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CryoLetters 29(1), 43-52 (2008)  CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOBIOLOGY, WILDLIFE CONSERVATION & REALITY 1

William V. Holt

Institute of , Zoological Society of London, Regent’s Park, London NW1 4RY Email [email protected]

Abstract

Conservation is about protecting and nurturing species so that they can survive, not only now but also into the future. Ideally this means protecting genetically diverse populations and not simply breeding a few individuals. In principle, cryobiology offers the means to help maintain genetic diversity by storing genetically important germplasm that could reinvigorate populations in the future. Unfortunately the technical problems associated with this ideal goal still provide a major barrier to the practical use of technology. Sometimes these are technical problems with the cryobiology, but lack of basic biological information about unusual species, coupled with difficulties in obtaining such information, means that progress will be possible with only a few species that are subject to intensive scrutiny. The opportunities nevertheless exist for cryobiologists and reproductive to make useful and global contributions to species conservation. I argue here that there are often two mutually suspicious groups of biologists, who do not interact or even understand each others goals. If conservation biologists and biotechnologists were more prepared to join forces and share their expertise, there would be much improved prospects for achieving lasting success in the conservation of a small, but well targeted, number of threatened species. Keywords: artificial insemination, genetic resource banks, biobanks, cloning.

INTRODUCTION

Since the discovery that eggs could be fertilized in a laboratory Petri dish and that spermatozoa and could be frozen indefinitely, technologically minded reproductive biologists have been passionate about using these tools in practical ways to produce offspring. Applications for alleviating human infertility problems are carried out extensively: there is now an amazing list of options for correcting even the most serious cases of reproductive dysfunction. Spermatozoa can be used for artificial insemination, eggs can be recovered and fertilized in the laboratory, spermatozoa can be recovered from the epididymis or even from the testis and injected directly into the egg cytoplasm. As a last resort, couples can opt for donor , donor eggs, or even both. These advances have led to massive amounts of media attention, but have also tended to create two serious mistaken impressions. The first is

1 Originally presented at the Symposium “ Validation, Safety and Ethical Issues Impacting the Low Storage of Biological Resources” Society for Low Temperature Annual meeting held in Derby, UK - 12– 14 September 2007

43 that it is the job of reproductive biologists to produce offspring using high-tech infertility ‘fixes’ and second, that the creation of life in a test tube is easy. These notions have caused problems in the conservation community, the first being that traditional conservation biologists are often suspicious of technology. Equally, naïve managers and the public also often suppose that problems of breeding endangered species can be overcome simply by directly applying clinical and agricultural techniques. There are many factors to consider before adopting this suite of techniques. Most importantly is the simple truth that we have too little basic information on most species. How can in vitro fertilization be performed if methods for inducing sperm capacitation or ovulation do not exist? How can assisted breeding proceed if we do not know about the timing of ovulation, the preferred site of sperm deposition or the minimum number of sperm required for conception? These (and many other) real issues must be solved before any high-tech progress can be made with wildlife species, and certainly argue the need for many more basic investigations. The aim of this review is to provide an honest assessment of the current and future potential of reproductive technologies for conservation and genetic management of small populations. As this article also aims to assess the role of cryobiology in conservation, I will concentrate on the potential value, and its limitations, to be gained from , storing and using cryopreserved gametes, embryos and somatic cells. However, these cannot be discussed without considering the general role of reproductive technologies in conservation.

REPRODUCTION, CRYOBIOLOGY AND PERCEPTION

There is a perception problem; not only about cryobiology, but about reproductive biology as a whole, as the disciplines are poorly understood by colleagues in the wildlife community. Reproduction is not even listed under ‘topics of interest’ in major journals devoted to biodiversity conservation (see, for example, publication guidelines for the journals and Animal Conservation ). This is very surprising as most reproductive biologists would argue quite naturally that species would quickly become extinct if reproduction ceased. Cryobiology is widely regarded as a subset of the reproductive toolbox and for that reason suffers from the same, or possibly worse, lack of recognition. What lies behind this seems to stem from the widely differing perspectives and backgrounds between reproductive scientists and conservation biologists. Conservation biologists usually think in terms of endangered populations rather than individuals, while the hi-tech approaches favoured by the technologists typically operate only at the level of the individual animal. When procedures such as artificial insemination and transfer are used to overcome breeding problems in zoos, they are usually providing small scale solutions which may be useful in the context of genetically managed breeding programmes, but which may be completely out of touch with the global status of a species. Neither side of this disciplinary divide should be self-righteous in the belief that their own views are unassailable, but it is clear to me, as someone working at the interface between these groups, that this is often the prevailing attitude. Biotechnologists tend to consider that because their methods are improving all the time, it should be easy to save species by reproductive technology, while the conservationists often discount technology as being of little relevance to the global situation. The reality of the situation must lie somewhere in between and I will attempt to find it in the next few paragraphs.

