J . Zoo/., Lond. (1996) 238, 531-544

Genetic resource banks in wildlife conservation

W. V. HOLT*, P. M . BENNETT*, V. VOLOBOUEV’IJ

*Institute of Zoology, Zoological Society of London, Regent’s Park, London NWI 4 R Y VSection de Biologie, Institut Curie, 26 Rue d’Ulm, 75231 Paris, CEDEX 05, France

A N D P. F. WATSON

The Royal Veterinary College, Royal College Street, London NW1 OTU

(Accepted 14 Decenzber 1994)

(With 2 figures in the text)

Recent advances in reproductive technologies for animal breeding, together with improvements in techniques for storage of gametes and embryos, have encouraged the view that the time is now appropriate for developing systematic policies of germplasm banking. Such activities would aim to support more conventional breeding programmes for threatened species, by providing the opportunity to store valuable genetic material for use on some future occasion. A number of pertinent issues should be addressed, however, before embarking upon the large scale imple- mentation of genetic bank programmes. This review raises and discusses some of the issues involved.

Contents

Introduction . . . . . . . . . . . . . . . . . . The principles and limitations of genetic resource banks Types of genetic resource banks . . . . . . . . . . Advances in techniques of assisted reproduction. . . .

Potential benefits for metapopulation management . . Organization and integration of genetic resource banks Current activities within the EC programme . . . .

Review of cryopreservation techniques . . . . . . Experimental initiatives within the EC project. . . .

Summary . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . . . .

Breeding programmes for threatened species . . . .

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Introduction

Genetic resource banking is the term given to the storage of gametes and embryos from threatened populations with the specific and deliberate intention to use them in a breeding programme at some future occasion. The concept has recently been embraced as an essential tool among the strategies for the conservation of the diverse wildlife of the planet (for example, see Wildt, 1992). While it is easy to appreciate the superficial appeal of such a project, some serious questions need to be answered before it can play an effective part in the overall plan. In particular,

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will this be a useful scientific activity in the conservation of species and habitats? This question is important in decisions concerning allocation of limited resources. However, it is difficult to answer at present because conservation biology is an emerging discipline with few rigorous evaluations of the reasons for particular successes or failures. This review describes the anticipated value of genetic resource banks. but also cautions that considerable forethought is needed to avoid expensive errors which could diminish considerably the value of the stored material.

The principles and limitations of genetic resource banks

The rapid progress of assisted reproductive technology in human medicine and farm animal production has been regarded by some as holding considerable promise for breeding endangered wild animals. It has been claimed that these techniques offer the potential to ease deleterious processes such its the rapid decline and fragmentation of populations, and the accompanying loss of genetic diversity. which precipitate the extinction of species (e.g. Ballou, 1984, 1992; Benirschke, 1984: Dresser. 1988: Holt & Moore, 1988; Mace, 1989; Reid & Miller, 1989). However, despite the sporadic demonstration that some of these techniques can be applied to breed non-domesticated species maintained in captivity. there have been few attempts to apply the technologies on a routine basis within the context of a captive breeding programme. Furthermore. they are not being used seriously to aid conservation programmes for threatened species in their natural habitats.

Despite this apparent failure. so far, to translate the potential offered by reproductive technology into practical and useful applications, it is clearly worth continuing to explore ways in which technological advances can be used to underpin and support conservation efforts. In this respect, one suggested activity is to undertake a programme aimed at preserving genetic material, specifically spermatozoa. oocytes. and embryos, from endangered and threatened species. At face value the goal of such a programme is simple and directly useful; it effectively lengthens the 'genetic lifespan' of valuable individuals. who can continue to contribute to the genetic diversity of populations until the stored material is exhausted, perhaps long after their death .

Before such a programme is initiated, i t is important to ensure that the stored material will be useful when required. For example. failure to check for the presence of microorganisms may inadvertently lead to disease transmission; use of inappropriate cryopreservation procedures may result in poor quality of stored gametes and embryos, whilst careful identification of the frozen samples and characterization of their origins is clearly essential. At present, successful and reliable cryopreservation procedures for oocytes of' any species except the mouse, hamster, and rabbit have yet to be developed. I t can be argued, therefore, that oocytes should not be considered useful material until further progress has been made. Similar arguments can also be used to exclude spermatozoa and embryos from many animal groups for which reliable cryopreservation techniques do not yet exist. Examples include most wild rodents, all marsupials, and surprisingly even domestic pigs, where reliable embryo freezing has not yet been achieved. These limitations demonstrate that both species, and type of germplasm to store, must be carefully considered and justified before embarking upon a genetic bank project.

