may be a way to conserve the genome of animals which cannot be used for the production of embryos, or which are otherwise unsuitable.

Collection of ovaries from slaughtered animals is relatively easy. They can be easily transported and oocytes are recovered by puncturing all follicles at the surface of an ovary whose diameter is between 1–6mm and by aspirating the fluids using a simple syringe with a fitted small canula. Cumulus/oocyte complexes are isolated from sediments obtained by allowing pooled follicle fluids to settle for several minutes. These complexes are washed and oocytes with a complete dense cumulus oophorus and a dark evenly granulated cytoplasm are selected. Fertilization can be achieved by using deep frozen semen which has to be subjected to a swim-up procedure (Parrish et al., 1985). 24 h after the in vitro fertilization procedure, oocytes are allowed to undergo further development by culturing them in vitro together with granulosa cells, for example (39°C, 5% CO₂, max. humidity). After 7 days of in vitro culture morula and blastocyst stages are available.

In our own experiments more than 30% from the oocytes matured and fertilized developed to morulae or blastocysts. This value was increased to 47% if the mean fertilization rate of 68% is taken into account (Berg and Brem, 1989). On average 15 oocytes can usually be obtained from both ovaries of one animal and up to 5 of them will develop to morulae/blastocysts.

A number of experiments have demonstrated that mature oocytes can be frozen and stored successfully in liquid nitrogen. Schellander et al. (1988) have shown that frozen thawed cattle oocytes can be used for in vitro fertilization successfully. At present a lot of experiments are on the way to establish freezing procedures for fertilized oocytes and early stage embryos. In summary, the conservation of genomes by using immature, mature or fertilized oocytes from slaughtered animals promises to be a very successful way in the near future for cryopreservation of genetic material of endangered breeds.

The storage of slices of ovaries in liquid nitrogen could also be a possible way for cryopreservation of genomes. Daniel and Juneja (1987) took ovaries from slaughtered cattle, cut them in longitudinal sections and froze them. After thawing, these ovary slices gave rise to living cells suggesting that oocytes may also survive this treatment. It may also be envisaged that thawed ovary slices might be transferable into suitable recipients to obtain oocytes which can be fertilized. We have done such experiments in mice using ovaries from transgenic donor mice but up to now we have not tried to freeze them. However, the transferred transgenic ovaries have resulted in live offspring from recipient animals after transplantation of demi ovaries in mice (Brem et al., 1989b).

2.4 Cells and nuclei

The nucleus of each cell of an animal contains the entire genetic information. At least in theory this opens up the possiblility to preserve the genetic information of individual animals in the form of cells or cell nuclei. Until now, however, it has been impossible to reactivate an entire organism from the genetic information contained in a single somatic cell. In contrast pluripotent embryonal stem cells or blastomeres derived from early embryonal stages can be used to create animals carrying the genetic information of these cells by making chimaeras by transfering the nucleus of such cells to enucleated oocytes.

Future developments in biotechnological research will eventually greatly advance techniques for reactivation of the genetic material in cells. Even if these techniques are not available within the next 10 or 20 years it seems worth while to store cells and/or nuclei now, in addition to storing genes, to maintain the possiblity of using these components later. An overview of the suitability of cells or cell lines for conservation and reactivation is shown in Table 1.

Table 1: Suitability of cells for cryoconservation and reactivation.

* ICM = inner cell mass of blastocysts

** PGC = primordial germ cells

3. Ex Situ preservation of genes

3.1 Isolated chromosomes

Recently it became feasible to isolate and enrich individual chromosomes. Application of flow-through cytophotometry allows both karyotype analysis and recovery of sufficient amounts of material by chromosome sorting. Flow-through cytophotometry essentially involves measurement of fluorescence emitted from chromosomes stained with suitable fluorescent dyes after excitement with monochromatic laser light. Fluorescent emissions are detected with light detectors and processed as electronic signals by a computer.

Apart from providing flow-through cytogrammes, the technique also allows determination of the amount of DNA per chromosome, and of base composition, and the degree of chromatin condensation. The suspension of chromosomes passes the laser beam in tiny droplets. These may be charged electrically according to the amount of fluorescence measured which, in turn, corresponds to individual chromosomes. The charged droplets can be deflected from their normal paths in an electric field, while the paths of uncharged droplets will not be affected. Enrichment of certain chromosome subsets by flow-through cytophotometry is also known as fluorescence-activated cell sorting.

A sorting frequency of approximately 100 specific chromosomes per second will yield ca. 400000 enriched chromosomes per hour. In some cases the purity of the enriched chromosome fractions may reach 99% (Lebo et al., 1984). Normal values are usually in the order of 80% (Cremer and Cremer, 1985). Sorted chromosomes can be stored in suspension and may be also processed immediately.

Some examples have demonstrated that isolated chromosomes may also be used directly for gene transfer. The technique is known as chromosome-mediated gene transfer. Animals carrying fragments stably integrated into the germ line so that stable strains can be obtained are called “transomic”. Procedures for introducing exogenous DNA into mouse eggs by injection of chromosome fragments have already been developed. Richa and Lo (1989) have dissected chromosome fragments from human metaphase spreads and microinjected them into the pronuclei of fertilized mouse eggs. Many of the injected eggs subsequently exhibited normal pre- and post-implantation development. Some embryos retained centromeric DNA as demonstrated by in situ hybridization analysis and after injection of non centromeric fragments a positive mouse was found. This procedure should facilitate incorporation of very large DNA fragments of more than 10 mega bases into cells and embryos without the need for cloned seqences (Richa and Lo, 1989).

3.2 Genomic DNA and/or cDNA Libraries

3.2.1Genomic DNA libraries

High-molecular weight DNA is prepared either from tissue or from blood. The use of material relatively devoid of connective tissue such as liver as starting material is an advantage. Suitable tissue samples can be obtained either as biopsy material from live animals or immediately after slaughtering. It is important that the material must be processed immediately if no suitable means of conservation are available. Otherwise the tissue may also be stored at -20°C, -80°C or in liquid nitrogen for an idefinite period. Establishment of a genomic DNA library requires several milligrammes of tissue. If the DNA is to be isolated from blood, leucocytes should be prepared first.