CRYOBIOLOGY, CONSERVATION AND REALITY

The most obvious application of cryobiology in species conservation is to provide a hedge against factors that bring about extinction. Not only that, but in an ideal world, the

44 objective of a conservation programme should be to enable a species to flourish into the future and even to undergo the natural processes of evolution. In this sense, extinction may simply be a natural end point for some species. However, the latest (2006) red list of threatened species (http://www.iucnredlist.org/ ) lists 1093 mammals, 1206 birds, 341 reptiles, 1811 amphibians and 1173 fishes. A moment’s reflection shows us that it would be completely impractical and unrealistic to consider applying reproductive technologies to all of these. For the mammals, only slightly more than 50 wild species (including some that are not threatened) have ever been produced by any reproductive manipulations involving artificial insemination or embryo transfer (22). Of these, only about 16 have been bred using either cryopreserved semen or frozen embryos. This number represents the result of about 25 years of effort; not only that, but examination of the species represented in this list shows that most successes were not followed up and consolidated. Equivalent analyses can be presented for the other vertebrate groups. Several bird species have been bred using frozen semen (8), but nothing like the 1206 species currently under threat and a number of fish species have been bred by artificial means. Intensive research in aquaculture has provided much detailed information about semen preservation in fish species (1), but these are almost exclusively food fish. There is a possibility that some of these techniques may work for threatened species but these will have to be examined on a case by case basis. Embryo freezing in fish is one of the major unsolved problems in cryobiology, so at present there is no possibility of using this technique. Why are there so few species in the list? Bear in mind that these are wild species and that there is no comprehensive body of literature that researchers can draw upon. Moreover, these are “threatened” species and, by definition, there are few opportunities to study their . Even if researchers and practitioners want only simple information such as the of the female reproductive tract, so important for carrying out inseminations or embryo transfers, they will mostly have to find out about these things by experiment and observation. More complex information, such as how best to inseminate frozen semen or to transfer embryos, is only available if the species has been subject to intensive and well integrated research programmes. Relatively few such research programmes have been sustained for long enough to provide useful data and these have mainly been driven by dedicated individuals who have been able to maintain their efforts through their own enthusiasm and perseverance. Key examples of such integrated research programmes include the establishment of a major genetic resource bank programme to support the reintroduction of the black-footed ferret in the USA (17), a major international research programme on small wild felids (28) and some sustained programmes of research into the reproductive biology of Australian marsupials (18, 19, 29, 30). By virtue of their classification as threatened, it is clear that the species in question do not exist in large numbers. Developing research programmes with such species is therefore problematic from the outset. There are several pertinent issues here: one major objection is that research carried out on few animals may be difficult to justify on scientific grounds. Ethics committees may be reluctant to sanction experimental studies that are too small to provide robust data on the basis that the effort will be wasted. Breeding programme managers may also be reluctant to subject their animals to reproductive procedures, especially when invasive methods are discussed. Moreover, the intensive style of research needed to establish artificial breeding methods means that animals need to be managed intensively, almost, but not quite, ruling out applications to species in their natural habitats. These considerations lead to the general conclusion that reproductive technologies are only applicable to a small number of species, these mostly being the larger animals that are held in captivity under close management. In turn, this leads to the second conclusion that techniques involving cryobiology are best used to support breeding programmes for the same species. To support that argument I would draw attention to the hundreds of bat and rodent

45 species that are classified as being under some degree of threat. It would be impractical to consider setting up artificial insemination programmes to conserve these species when, given the right facilities, it is certainly easier to breed them by natural means or to protect their habitats. To counterbalance this plea for a degree of realism from some of the biotechnologists, I suggest that conservation biologists rarely recognise opportunities to invite reproductive specialists and cryobiologists to help with their species survival plans. This is a pity and has led to a number of missed opportunities. For example, a number of important reintroduction programmes have been undertaken over the last twenty years or so. A small population of Père David's deer bred at several zoos and wildlife parks in the United Kingdom were re- introduced to the Central Yangtze in 1985 by the Chinese government and in 1986 by the World Wildlife Fund. From this founder population of thirty nine individuals, the number has increased steadily. A recent count put the population at 2,500 individuals in three national reserves. This is one of those rare examples where the technology for artificial insemination with frozen semen is well established for the species concerned (31). It would have been feasible to collect and store semen from the founder animals before they were sent to China so that the genetic diversity of this group could have been optimised from time to time if genetically important individuals failed to breed. Such a strategy would have helped to stave off the deleterious effects of inbreeding, which are a well known consequence of small population size. In this particular case there does not appear to be any problem with breeding and the population is obviously thriving. This is not always the case, however; the extant 40 bison constituting the Texas State Bison Herd are directly and exclusively descended from a bison herd assembled in the 1880s, and represent a historically and genetically valuable resource. The herd is under close genetic scrutiny and, because it is so small, the managers are concerned about the potentially destructive effects of inbreeding. Ecological modelling (12) simulations have shown that the judicious but limited application of artificial insemination to manage the herd would significantly help in reducing the rate of heterozygosity loss, although it would not stop this process indefinitely. Theoretically these effects would be reduced even further if semen samples had been collected and frozen at the earliest opportunity for use in artificial insemination. In fact, this strategy, which would constitute the establishment of a genetic resource bank for the species, could be implemented at any time. A genetic resource bank for a different bison herd has previously proposed and implemented (25), although in this case the theoretical framework for its use was not as well developed. Detailed theoretical modelling of the optimal strategies for the establishment and optimal usage of genetic resource banks of gametes and embryos from other large mammals have shown that this is not as simple as first supposed (13).