In parallel with a germplasm bank, a collection of associated cells and sera would provide helpful. even essential. backup. Indeed, any conservation programme would benefit from such a collection. Cells such as fibroblasts. which are easily obtained from small skin or muscle samples, would be available for DNA and protein analysis when needed. Since fibroblasts can be cultured

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from small numbers of cells, and the cultures themselves cryopreserved and multiplied, any future DNA analysis would not involve destruction of the germplasm stocks. Thus unforeseen questions about the origin and relatedness of individuals and the evolutionary systematics of the taxa under consideration could be answered adequately in the future. Similarly, future questions about the disease status of samples could be investigated if parallel samples of serum were collected together with germplasm. Unfortunately, serological testing would inevitably involve the destructive use of serum samples, and therefore decisions will have to be made in advance about the number and volume of samples to be stored.

Even a preliminary consideration of genetic resource banking quickly reveals that many complex questions have to be considered prior to implementing a serious long-term programme. A group of biologists interested in these issues met in February and March 1994. Under a two year research programme funded by the European Commission (DGXII), this group aims to review the current status of various reproductive and cryopreservation technologies, to formulate guidelines for establishing new genetic resource banks, and to make recommendations for future research and actions that will direct these activities so they meet conservation goals. As part of this process, participating laboratories will carry out model experiments and collaborative investigations. In this paper, we provide a brief summary of the background to the project, and describe the activities that will be undertaken following this first meeting.

Types of genetic resource banks

Genetic resource banks can be organized for different purposes and have multiple uses. The primary focus of the first meeting was the storage of germplasm, i.e semen, eggs, and embryos, and its use in animal breeding programmes. Storage of germplasm offers considerable benefits in animal breeding programmes (see below) which are unavailable through the use of somatic cells and tissues alone.

Genetic resource banks of frozen or fixed somatic cells from threatened species have important conservation merits, however. For example, molecular and cytogenetic analyses of these materials can assist in defining the pedigrees of individuals, the distinctiveness of populations, and the evolutionary relationships between species. The same approach, i.e. cell banking and appropriate analyses of cell lines, may serve as a useful tool for the rapid characterization of species as yet undiscovered. Some estimates predict that these are even more numerous than those species already described (Groombridge, 1992). With the advent of the polymerase chain reaction (PCR) method of DNA amplification, museum specimens of tissue and bone can be regarded collectively as a genetic resource bank for research. As such they are important sources of material for basic and applied research in evolutionary systematics, biotechnology, medicine, and agriculture.

A number of specialized cell banks have been set up with scientific research as the main focus. These include the European Collection of Animal Cell Cultures (ECACC) at Porton Down (Salisbury, UK) which accepts novel cultured cell lines, stores them, and redistributes them when required by researchers. Similar cell culture collections exist in other countries (for details, see the results of a survey by Dessauer & Hafner, 1984). Very few cell lines from endangered species are stored in these collections, however. One direction within the current project is therefore the identification of centres throughout the world which do, in fact, hold established and fully characterized cell lines from wild and endangered species.

A number of centres have already established genetic resource banks which store frozen

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germplasm. Mouse embryos from the numerous mutant strains generated through several decades of research are stored in this way, and when required they can be recovered and transferred to recipient animals. Detailed knowledge of mouse reproductive physiology has enabled this system to function effectively, and serves to highlight the goals for similar projects with other species.

Genetic resource banks also exist for domestic animals of agricultural importance. Dairy cattle are routinely bred by the use of artificial insemination with frozen semen, and cattle breeding centres with stocks of frozen semen therefore exist throughout the world. The success of this system is partly fortuitous in that high conception rates are obtainable with relatively low numbers of viable spermatozoa, but has also developed because the physiology of the bovine female reproductive cycle is well documented. Some pig-breeding companies have recognized the need for long-term storage of spermatozoa from important genetic lines and this has prompted them to formulate genetically based policies for semen storage. Although these stocks are mainly privately owned, they constitute true genetic resource banks as their potential usage is viewed on a long-term basis. The Rare Breeds Survival Trust in the UK implements similar policies for rare cattle and pig breeds of potential agricultural value. No similar programmes currently exist for endangered species.