Since high-molecular weight DNA (Mr = 3 x 10⁷) is required for the preparation of genomic libraries, the DNA must be treated very carefully. Suitable techniques have been developed yielding DNA with molecular masses between 4 × 10⁶ and 500 × 10⁶, which can be cleaved subsequently with restriction endonucleases. Naked recombinant phage or cosmid DNA cannot be introduced satisfactorily into bacteria. It is therefore necessary to package the DNA into empty phage heads in vitro and to subsequently infect bacterial cells. The principle of in vitro packaging is based essentially on the ability of DNA fragments carrying cos sites at their 5'- and 3'-ends to be packaged in vitro in the presence of empty phage heads and packaging proteins. This process requires that the molecules are at least 38 kb in length and not longer than 52 kb. Packaged phage heads are matured into infectious phage particles in vitro in the presence of gene products W and FII and phage tails. All proteins required for packaging can be obtained from strains BHB2688 and BHB2690 of E.coli. Packaging mixes are prepared once and can be stored at -80°C. Depending upon the vector there are a number of E. coli strains which may be used for infection. Infection is achieved by incubating a mixture of infectious phage particles obtained by in vitro packaging of recombinant DNA molecules with pre-treated sensitive cells. This step is followed by amplification of the library after the litre has been determined.

Depending upon the vector (or cosmid) used the established gene library can be stored as a suspension culture at 4°C for several years or as a glycerol stock at -80°C. for a long period. In yeast it is possible to establish so called YAC libraries containing DNA-fragments up to several hundred kb long.

3.2.2cDNA libraries

The starting material for the establishment of a cDNA library is mRNA isolated either from pieces of tissue or whole organs. cDNA libraries are not complete collections of the entire genetic information of an organism, rather they represent the current state of gene expression in tissues or organs. It is therefore critical to keep in mind the current state of activity of a particular organ or tissue when cDNA libraries are prepared. A decisive step in cDNA synthesis is the formation of a double-stranded DNA copy of the mRNA. This requires the starting mRNA to be of the highest possible quality. The isolated total RNA contains considerable amounts of ribosomal RNA (rRNA) and transfer RNA (tRNA) while the proportion of messenger RNA (mRNA) is comparatively low.

Most mRNAs are characterized by a polyadenylated 3'- end which is known as poly (A) tail. This sequence is lacking in other structural RNAs. Separation of mRNA from other RNA species can be achieved by poly (A)+ selection. Total RNA is first heatdenatured to remove any secondary structures and subsequently passed over an oligo (dT) cellulose column. At high salt concentrations RNA containing poly (A) ends will bind to oligo (dT) cellulose while RNA without tails will pass through the column. Polyadenylated RNA can then be eluted from the column by removing the salt. Column chromatography is repeated several times to reduce contamination with other RNA species.

Another protocol involves the use of poly (U) sepharose instead of oligo (dT); it binds longer stretches of nucleotides and liquid passes much quicker through the column.

As a rule of thumb one may keep in mind that approximately 1% of the input RNA should be recoverable as poly (A)+ RNA. This mRNA should produce a smear during gel electrophoresis with sizes starting at approximately 20 kb and becoming progressively smaller. Highest intensities should be found in a region between 5 and 10 kb.

The first strand of the cDNA is synthesized enzymatically by reverse transcriptase with poly (A)+ mRNA as starting material. The second strand and hence the finished cDNA product is synthesized by a combination of treatments with RNase H, DNA polymerase I and E. coli ligase. Several different protocols have been developed for the synthesis of the second strand.

3.3 Isolated Genes

The isolation of genes or coding regions of a gene (cDNA) is usually achieved by screening a gene library. It is possible to screen many recombinant clones simultaneously by growing individual clones of a gene library on agar plates and transferring them to nitrocellulose filters.

A critical parameter governing the screening process is the number of clones that must be analysed to find a particular gene. This number should be calculated on the basis of theoretical considerations before the experiment is carried out. One problem that frequently occurs is the selective loss of certain clones and the over-representation of other clones after amplification of the library. If a desired clone cannot be detected in the library it will be necessary to screen another library that has been established independently.

References

Berg, U. and Brem, G. (1989). In vitro production of bovine blastocysts by in vitro maturation and fertilization of oocytes and subsequent in vitro culture. Zuchthygiene 24, 134 – 139.

Brem, G., Brenig, B., Müller and Springmann, K. (1989a). Ex situ cryoconservation of genomes and genes of endangered cattle breeds by means of modern biotechnological methods. FAO Technical Papers, Animal Production and Health Nr. 76, Rome.

Brem, G., Baunlack, E., Müller, M. and Winnacker, E.-L. (1989b). Transgenic offspring by transcaryotic implantation of transgenic ovaries into normal mice. In Molecular Reproduction and Development, in press.

Cremer, T. and Cremer, C. (1985). Chromosomenspezifische DNA-Bibloiotheken in der Humangenetik. In Molekular-und Zellbiologie. Aktuelle Themen (eds Blin, Trendelenburg, Schmidt). Springer Verlag.

Daniel, J.C. and Juneja, S.C. (1987). Cryopreservation of sliced bovine ovaries. Theriogenology 27, 220.

Lebo, R.V., Gorin, F., Fletteric, R.J., Kao, F.-T., Cheung, M.C., Bruce, B.D. and Kan, Y.W. (1984). High-resolution chromosome sorting and DNA spot-blot analysis assign McArdle's syndrome to chromosome 11. Science 225, 57–59.