INTEGRATED CRYOBIOLOGY AND WILDLIFE CONSERVATION PROGRAMMES

The black footed ferret reintroduction cited (17) is one of the few examples in which conservation biologists, reproductive technologists and cryobiologists have genuinely collaborated to re-establish a species once thought to be extinct. The reintroduction programme was initiated after several years in which a small group of animals had been rescued from a remnant wild population and bred in the laboratory using surgical methods of artificial insemination. This was an intensive effort and it is to the credit of all concerned that a genetic bank of frozen semen currently exists to support the continued captive breeding programme and, by extension, the reintroduction programme. As cryopreservation of black- footed ferret spermatozoa is not straightforward an important aspect of this programme was the development of a workable technique for both semen collection and freezing. Early

46 success was obtained when JoGayle Howard and her colleagues at The National Zoo in Washington DC (16) used a semen diluent based on 11% (w/v) lactose and 4% (w/w) . She inseminated ten females using a laparoscopic technique, directly into the uterus, and obtained 7/10 conceptions. Subsequent studies have led to improvements in the methodology, but this was certainly a good start. A few other conservation programmes involving collaboration between cryobiologists and wildlife biologists are currently in progress. One noteworthy effort by the Cheetah Conservation Fund (CCF) to establish a semen bank for wild cheetah in Namibia (5); here at least, some of the technical problems involved with semen freezing seem to have been solved but it is worth mentioning that cheetah and several other large felid species show an extensive range of sperm abnormalities that are certainly unhelpful in terms of semen cryopreservation. Some conservation strategies in Australia, where many species of marsupial are under threat, have involved cryobiologists and reproductive scientists from the outset, because there seems to be a greater willingness there to regard semen and embryo cryobanking as potentially useful strategies to bolster more traditional measures such as habitat protection (23). This visionary approach has been seriously impeded by technical problems: there is currently only one marsupial species (the koala) being routinely produced by artificial insemination. This represents the outcome of a considerable research effort by Dr Steve Johnston and his colleagues at the University of Queensland; the latest birth takes the total number of offspring to more than thirty produced using fresh semen. This level of success means that the insemination techniques are well established (19), but unfortunately there is still a major problem with the survival of koala semen after cryopreservation. Although these cells survive extremely well when chilled, once they have been frozen and thawed in 14% glycerol (the optimal method identified to date) they become unstable and the sperm heads undergo swelling and decondensation of the DNA. This problem is currently unsolved, although alternative may offer some improvements. This lack of success with semen freezing in the koala is mirrored by an even more serious failure of semen cryopreservation technology in macropods (kangaroos and wallabies). Efforts to develop semen cryopreservation protocols for these species have run into major problems because there is a need for excessively high glycerol concentrations (>17% v/v) in order to obtain any significant level of sperm survival. However, after thawing, the sperm plasma membrane disintegrates during the rewarming process (14). Efforts to discover the mechanism of this effect have so far been unsuccessful (20, 21). One of the best examples of cooperative and integrative science to restore wild bird populations has involved the cranes. Following grassland and wetland loss/degradation, seven of the 15 crane species living today are threatened with extinction, and several subspecies are critically endangered. In 1941, the migratory population of the whooping crane was estimated at just 15 birds. Through collaborations of the Patuxent Wildlife Centre, US Fish & Wildlife Service, the Canadian Wildlife Service and the International Crane Foundation, substantial progress has been made in recovering this species. There now are about 190 whooping cranes in the Aransas-Wood Buffalo population, 75 in the Florida non-migratory flock and about 120 adult birds in captivity. This population growth has largely been the result of systematic studies to understand bird behaviour, , husbandry, chick rearing and medical requirements as well as basic and applied reproductive biology (8, 9). Especially important has been the development of semen collection and AI that have been essential to the effective management of many species, but particularly to the Mississippi sandhill crane (10).