Fears that prolonged storage of gametes or embryos in liquid nitrogen may lead to loss of fertility or an increase in genetic abnormalities appear to be groundless. The fertility of ram semen was found to be undiminished after 16 years of storage (Salamon, Maxwell & Evans, 1985). Indeed. i t has been calculated that 50% of cells would be killed by background cosmic radiation only after 3000- 10 000 years. Surprisingly. the foetal abnormality rate after artificial insemination in humans has been consistently lower than in the general population and the abortion rate is also reduced (Sherman, 1978). Effective and careful long-term management of the stored samples is therefore probably the most important influence determining the main- tenance of gamete and embryo quality after freezing.

At present. the activities of existing genetic resource banks for endangered species are either non-existent or are not co-ordinated on a national or international basis. One of the main aims of this project is to establish common goals and standard procedures so that these activities can be systematically planned, co-ordinated, and monitored on an international basis.

Advances in techniques of assisted reproduction

Considerable effort has been expended on establishing optimum methods of semen handling and preparation that maximize the chances of successful insemination when the semen sample is thawed. These technological advances have mainly been the result of research and development in agricultural, fisheries. and clinical laboratories where direct economic or social advantages are easily identifiable. Conservation biology can benefit from the main thrust of these studies, although specific problems with individual species remain to be investigated. Progress on technical issues, such as optimization of freezing procedures, has now reached the point, however. where i t is realistic to consider germplasm storage from selected species as an additional tool for supporting the viability of endangered populations (Holt & Moore, 1988). Before undertaking such a venture it is essential to consider limitations to the eventual efficient use of stored materials. Such limiting factors are partly centred around the current status of reproduc- tive technology as outlined below.

For mammalian species the effectiveness of artificial insemination and embryo transfer using

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frozen materials are determined as much by variations in reproductive physiology between species as by technical problems that arise during the cryopreservation process. The timing of inseminations, oocyte recovery, and embryo transfer is particularly crucial, requiring accurate knowledge of the female reproductive cycle (for review, see Hodges, 1992). This is lacking for many species (Holt & Moore, 1988). The cryopreservation step imposes a major stress, however, upon the viability of spermatozoa, significantly shortening their fertile lifespan in the female reproductive tract (Mattner, Entwhistle & Martin, 1969). Small errors in insemination timing, perhaps only by a few hours, could easily mean that fertile spermatozoa and oocytes are never present at the same time. Similarly, in contemplating oocyte recovery, in vitro fertilization, and embryo transfer, it is essential that oocytes are recovered, and embryos replaced, at precisely timed stages of the reproductive cycle. It is clear, therefore, that semen banking will be most effective at present for species whose reproductive physiology is well characterized, and where appropriate techniques exist for manipulating or monitoring the female reproductive cycle. The implication of this argument is double-edged. On one hand, gene bank projects are most likely to succeed with the most highly studied species, whilst on the other, the most endangered, and least studied, species are also the least likely candidates to benefit from the technology. One possible way out of this dilemma would be to set up a ‘foresight’ exercise, where candidate species are identified as those mostly likely to be threatened in the future, but where study groups can still be assembled for basic biological study.

Farm animal production systems routinely use reproductive technologies such as artificial insemination and embryo transfer to maximize desirable gene lines. At present, these techniques are not routinely used with non-domesticated species. For genuine germplasm banks to succeed in practice, it will be necessary, however, to develop the appropriate reproductive technologies to a high level of sophistication, where managers of captive breeding programmes could call upon the use of particular semen samples or embryos with reasonable confidence of success.

Methods for semen collection, transport and insemination are particularly well-established in birds (especially cranes, raptors, and gamebirds), and considerable work has been undertaken to establish similar procedures for mammals (for reviews, see Watson, 1990; Holt, 1992). Conse- quently, artificial insemination using frozen/thawed semen is, at present, the most practical method for utilizing genetic resource banks. In principle, embryo storage and ultimately oocyte storage should have a valuable role, but for exotic mammalian species there have been few reports of successful embryo cryopreservation and transfer, followed by a birth. Techniques of mammalian oocyte storage, and maturation of oocytes from slices of frozen ovarian tissues are in progress (for review, see Whittingham & Carroll, 1992), but, except for laboratory mice, these are very much at the research stage.