Lopes, RF., Rodrigues, J.L. and Christmann, L. (1989). In vivo survival of mouse embryos frozen by direct plunging into liquid nitrogen and vitrification Theriogenology 31, 219

Massip, A., Van der Zwalmen, P. and Ectors, F. (1987). Recent progress in cryopreservation of cattle embryos. Theriogenology 27,69–79.

Parrish, J.J., Susko-Parrish, J.L. and First, N.L. (1985). In vitro fertilization of bovine oocytes using heparin treated and swim up separated frozen thawed bovine semen is repeatable and results in high frequencies of fertilization. Theriogenology 23, 216.

Polge, C, Smith, A.U. and Parkes, A.S. (1949). Revival of spermatozoa after vitrification and dehydration in low temperatures. Nature 164, 666.

Rall, W.F. and Fahy, G.M. (1985). Ice-free cryopreservation of mouse embryos at -196°C by vitrification. Nature 313, 573–375.

Richa, J. and Lo, C.W. (1989). Introduction of human DNA into mouse eggs by injection of dissected chromosome fragments. Science 245, 175–177.

Robertson, J.L., Minhas, B.S., Randall, G.W., Dodson, M.G., Palmer, T.V. and Ricker, D.D. (1989). Ultrarapid freezing of mouse embryos with DMSO and trehalose. Theriogenology 31, 250.

Scheffen, B., Van der Zwalmen, P. and Massip, A. (1986). A simple and efficient procedure for preservation of mouse embryo by vitrifivation. Cryo-Letters F, 260–269.

Schellander, K., Brackett, B.G., Führer, F. and Schleger, W. (1988). In vitro fertilisation of frozen thawed cattle oocytes. 11th International Congress on Animal Reproduction and Artificial Insemination, Dublin 1988.

Van der Zwalmen, P., Touati, K., Ectors, F.J., Massip, A., Beckers, J.F. and Ectors, F. (1989). Vitrification of bovine blastocysts. Theriogenology 31, 270.

CRYOGENIC PRESERVATION OF WILD ANIMAL GERMPLASM

M.H. Woodford ¹

¹ Veterinary Specialist Group, Species Survival Commission, IUCN Apt B-709, 523 St NW, Washington DC, USA.

1. Introduction

Throughout recent years the rate of global biotic impoverishment has greatly accelerated. The pace at which this phenomenon has occurred is unmatched in the past 65 million years. Exponential growth in human populations and even greater increases in the rate of consumption of the world's natural resources have led to the loss of many species and habitats (Wilson, 1988). If this trend continues, by the year 2050 we may see the loss of up to one quarter of the world's species (Reid and Miller, unpubl.), an end to the rise in agricultural yields (Plucknett and Smith, 1986) and major disruptions in ecosystem functions including the loss of valuable water sheds, loss of range productivity, diminished fishery production, and potentially dramatic changes in the climate and hydrology of entire regions (Salati and Vose, 1983).

Unfortunately at this late hour it is no longer enough merely to slow the inexorable loss of biodiversity; global biotic impoverishment must be brought to a halt and the processes reversed.

The number of concerned organizations and the diversity of their work has increased in recent years but the activities of these individuals and institutions are still too fragmented and limited to bring about the required fundamental changes.

However, the prospects for change are greater now than of late. Worldwide, concerned organizations are coming to recognize the pressing need for improved coordination of efforts and the advantages of a unified strategy. Other reasons for cautious optimism are the rapid advances in the science of conservation biology and the dramatic increase in public, political and academic awareness of the problems of loss of biodiversity and its potentially severe effects on future human well-being.

It is in this context that it would seem wise and appropriate to propose that wild animal genetic material should be allocated a place alongside the germplasm of domestic stock in the newly established FAO Regional Animal Gene Banks. At present very little wild animal germplasm has been cryopreserved and, such as there is, can be found in the laboratories of a few American and European zoological gardens where the very necessary basic research is being conducted. The extension of cryopreservation facilities for wildlife material to include the FAO international network would present a great advance in the worldwide effort to conserve biodiversity, and it is encouraging to note that at the Tenth Session of the FAO Committee on Agriculture (COAG) in April 1989 and at the Ninety-Fifth Session of the FAO Council in June 1989, it was recommended that FAO “be urged to study more closely the possibilities of joint programmes and cooperation with other bodies whose principal interest lies in wildlife because of the close association between domestic animal and wildlife genetic resources…”.

Many of the most highly vulnerable endangered species are to be found in developing countries which will not have the money or the expertise to establish their own gene banks in the foreseeable future. Politically, too, it would seem a good idea to store the genetic material of selected wild animals as near to the country of origin as possible and to involve the national conservation authorities in its collection, maintenance and eventual deployment. Important though the establishment of wild animal (and plant) gene banks may be for the conservation of biodiversity, it is unlikely that many of these could be established by the wildlife authorities on a global basis due to the expense and shortage of skilled staff. An opportunity to share an existing (or proposed) facility with FAO is thus obviously extremely attractive.

The World Resources Institute (WRI) and the International Union for Conservation of Nature and Natural Resources (IUCN) have proposed a programme aimed at maintaining the maximum possible diversity of species, habitats and ecosystems while using them wisely to provide for human needs now and in the future. The specific objectives of this programme include:

• stimulating greatly increased international cooperation on the conservation of biodiversity;

• spurring institutional and policy reform to address the root causes of biotic impoverishment and to encourage sustainable use of ecosystems and species;

• fostering agreement on a global strategy and long term work plan to save, study and use biodiversity; and

• promoting the development and application of new funding mechanisms for efforts to conserve biotic resources. World Resources Institute “Gift for the Future” Programme)

Unfortunately it has now been realized that very few National Parks and other designated protected areas are big enough to support adequate populations of key species without the risk of a slow erosion of genetic variability.

One way of correcting this situation is by the regular introduction of fresh germplasm, either in the form of live individual animals from unrelated distant sources (where possible) or indirectly using cryopreserved material. Few, if any, zoos have the space or the funds to keep the minimum requirement of 50 “effective” breeding animals of one species for ensuring that genetic variance is not lost and none could host the minimum required 500 “effective” breeders to allow for ‘speciation’ to take place in response to environmental change. It is these circumstances that makes the possibility of establishing a wild animal gene bank on a global scale an important component of any international effort to conserve biodiversity.

When the IUCN published the World Conservation Strategy in 1980 a major feature was the recognition of the importance of biodiversity, both genetic and ecological, as “the raw material for much scientific and industrial innovation …”. Meanwhile, the Convention of International Trade in Endangered Species (CITES) has listed over 200 mammalian species as being threatened with extinction. In consequence, it seems inevitable that the management of wildlife stocks in both zoos and wild protected areas will demand the development of the latest techniques for assisting captive propagation, and already much interest is being generated in the field of wild animal reproductive studies.

Building on the foundations laid by the agricultural industry, researchers in the field and in zoos have begun to study the reproductive processes of wild animals and to use and adapt the technologies of domestic animal embryo transplant and artificial insemination to enhance the reproductive potential of both captive and free living wild animals. A major problem facing wild animals, captive and free living, is the loss of genetic diversity due to inbreeding. This is particularly serious in those species whose numbers have been so depleted that they can be said to be passing through a “genetic bottleneck”. An example of this is the plight of the douc langur (Pyathrix nemaeus ) a Southeast Asian primate which is now almost extinct in the wild and whose numbers in captivity are so low that the genetic diversity required for continued survival no longer exists (Gorman, 1980). The inbreeding problem in zoos which has resulted in decreased fertility, high neonatal mortality and frequent birth defects has been addressed by the International Species Inventory System (ISIS), an organization which catalogues and computerizes reproductive information from nearly 200 zoological institutions worldwide. ISIS acts as a clearing house for participating zoos which wish to exchange blood lines and it is hoped that when wild animal semen and embryo banks become a reality, cryopreserved germplasm will be catalogued in the ISIS computer so that genetic material can be transported between zoos (rather than live animals themselves) for the introduction of new blood lines.

Current wild animal reproductive research in zoos is directed towards maintaining and improving genetic and species diversity and the key to the future for many species now threatened with extinction will surely be the development of advanced reproductive technology including embryo transfer, artificial insemination and germplasm cryopreservation.

However, the application of these technique to wild species, captive and free living, is still in its infancy and is fraught with difficulties.

2. The technologies

2.1 Artificial Insemination and Frozen Semen

Artificial insemination and the use of frozen semen in the cattle industry have been widely practiced reproductive procedures for many years, and it was hoped that adaptations of these techniques might be useful in the propagation of captive wild animals. Unfortunately, it was not at first appreciated how little is known about the basic reproductive processes of wild animals and although many zoos have collected and frozen semen from a wide range of species the results have not been encouraging. Nevertheless, in spite of many problems, artificial insemination using fresh and frozen semen has been successfully performed in some cases (Holt and Moore, 1988) (Table 1).

Much more semen has been collected from wild species than has actually been evaluated and used. The first artificial insemination in a wild species with previously frozen semen occurred in 1973 with a wolf (Seager, 1981). This was followed by the successful insemination of a gorilla (Douglass and Gould, 1981). Several attempts have been made to inseminate wild-caught felidae (cats) but most have ended in failure (Dresser et al., 1982b). London Zoo finally produced a puma in 1980 by surgical artificial insemination with fresh semen (Moore et al., 1981) and in 1981 Cincinnati Zoo was successful in producing a Persian leopard by non-surgical artificial insemination with fresh semen (Dresser et al., 1982b).

Table 1: Successes in artificial insemination of captive wild species.

* frozen semen

Many problems are associated with artificial insemination in wild animals. Firstly, it requires the use of anaesthesia, always a risk, for both the semen collection from the male and the insemination of the female. Then the fertility of semen obtained by artificial collection techniques is often less than that produced by natural ejaculation, and sperm usually begin to deteriorate soon after collection. Furthermore, the timing of the insemination to coincide with ovulation requires a detailed knowledge of the female reproductive cycle. Fortunately the use of hormones such as prostaglandins and progesterone to bring the female into oestrus can allow the insemination to be planned ahead. This procedure has been used successfully for addax (Densmore et al. , 1987), scimitar-horned oryx (Oryx dammah) and Pere David's deer (Elaphurus davidianus). However, the suitability of the use of prostaglandins has yet to be confirmed for each new species, since outside the ungulates, some species do not respond to prostaglandins at all.

2.2. Semen cryopreservation

A great deal of work needs to be done on the cryopreservation of the semen of exotic animals. To date, the sperm of at least 200 different species has been frozen, but very little of it has been thawed and tested (Seager, 1981). From the semen that has been tested, cryobiologists have found that sperm from each species needs to be extended and frozen under slightly different conditions to produce optimal results (Howard et al., 1981). Extenders used to preserve the collected semen basically consist of a buffered solution that contains a cryoprotectant (e.g. glycerol), antibiotics and either egg yolk or milk. Many variations have been tried in the basic recipe for semen extender. The agricultural industry has found that the optimal extender for a given species appears to be highly species specific and this is also proving true for wild animals. It has been suggested that a clue for semen preservation may be found by studying certain female reptiles which are able to keep sperm viable in their bodies for up to six years after mating (Cherfas, 1984).

The use of frozen semen is of great value for improving the genetic diversity of a captive population of wild animals and as with embryo transfer, the risk and cost of transporting semen between donor and recipient is far less than transporting a male animal for natural service. In the same way, the use of frozen semen can overcome quarantine restrictions and lessen the risk of disease transmission.

2.3. Embryo transfer

The application of embryo transfer (ET) to wild species is a relatively recent event (Table 2).