Looking further forward, the latest developments in reproductive technology might be employed to maximize the chances of using genetic material. Intracytoplasmic sperm injection (ICSI) is currently gaining popularity as an alternative to IVF for infertile human couples. For ICSI, individual spermatozoa are injected directly into oocytes. In principle, this offers a means of using poorly preserved spermatozoa, provided a supply of oocytes is available. It is by no means clear, however, what governs the success of this technique, and highly motile spermatozoa still seem to have an advantage in generating viable embryos. Embryonic stem cells offer another potential avenue for generating embryos, provided the cell lines can be generated. These cells are derived from the inner cell mass line of mammalian blastocyst, and must be cultured in vitro then fused with ‘immortalized’ cells to produce a cell line which is capable of regenerating morpho- logically normal two- to four-cell embryos. If transferred to oviducts, these embryos are capable

536 W . V . HOLT ET . 4 L .

of development (for review, see Moor, Sun & Galli, 1992). At present, this technology is still very much at the initial stages. and it is unclear whether it will eventually become a practical technique for anything but common species. However. developments in this field might eventually lead to the generation of embryonic cells from stored somatic cell lines.

Considerable interest is currently being generated by the prospect of gene banks for aquatic species. It is possible to preserve spermatozoa from some fish species (e.g. salmonids, Scott & Baynes. 1980; Tilopin, Rana & McAndrew, 1989), and progress is being achieved with embryo cryopreservation in aquatic invertebrates such as rotifers, blue mussels, and oysters. This research is being driven by the requirements of industrial aquaculturists, who already keep live 'wet' gene banks of their stock. Within the current project, Drs Krishen Rana and Gordon McGregor Reid are investigating the feasibility and logistics of setting up a gene bank for the endangered cichlid fish of lake Victoria using cryopreserved spermatozoa.

Breeding programmes for threatened species

Over the last 15 years, there has been a great increase in the number of organized captive breeding programmes for threatened species (IU DZG/CBSG, 1993). Many of these programmes aim to provide animals for reintroduction to their natural ranges if this proves desirable. Considerable efforts have been made to provide a theoretical framework, mainly drawn from the field of population biology, in which to operate these breeding programmes (Lacy, 1987; Foose & Ballou, 1988). Small isolated populations are prone to processes such as the loss of genetic diversity and fluctuations in size, age structure, and sex ratios. Many of these captive breeding programmes have formal plans to manage small captive populations, which attempt to minimize these and other processes that are believed to cause extinction. For example, a common goal is to maintain a minimum of 90% genetic diversity in a demographically stable population for 200 years (Soule r t al., 1986). A range of management strategies have been established which aim to achieve this. For example, it is common for co-ordinators of captive breeding programmes to calculate statistics that estimate the degree of relatedness between individuals and subse- quently to plan niatings co-operatively between zoos and other organizations that aim to minimize inbreeding. More complex methods for estimating gene losses from wild-caught founder animals are also used, and management strategies are employed to minimize predicted loses of rare alleles. Inbreeding itself poses a threat to the viability of small populations. For example. Templeton & Read (1983, 1984) developed a captive breeding programme for Speke's gazelle (Gmzella speliei 1, starting with only one male and three females. They found that initial inbreeding depression was severe, leading to the death of 80% of live-born individuals before maturity. Templeton ( 199 1) later noted that the inbreeding depression was successfully reduced by a managed breeding programme, and no longer threatens the herd.

At present, the achievements of these programmes, as measured by captive-bred animals returned to the wild. have been modest (IUDZG:'CBSG, 1993). This situation, together with the long time-scales of these programmes, make it difficult to evaluate the effectiveness of the population management strategies employed to maximize factors such as genetic diversity. Nevertheless, a number of workers have emphasized the potential benefits of using resource banks of frozen germplasm in threatened species breeding programmes (e.g. Ballou, 1984, 1992: Benirschke. 1984; Dresser, 1988; Holt & Moore, 1988; Mace, 1989; Johnston & Lacy, 1991; Rall, 1991; Mace, Pemberton & Stanley, 1992). Furthermore, an action plan for the establishment of a genome resource bank for tigers was prepared by Wildt et al. (1993) under the joint auspices of

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the American Association of Zoological Parks and Aquaria (AAZPA) and the IUCNjSSC Captive Breeding Specialist Group (CBSG). Many of the issues raised within this action plan are the same as those discussed within this review.