Synchronization of the donor and recipient animals in ET is accomplished by precisely timed injections of prostaglandins. Superovulation of the donor is brought about by the injection of follicle stimulating hormone (FSH). Superovulation has been fairly successful in wild ungulates but optimal drugs and dosages have yet to be tested for most other species. As many as 31 embryos have been collected from one FSH-stimulated eland cow (Dresser, 1983). On the other hand, hormone-induced superovulation has been less than successful in the wild equids, zebra and Przewalski's horse (Hearn and Summers, 1988). It seems that there is a great deal of variation to the response to superovulation hormones amongst species and more work is needed to clarify this.

In felines, superovulation of donors and synchronization of donors and recipients is complicated because most cats are induced or reflex ovulators, and usually do not ovulate without the stimulation of copulation. Human chorionic gonadotrophin (HCG) has been administered to domestic cats to provoke ovulation sometimes with stimulation by a vasectomized male (Bowen, 1987). Very little work, however has been done on wild cats (Bowen et al., 1982; Reed et al., 1981)

It is hoped that domestic cats may be able to serve as surrogates for incubating embryos from all endangered wild cat species such as the black-footed cat (Felis nigripes). The very recent success by Dresser at Cincinnati Zoo when ova recovered from an Indian desert cat, fertilized in vitro and transferred to a domestic surrogate which resulted in the birth of a kitten bodes well for the future.

Table 2: Successes in embryo transfer (ET) in captive wild species

The development of embryo transfer techniques is of great importance for the maintenance of genetic diversity in captive populations and the ability to introduce new genetic material into a captive population through transfer of non-local embryos is far preferable to the translocation of live adult animals. Some research in zoos is also directed at interspecies embryo transplants to surrogates of more common species, thus greatly increasing the reproductive potential of the donor species.

Interspecies embryo transfers have had some limited success in wild animals but much more research is needed. Intergeneric embryo transfers, e.g. eland to domestic cow (Dresser et al., 1982a), water buffalo to domestic cow (Drost, 1983) as opposed to interspecies, e.g. tiger to lion (Reed et al, 1981), have, however, so far not been successful.

2.4. Cryopreservation of embryos

The first successful freezing of mammalian embryos was reported in 1972 (Whittingham et al, 1972; Wilmut, 1972). Since then more than 300 articles and 100 abstracts have been published on the subject.

The freezing of mouse, rabbit and domestic cattle embryos is now a routine procedure and altogether embryos of 11 mammalian species have been successfully frozen (success means that live young have been born from frozen embryos).

Because of the extremely limited supply of experimental material, few attempts have been made to transfer previously frozen wild animal embryos into recipients. However, some limited success has been achieved (Cherfas, 1984; Dresser et al., 1984a,b; Hearn and Summers, 1986; Kramer et al, 1983).

Cryopreservation will undoubtedly prove to be an important adjunct to reproductive research in wild animals. Geneticists Thomas Foose and Ulysses Seal have calculated that a population of 250 properly managed animals of a particular species can theoretically preserve 95% of the original genetic diversity of the group after 50 generations or 400 years (Myers, 1984). The world's zoos, unfortunately, have limited facilities and can rarely accommodate large enough numbers of each species. Successful cryopreservation will allow the problems of limited numbers and space to be overcome by permitting the maintenance of the desired genetic diversity in liquid nitrogen.

Embryos, semen and ova containing new genetic material could be recovered from the wild, frozen, and brought back to improve the genetic health of captive populations but at present government restrictions in many countries prohibit the import of embryos from other areas. It is hoped, however, that changes in these laws, as a result of further research, may eventually come.

3. Disease transmission hazards

The diseases transmissible by semen and embryo transfer have been reviewed by Hare (1985) and Singh (1988). Disease transmission between different populations can be greatly reduced by embryo transfer, because an intact embryo collected from a diseased mother is often free of the bacterial or viral disease agent and so does not transmit the disease to the foster mother. The surrogate mother, too, may confer passive immunity to her offspring that develops from a transferred embryo. This passive immunity may be transplacental or via the colostrum, and can prove of value in producing offspring passively immune to local endemic diseases.

If the results of research on domestic animal embryos can be extrapolated to wild animals it would seem that the potential of embryos to transmit infectious disease is considerably less than that of semen or live animals. In fact, it remains to be established whether, under field conditions, disease transmission will ever occur via embryo transfer. Sufficient research has been carried out with embryos from bovine leukaemia virus-infected donors and foot and mouth disease virus donors to determine that these viruses will not be transmitted via embryos, provided they are handled properly (Singh, 1988). Research is at present in progress to determine whether similar conclusions can be reached for other viruses but much more work will be required before an opinion can be given on the safety of embryo transfer for all pathogens in all domestic species.

Little, if any, research has been done on the disease transmission potential of cryopreserved germplasm derived specifically from wild animals. Semen clearly has great potential for the spread of infectious diseases but semen also provides an excellent means of disease control when it is collected and transferred artificially using strict aseptic techniques because direct animal contact is avoided, the health status of the donor can be predetermined and, if the semen is frozen, aliquots can be tested for the presence of micro-organisms.

Embryos have less potential for the transmission of infectious agents than does semen. At the developmental stage at which they are usually transferred embryos are protected by a relatively thick capsule, the zona pellucida. Therefore, for an embryo to be infected at the time of transfer the causal agent has either to infect the gamete(s) or penetrate the zona pellucida from the environment. Available evidence suggests that such agents are likely to be few in number. For the embryo to transmit infectious disease it has to be either infected with a minimum infective dose or act as a carrier through agent association with the zona pellucida. Preliminary in vitro evidence indicates that while many agents are removed or inactivated by washing or other treatments, a few are not. For the latter, critical questions to be answered are whether the embryo is ever exposed to the agent in the reproductive tract of the infected donor animal and, if it is, whether it is capable of carrying over a minimum infective dose. Current regulations to control disease transmission by embryos of domestic animals are based largely on the philosophy that if the donors are free from specified diseases, their embryos will be similarly free. Evidence to date suggests that this approach may be unnecessarily restrictive in regard to a number of diseases (Hare, 1985). It is to be expected that the principles summarized here will be applicable to cryopreserved wildlife germplasm but that while the results of current research on diseases of domestic animals transmissible by semen and embryo transfer techniques can probably be safely extrapolated to include fresh and cryopreserved wildlife genetic material, care will be needed to ensure that specific wildlife diseases are not transferred.

The problem with experimental work involving farm animals is often to obtain sufficient numbers with which to calculate the probability of disease transmission. This problem is obviously infinitely greater when one considers the wildlife situation. However, it should be possible to screen wild animal donor populations for disease agents while collecting the germplasm for cryostorage. It is further suggested that parallel serum samples should be collected and stored alongside the genetic material so that retrospective screening can be carried out as and when new techniques are developed or new disease problems emerge. Wildlife germplasm collected from zoo animals would, of course, benefit from the quarantine and zoo-sanitary procedures in force at the zoological garden.

4. The collection and storage of wild animal germplasm

The skills and technology required for the collection and storage of wild animal germplasm are essentially the same as those employed for domestic animals. Initially much of the material would be collected under ideal conditions in the laboratories of zoological gardens and some of it would be stored in the facilities provided at their specialist laboratories.

However, some material would need to be collected from the wild. It is envisaged that this operation would be carried out by specially trained and equipped teams probably emanating from the reproductive physiology departments of major zoos and universities. In cases where it is undesirable further to deplete wild stocks by removing more individuals from the wild (e.g. the Siberian tiger) it would be possible chemically to immobilize a wild male, collect its semen by electro-ejaculation, evaluate, freeze and store the material, administer the specific antagonists to the immobilizing drugs to the animal and release it back into its wild environment.

The semen could then be transported and used to inseminate several captive females (Seager, 1983).

Genetic material could also be recovered from wild animals which are the subject of cropping or reduction programmes and in this instance it should be possible to recover oocytes from the ovaries of slaughtered females by aspiration biopsy. The oocytes could then be matured in vitro prior to cryostorage or fertilized in vitro and stored as zygotes.

5. Sources and selection of wild species for cryopreservation of germplasm

Zoos and wildlife conservation areas are the greatest source of wild animal genetic material for cryopreservation. Selection of suitable species will depend upon the further development of successful technologies for the preservation of wild animal germplasm on a species-by-species basis.

It is suggested that in the first instance the selection of priority species should be made on a regional basis by a panel of experts representing international and national conservation authorities. Priority might be given for economical as well as ecological reasons to the wild relatives of domestic livestock whose genes may be of value for future infusion into domestic breeds. It would also seem practical first to exploit existing basic techniques for use in wild artiodactyla and perissodactyla rather than carnivores or aquatic mammals for which little information is available (Wildt et. ah, 1986).

In order to maintain maximum genetic diversity, germplasm should be collected, where possible, from animals still in a wild situation where the donor population is still large.

6. Ownership of cryopreserved wild animal germplasm

It is proposed that wild animal germplasm stored in an FAO Regional Animal Gene Bank should be owned by the government of the country of origin.

7. Access to the material

Since the coordinated collection and cryostorage of wild animal germplasm should be an integral part of an international global conservation effort to conserve biodiversity, it is logical that the access to and the deployment of the preserved germplasm should be managed and coordinated by the owning government in consultation with IUCN and FAO.

8. Other gene banks for wildlife germplasm

A small number of zoos throughout the world are engaged in research into the technology of cryopreservation of wildlife germplasm. In the United States the largest embryo bank for exotic animals is at the Center for Reproduction of Endangered Wildlife, Cincinnati Zoo, where the embryos of seven species are held in cryopreservation along with 50 species represented in frozen semen. Other smaller gene banks are located at the National Zoological Park, Smithsonian Institution, Washington, D.C., the College of Veterinary Medicine, Texas A & M University, the Zoological Society of London and the Center for Reproduction of Endangered Species at San Diego Zoo.

It would be desirable, where possible to maintain duplicate material in several widely spaced geographic regions so as to insure against accidental loss.

9. Replenishment of material

Replenishment of stored wild animal germplasm would of necessity be on an opportunistic basis and would depend upon: (i) the planned conservation strategies for the species concerned; (2) the source of the germplasm and the difficulty in replenishing it; (3) the storage life with the method used; and (4) the amount of genetic change that is taking place in a population included in storage.

10. Future uses of wildlife germplasm

Wildlife genetic material stored in an FAO Regional Animal Gene Bank would be put to the following uses:

1. To help eliminate the risk and expense of shipping captive wild animals for breeding purposes.

2. To inseminate females in the absence of the male and thus eliminate the problem of the incompatible pair.

3. To reduce the hazard of disease transmission from the introduction of new animals for breeding.

4. To introduce new blood lines into captive gene pools (and, indirectly, into wild ones).

5. To provide an insurance against catastrophes and natural disasters.

6. To provide genetic material from captive or wild stocks for infusion into genetically static domestic breeds for the promotion of hybrid vigour and to introduce new and economically valuable traits.

7. To allow the rapid multiplication of an endangered species by interspecies embryo transplant.

   (Seager, 1983; Wildt et al, 1989)

11. Sample size

Sample size in genetic programmes will be influenced by both genetic considerations and cost. For a dominant gene, semen to produce 20 viable offspring (possibly 100 units) should suffice but somewhat more semen (possibly 200 units) may be needed to preserve a recessive gene. The preservation of quantitative variation within a population would require about 100 units of semen from each of 10 to 20 unrelated males. (A unit of semen is the amount appropriate for one insemination.)