Potential benefits for metapopulation management

Given that insufficient space will always be available for storage of material, the nature of the stored material must reflect the goals of the banking programme. It is therefore essential to identify these goals before proceeding further with any technical work. However, identification of the goals themselves can be problematic; if the purpose is to preserve 90% of the genetic diversity in a population, a suitable sampling strategy would be very different from simple efforts to hold representative individual samples. In this context, even the choice of a subject species poses difficulties. Would the goals of genetic banking be more effectively attained by choosing species which still exist in large numbers? Conversely, is it already too late to use genetic resource banking effectively for highly endangered species?

What are the potential benefits of using genetic resource banks for supporting threatened animal populations? It should be emphasized that genetic resource banks and assisted repro- ductive techniques cannot be used to replace living animal populations whether in the wild or captivity. Most workers have emphasized the increased opportunities they allow for integrating the management of isolated populations (wild and/or captive) of threatened species in planned species recovery programmes, a process which has been termed ‘metapopulation management’. While these benefits will usually only accrue if living animal populations can be maintained so that stored material can be used, it has been suggested that some techniques, such as interspecific embryo transplantation, may have an important role when living populations have declined to critically low numbers or gone extinct.

A range of potential benefits of using genetic resource banks of frozen germplasm for conservation programmes requiring metapopulation management have been identified. These include:

0 Storing germplasm from a relatively small number of individuals per species, carefully chosen on the basis of pedigree analysis and molecular screening, enables a high proportion of genetic diversity to be preserved indefinitely, thereby providing some protection against catastrophes.

0 A large number of species can be banked and is limited only by the number of containers in the bank( s).

0 Managed breeding or recovery programmes are facilitated because transportation of frozen gametes has many advantages over moving live animals. For example, the opportunities for gene flow within and between small isolated wild and/or captive populations will be greatly enhanced.

0 Storage of germplasm from animals of known provenance, which have been genetically characterized by karyotype and biochemical or molecular markers, can secure the integrity of a genepool against the threat of introgression.

0 The lifespan of an individual is extended for as long as its germplasm is viable and the genetic resource bank is maintained. This means that generation times can be extended to reduce genetic drift, and therefore smaller population sizes are required to meet genetic goals. It has been argued that more space is therefore made available for breeding threatened species in captivity.

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0 Attempts to maxiniize genetic diversity can be improved by, for example, storing and using germplasm from genetically unrepresented potential founder animals, descendants of under- represented founders. or doomed wild animals. Inbreeding can be minimized by restoring germplasm to unrelated or the most distantly related animals.

0 Effective population sizes (Ne) can be managed by using stored germplasm to help equalize family sizes. by manipulating age-specific fertility rates, and by attempting to control sex ratios.

0 Unlike the situation in domestic animal production, artificial selection must be avoided in breeding programmes for threatened species because it reduces genetic diversity. Stored germplasm of donor animals has a 'hidden phenotype' and this would help to minimize the effects of keeper selection in living populations. Likewise, some of the effects of local adaptation to unnatural habitats could be countered.

0 Husbandry problems, such as incompatible pairs that fail to breed, could be circumvented. 0 As further advances in reproductive biology and biotechnology are made, and as long as stored

germplasm exists, there will be the potential for treating infertility in genetically important animals, screening for disease, restoring 'lost genes' or choosing the sex of offspring.

Many of these potential benefits would be substantial. In particular, the economic, legal, administrative, disease, and welfare advantages of transporting frozen germplasm instead of live animals would reap enormous immediate benefits in wildlife conservation programmes. In the longer term. the increased opportunities for gene flow between isolated populations will be vitally important for the success of many species recovery programmes. However, many of these perceived advantages can only be realized in conservation programmes if attempts are made to establish systematic procedures for operating genetic resource banks.

Organization and integration of genetic resource banks

For genetic resource banks to be effective, i t will be necessary to develop systematic methods for evaluating many questions. For example: how many banks are required to meet the goals of a conservation programme? (This deceptively simple question presupposes that the goals are well defined, which may not necessarily be true.) Where should the bank(s) be physically kept? How long will they need to be securely maintained? Which organizations should finance and administer them? What criteria should be used to select species for banking? From how many populations should material be collected and how should they be defined? How do we select which individuals should be donors and recipients? How many donors will be required to meet defined genetic and demographic objectives? Should stored material be used within each generation? What current legislation limits the international transportation of frozen germ- plasm?