Storage of frozen embryos would be desirable where it is important to preserve the capability of reconstituting a species or strain and maintaining it with low inbreeding. At least 20 offspring of each sex has been suggested as a reasonable sample for domestic pigs, sheep and goats and twice that number of females for cattle. At present no estimates are available for wildlife. If the pregnancy rate following transfer of frozen-thawed embryos is 30% and survival of the resulting offspring to breeding age is 80% then 167 to 333 embryos per stock should be preserved. The sample should represent 20 or more unrelated or only slightly related parents. (Animal Germplasm Preservation and Utilization in Agriculture, Council for Agricultural Science and Technology, Report No: 101, Sept., 1984).

12. Storage costs

It is suggested that the storage costs of cryopreserved wild animal germplasm stored in FAO Regional Animal Gene Banks should be met by such agencies as the International Conservation Financing Program, the World Bank and non-governmental organizations like the World Wildlife Fund and other charities.

13. Future prospects

Since preservation of genetic material from a species is one of the keys to ensuring diversity, the development of reproductive technology of exotic species, such as the cryopreservation of gametes, embryo transfer and artificial insemination, should be emphasized and supported. Ex situ wild animal conservation programmes that are dependent upon the long term preservation of genetic variation should apply this technology as it becomes available in view of the increased realization that captive breeding programmes are essential to prevent many species from becoming extinct. Loss of genetic diversity could also limit the potential of a population to adapt to new environments when reintroduced to the wild.

A large amount of basic research is urgently needed before application of this new technology will be routine for maintaining captive populations. It is hoped that the urgency will be recognized by many more scientists, worldwide, than at present and that ex situ conservation programmes will become the nuclei of genetic material for dwindling populations of wild animals. Extinction for some may be softened by the “frozen zoo concept”, which may turn out to be the single most important reproductive technology developed for wild animals during this decade. Its effects will reach centuries into the future for many species (Dresser, 1988).

14. Conclusion

The use of reproductive biotechniques such as cryopreservation of semen and embryos as a means of preserving and maintaining genetic diversity is currently the focus of great interest in international conservation circles.

The cryopreservation and storage of haploid gametes, diploid embryos and cell cultures at low temperatures (- 196°C) offers unique opportunities for facilitating the propagation of wild species and ensuring the conservation of genetic diversity. The development of banks of cryopreserved germplasm will:

• reduce the number of animals needed to ensure high levels of genetic diversity;

• facilitate the infusion of germplasm from wild populations into captive breeding programmes;

• provide insurance against the loss of diversity from epidemics, natural disasters and social/political upheavals.

Similarly, DNA and tissue banks will permit retrospective genetic analysis of founder animals. Research is needed to develop simple, alternative preservation approaches including vitrification, freeze drying and cold storage.

The widely dispersed geographic distribution of species requires international research programmes that will be conducted in many countries. To facilitate these efforts, formal international agreements and collaborative projects will need to be encouraged.

While most work has focussed on male gametes due to the relative ease of collecting and using spermatozoa, recent research indicates that preserving male gametes alone may not yield the desired results because (i) mitochondria and other cytoplasmic inheritance factors are transmitted only from mother to offspring; and (ii) functional expression of some nuclear genes is determined by whether the gene was inherited from the male or the female parent.

The cryopreservation and use of oocytes and/or embryos from female founders provides the only means of conserving maternally transmitted genetic variability (Rall et al., 1989).

The practical application of embryo cryopreservation to the propagation of wild animals will require:

1. a suitable number of fertile founder animals;

2. an extensive understanding of the behaviour and reproductive physiology of the species for the control of reproductive cycles;

3. safe anaesthetic techniques for manipulative procedures;

4. systematic sampling of female founders based on sound genetic criteria;

5. a fertile population of embryo recipients of the same or taxonomically similar species; and

6. secure, long term storage facilities. (Rall et al., 1989)

It should perhaps be stressed that while artificial insemination, embryo transplant and cryopreservation techniques may, in future, come to play an increasing role in the preservation of endangered species, it is doubtful whether an artificial breeding programme alone would be sufficient to save a species from extinction over an extended period. Conservation and enhancement of the species habitat and the maintenance of an adequate gene pool are also of vital importance and artificial breeding techniques can be useful only as part of an overall conservation programme.

Acknowledgements

The author wishes to thank Drs. B.L. Dresser, D.E. Wildt and W.F. Rall for most helpful discussions during the preparation of this paper.

References

Balmaceda, J.P., Heitman, T.O., Garcia, M.R., Pauerstein, C.J. and Pool, T.B. (1986). Embryo cryopreservation in cynomologus monkeys. Fertil. Steril. 45: 403–406.

Bennett, S.D. and Foster, W.R. (1985). Successful transfer of a zebra embryo to a domestic horse. Equine vet. J. Suppl. 3: 53–62.

Bowen, R.A. (1987). Fertilization in vitro of feline ova by spermatozoa from the Ductus deferens. Biol. Reprod. 17: 144–147.

Bowen, M.J., Platz, C, Brown, C.D. and Kraemer, D.C. (1982). Successful artificial insemination and embryo collection in the African lion (Panthera leo). Am. Assoc. Zoo. Vet. 57–59.

Cherfas, J. (1984). Test tube babies in the zoo. New Sci., 16–19.

Densmore, M.A., Bowen, J., Magyar, S.J., Amoss jr, M.S., Robinson, R.M., Harms, P.G. and Kraemer, D.C. (1987). Artificial insemination with frozen thawed semen and pregnancy diagnosis in addax (Addax nasomaculatus). Zoo. Biol. 6: 21–29.

Douglass, E.M. and Gould, K.G. (1981). Artificial insemination in lowland gorilla (Gorilla g. gorilla). In Animal Proc. Am Assoc. Zoo. Vet.

Dresser. B.L. (1986). Embryo transfer in exotic bovids. Int. Zoo. Yearb. 24/25: 138–142.

Dresser, B.L. (1988). Cryobiology, embryo transfer and artificial insemination in ex situ animal conservation programs. In Biodiversity (eds. E.O. Wilson and F.M. Peter). Nat. Acad. Press, Washington, D.C.

Dresser, B.L., Kramer, L., Pope, C.E., Dalhausen, R.D. and Blauser, C. (1982a). Superovulation of African eland (Taurotragus oryx) and interspecies embryo transfer to Holstein cattle. Theriogenology, 17: 86 (Abstr.).

Dresser, B.L., Kramer, L., Reece, B. and Russell, P.T. (1982b). Induction of ovulation and successful artificial insemination in a Persian leopard (Panthera pardus saxicolor). J. Zoo. Biol. 1(1): 55–57.

Dresser, B.L., Kramer, L., Dalhausen, R.D., Pope, C.E. and Baker, R.D. (1984a). Cryopreservation followed by successful embryo transfer of African eland antelope. In Proc. 10th Int. Cong. An. Reprod. and Art. Insem. pp. 191–193.

Dresser, B.L., Pope, C.E., Kramer, L., Kuehn, G., Dalhausen, R.D., Maruska, E.J., Reece, B. and Thomas, W.D. (1984b). Successful transcontinental and interspecies embryo transfer from bongo antelope (Tragelaphus euryceros) at Los Angeles Zoo to eland (Tragelaphus oryx) and bongo at the Cincinnati Zoo. In Ann. Proc. Am Assoc. Zoo. Parks and Aquar. pp. 166–168.

Dresser, B.L. et al (1989). In Cat News, published July 1989 by SSC/IUCN Cat Specialist Group.

Drost, M. (1983). Reciprocal embryo transfer between water buffaloes and cattle. In Proc. Ann. Meet. Soc. for Theriogenology pp. 63–71.

Foster, W.R. and Bennett, S.D. (1984). Non-surgical embryo transfer between a Grant's zebra and domestic quarter-horse: A tool for conservation. In Ann. Proc. Am. Assoc. Zoo. Vet., pp. 181.

Gorman, J. (1980). Brave new zoo. Discover, 1: 46–49.

Hare, W.C.D (1985). Diseases transmissible by semen and embryo transfer. O.I.E., Paris, 36 pp.

Hearn, J.P. and Summers, P.M. (1988). Semen banking - Is it not feasible for captive endangered species? Oryx, 22: 3, July 1988.

Howard, J.G., Pursel, V.G., Wildt, D.E. and Bush, M. (1981). Comparison of various extenders for freeze-preservation of semen from selected captive wild ungulates. J. Amer. vet. med. assoc. 179: 11, 1157–1161.

Kraemer, D.C., More, G.T. and Kramer, M.A. (1976). Baboon infant produced by embryo transfer. Science 192: 1246–1247.

Kramer, L., Dresser., B.L., Pope, C.E., Dalhausen, R.D. and Baker, R.D. (1983). The non-surgical transfer of frozen-thawed eland (Tragelaphus oryx) embryos. In Ann. Proc. Am. Assoc. Zoo. Vet., pp. 104–105.

Kydd, J., Boyle, M.S., Allen, W.R., Shepherd, A. and Summers, P.M. (1985). Transfer of exotic equine embryos to domestic horses and donkeys. Equine Vet. J. Suppl. 3: 80–83.

Moore, H.D.M., Bonney, R.C. and Jones, D.M. (1981). Induction of oestrus and successful artificial insemination in the cougar (Felis concolor). In Ann. Proc. Am. Assoc. Zoo. Vet. pp. 141–142.

Myers, N. (1984). Cats in Crisis! Int. Wildl. 14: 42–52.

Plucknett, D.L. and Smith, N.J.H. (1986). Sustaining agricultural yields. Bioscience 36: 40–45.

Rall, W.F., Schiewe, M.C., Bush, M. and Wildt, D.E. (1989). Cryopreservation of oocytes and embryos collected from wildlife species. Soc. for Cryobiology, 26th Ann. Meet. June, 1989.

Reed, G., Dresser, B.L., Reece, B., Kramer, L., Russell, P., Pindell, K. and Berringer, P. (1981). Superovulation and artificial insemination of Bengal tigers (Panthera tigris) and an interspecies transfer to the African lion (Felis leo). In Ann. Proc. Am. Assoc. Zoo. Vet. pp. 136–138.

Reid, W.V.C. and Miller, K.R. (unpubl.). Keeping options alive: The scientific basis for the conservation of Biodiversity. WRI, Washington, D.C.

Salati, E and Vose, P.B. (1983). Depletion of tropical rain forests. Ambio 12: 67–71.

Seager, S.W.J. (1981). A review of artificial methods of breeding in captive wild species. Dodo, Jersey Wildl. Preserv. Trust. 18: 79–83.

Seager, S.W.J. (1983). The breeding of captive wild species by artificial methods. Zoo. Biol. 2: 235–239.

Singh, E. (1988). Potential of embryos to control transmission of disease: A review of current research. Animal Disease Research Inst., Nepean, Ontario, Canada.

Stover, J., Evans, J. and Dolensek, E.P. (1981). Interspecies embryo transfer from gaur to domestic Holstein. In Ann. Proc. Am. Assoc. Zoo. Vet. pp. 122–124.

Whittingham, D.G., Leibo, S.P. and Mazur, P. (1972). Survival of mouse embryos to -196°C and -269°C. Science 178:411–414.

Wildt, D.E., Schiewe, M.C., Schmidt, P.M., Goodrowe, K.L., Howard, J.G., Phillips, L.G., O'Brien, S.J. and Bush, M. (1986). Developing animal model systems for embryo transfer technologies in rare and endangered wildlife. Theriogenology, 25: 1, 33–51.

Wildt, D.E., Howard, J.G., Bush, M. and Rall, W.F. (1989). Strategies for cryopreservation of spermatozoa from wildlife specie. Soc. for Cryobiology, 26th Ann. Meet. June 1989.

Wilmut, I. (1972). The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. 11: 1071–1079.

Wilson, E.O. (1988). The current state of biological diversity. In Biodiversity (ed. E.O. Wilson). Nat. Academ. Press, Washington, D.C.