Underlying the concept of the genetic resource bank for wild, endangered species is the somewhat optimistic belief that animal populations will thereby be assisted to survive until suitable habitats can be found for reintroduction. This forward looking approach has a considerable influence upon the way in which the banks should be organized. If the extant living zoo populations are considered as live genetic resource banks, they were originally derived from wild, threatened, and fragmented populations. Captive animals breed in zoos through many generations (see Fig. 1). but the number of founder animals upon which the captive population is

GENETIC RESOURCE B A N K S

Current scenario for assisting conservation efforts by

captive breeding

Threatened fragmented

wild populations

I I

I

539

FIG. 1 . Schematic diagram showing interrelationships between different animal populations involved in captive breeding and reintroduction programmes. Several zoo-bred generations separate the founder populations from those eventually destined for reintroduction. The original wild founders may have been deliberately recruited into captive breeding programmes for species conservation.

W V HOLT E T A L

Integrated management of wild and captive populations using a genebank

t - _

,

/ , Reserve bank

' C o r n d o r s { # [ Wild bank 'r, Captive bank

4

. .

Threatened f ragrnented

wild populations

Co-ordinated captive

breeding programmes

FIG;, 2 Suggested srrtiteg) for integrating genetic resource banks into the breeding and management of threatened specie\. I t i \ cn\isaged tha t material from captive and wild sources should be physically separated. 'Wild' banks could be regarded AS analogous 10 \\lidlife corridors. where some interchange of genetic material with captive animals could be pcrmitied. A reserie hank of stored material Lrould only be accessed in cases of disaster or emergency.

based is frequently small. Genetic drift and inbreeding may alter the captive animals such that they eventually differ significantly from the original founders, for example in body weight, colorat ion, disease. and parasite resistance. Germplasm derived from such a captive population should probably be physically separated from that obtained directly from wild populations. This is further emphasized when it is considered that the disease status of wild animals will probably be unknown. whilst conversely. captive animals are routinely screened for infections as part of normal husbandry. To make optimal use of the genetic resources contained within germplasm banks. it will ultimately be necessary to transfer germplasm from captive populations to the wild and rkr wrm. Perhaps i t will therefore be necessary to set up an intermediate population of animals. with thc equivalent of 'wildlife corridors' between them and the truly wild population

GENETIC RESOURCE B A N K S 54 I

(Fig. 2). This mechanism would allow wildlife managers to control the ultimate interaction between differently derived populations.

Organizations such as the F A 0 (United Nations, Food and Agriculture Organization), EEAP (European Association of Animal Production) and the IUCN/SSC Conservation Breeding Specialist Group have begun to address these questions. Computer simulations that model the outcomes of varying decisions about collection, storage, and utilization of germplasm on, for example, the proportion of genetic diversity retained in the target species, appear to offer great promise (Johnston & Lacy, 1991). However, these models will need to be tested empirically for a wide range of species.

Current activities within the EC programme

A number of activities are currently being undertaken to help assess optimum procedures for the organization of genetic resource banks. The group is addressing the following issues either by reviews of current procedures or by establishing them via model trials and experiments for a number of threatened species. The current activities are as follows:

Review of cryopreservation techniques

A detailed survey of current practices and their success for the cryopreservation of gametes, embryos and oocytes in all major animal groups is being undertaken. Attention will be focused upon season and method of collection, techniques for dilution and handling, cryoprotectant / diluent composition, methods for packaging samples, cooling and thawing procedures, post- thaw handling, and methods for evaluating the viability of gametes and embryos.

As a result of this investigation, it is anticipated that a code of practice will be formulated to provide detailed guidelines for the collection, handling, and storage ofsamples. It will be possible to make certain recommendations about the suitability of various cryopreservation procedures for particular tissues and cells from different species. Where such information is unavailable, it is important to know whether this is through lack of investigations or because standard procedures have been tried but found to be unsuitable.

Experimental initiatives within the EC project

A small number of practical activities are being undertaken as part of this project to investigate these and other questions:

I . Experiments are being undertaken at The Royal Veterinary College, London, to assess methods for controlling the transmission of micro-organisms at very low temperatures. As far as we can ascertain there is no information about the likelihood of viral or bacterial transmission between frozen samples within a liquid nitrogen container. Such knowledge is crucial to ensure that accidental contamination of samples does not occur during storage.

2. A model genetic resource bank is being established jointly by Chester Zoo and The Institute of Aquaculture, Stirling, for threatened Lake Victoria cichlid fish, some of which are already extinct in the wild. Cryopreservation of spermatozoa has been achieved for these species, but can its use make an impact on the genetic diversity of the live population?

3 . Artificial insemination using frozen semen is being attempted on a species of threatened

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gazelle ( G o d / t i tlrrriin 117h01.r) in Spain (Estafion Experimental de Zona Aridas, Almeria). This will test the practicality and success of adapting standard sperm cryopreservation protocols for use in a non-specialized centre, and employing laparoscopic insemination techniques in conjunction with artificial control of the reproductive cycle. If successful, semen samples from genetically important individuals will be stored in Almeria. Subsequently, it is planned to test the feasibility of importing semen into the UK from Spain for use in an artificial insemination programme. This will demonstrate whether or not animal health restrictions would seriously impede the realistic international exchange of genetic resources for wildlife conservation. A model programme involving semen freezing, transport and iiz vitro fertilization and artificial insemination in callithrichid monkeys is being undertaken jointly between Thc Institute of Zoology, London. and the Deutschprimatenzentrum, Goettingen, Germany. Semen freezing in these species is problematic because of the small volumes involved, low sperm survival rates and specialized post-thaw handling methods. For sperm banks to operate reliably, it is essential that techniques are not totally dependent upon the skills of certain laboratories or individuals, and such inter-centre collaboration will test this aspect. Software is being developed that helps to define the information that will be essential for effective management of genetic resource banks. and how local and international databases should be designed for integrated planning and monitoring of genetic resource banks to meet conservation goals. Files of complete catalogues of vertebrate species, with their current status according to IUCN, CITES. and ESA lists, are being created at the Museum National d'Histoire Naturelle in Paris. These files will contain information on the distribution of species and subspecies in European zoos. Such a file has already been completed for mammals and is in progress for birds. A survey is being conducted to identify existing European cell banks, which at present are largrly unco-ordinated. The aim of this work is to examine ways in which co-ordination can be achieved in the future. The Institute Curie in Paris. whose cell and tissue bank contains over 3000 cryopreserved cell lines. and tissue samples from over 300 mammalian species. is developing a computerized catalogue system. This is intended to serve as a model system, and will help to identify the information needed for accurate description of samples. A protocol for the genetic characterization of cell lines. including cytological, biochemical, and molecular analyses, is being developed for the unequivocal identification of specimens and species.

Summary

Genetic resource banks. in combination with assisted reproductive techniques, offer the prospect of substantial benefits in species conservation programmes that require active manage- ment of isolated populations. Currently, artificial insemination using frozen semen offers the greatest practical benefits. Systematic procedures are required for establishing and managing genetic resource banks and a number of projects on threatened species are in progress within the EC that will assist in defining these guidelines.

u'c \soLild like tco tl iank the European Commission (DGXII) for providing financial support towards this project.

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Appendix

List of' irzst i t i i t iot is pcir t ici)(i t ;rig ir i r l i is piogrmnine

CSIC. Museum of Natural Sciences. Madrid. Spain. CSIC. Estacion Experiniental de Zoiia Aridas. Almeria, Spain. Dept. Reproductive Biology, German Primate Centre, Goettingen. Germany. Faculteit der Geneeskunde. Afdeling Biochemie. Katholieke University, Leuven, Belgium. Faculty of Veterinary Medicine. University of Utrecht, Netherlands. Institut Curie. Section de Biologie. 26 Rue d'Ulm. Paris. Institute of Zoology. University of Genova. Genova. Italy. Institute of Zoology. Regent's Park, London. UK. Marwell Preservation Trust. Winchester. UK. Museum National d'Histoire Naturelle. Laboratoire Mamniifere et Oiseaux, Paris, France. North of England Zoological Society. Upton. Chester. UK. Royal Veterinary College. London. U K . University of Stirling. Institute of Aquaculture, Stirling. Scotland, UK. University of Alcala de Henares. Alcala de Henares. Madrid, Spain.