47 CRYOBIOLOGY AND CAPTIVE BREEDING PROGRAMMES

Many captive breeding programmes are managed on a cooperative basis by zoos and wildlife parks throughout the world. Their aim is to maintain as much genetic diversity as possible in small and dispersed captive populations, thereby helping the survival of the species concerned. Some conservation biologists dispute the conservation value of these activities, arguing that by virtue of their small size, the populations are doomed to extinction. The Père David deer reintroduction example cited above shows that this argument is not entirely true. Indeed, reintroduction of the Mauritius kestrel was achieved successfully from a population of only four captive bred individuals. Clearly, such a small founder population will be highly inbred (11) and susceptible to new threats. Cryobiology should achieve its greatest level success within these captive breeding programmes, but that is unfortunately not true. These programmes are organized by consortia of zoos and aquariums throughout the world under the auspices of various organizations such as the European Association of Zoos and Aquariums (EAZA) and the American Association of Zoos and Aquariums (AZA). Individual specialists attend meetings to discuss the optimal breeding plans for their particular species of interest and breeding activities, successes and failures are recorded in studbooks. Clearly, the use of genetic resource banks of frozen sperm and embryos could be incorporated into these breeding plans, but at present I do not know any examples for which this is the case, other than the black-footed ferret example discussed above. The poor record of success with artificial breeding methods, and especially with frozen germplasm, means that such committees (known as Taxon Advisory Groups or Species Survival Plans) cannot rely on the use of these technologies to carry out their plans. Having said this, it is nevertheless true that individual zoos may, and have, attempted genetic enrichment of their populations by the importation of semen from elsewhere. These attempts have met with mixed success, and the outcomes are largely dependent upon the amount of effort invested and the expertise of the individuals concerned. Dr Bill Swanson and his team based in Cincinnati zoo have had considerable success with the importation of felid embryos into the USA from Brazil (28), and they have coupled this with training programmes for Brazilian veterinarians. However, other zoos who have attempted artificial insemination with wild felids have discovered that protocols for the successful induction of ovulation are still insufficiently effective and reliable to support routine success. Prime examples include attempts to breed tigers, snow leopards and clouded leopards by artificial insemination. A summary of individual protocols is outside the scope of this chapter, but a comprehensive and up-to-date compendium of protocols for various reproductive technologies in non-domestic species and companion animals is freely available on the CANDES (Companion Animals, Non-Domestic and Exotic Species) subcommittee section of the International Embryo Transfer Society (IETS) website (http://www.omahazoo.com/iets/ResourceManual2006.pdf ). Animal health issues are of considerable importance in the context of germplasm transport between countries. Quite rightly, regulatory authorities are concerned to ensure that if semen or embryos are imported, they do not bring any disease with them. Normally this involves subjecting the animals to a battery of prescribed tests before, during and after the planned period of gamete or embryo collection. Most attention is focused on animals related to common agricultural species, and so the usual requirement is to confirm that animals are not infected with for foot and mouth disease, bluetongue, and several other common conditions that could infect the recipient female, cross-infect nearby cattle, sheep and pigs and then devastate the national farming industry. This testing requirement, which must be accompanied by veterinary health certification, has direct implications for the establishment of genetic resource banks for captive breeding. It is impractical to collect some semen, perhaps from a gazelle or oryx, store it for several years in one country and then

48 expect to be able to send it abroad. The semen will probably have been collected without the requisite health testing, the certificate will not be available, and there is a strong likelihood that the samples will have been stored in alongside other samples from different species. The simple message here is that banks of frozen germplasm should be species-specific and ideally established and maintained as near as possible to the population of interest. This is not helpful if attempting to manage the genetics of several subpopulations, or metapopulations, which span national boundaries. In fact, paradoxically it is probably still easier to transport whole animals between these populations than to send canisters of semen. The IETS is currently addressing this problem and attempting to demonstrate logically that with detailed knowledge of disease transmission risk factors, environmental conditions and technical procedures, the real risks of disease transmission by germplasm transport can be minimized. The risk factors include not only the health status of the semen or embryo donor itself, but the egg yolk used in semen diluents, the liquid nitrogen used for freezing and the shipping container itself. Risks of bovine spongiform encephalopathy (BSE) in the UK have also meant that if bovine serum albumin is used in semen diluents or culture media, it should come from a nominally BSE-free country such as New Zealand.

SOMATIC CRYOBANKS AND CONSERVATION

Cryostorage of somatic tissues and viable cultures in “biobanks” is being increasingly undertaken throughout the world. In most cases, these activities are being carried out without any intention of using the materials for animal breeding or conservation; rather, the samples are intended for phylogenetic and evolutionary research in the future when the species are finally extinct. One example of such a bank is “The Frozen Ark” project (http://www.frozenark.org/ ), which was founded by a UK consortium with representation from the University of Nottingham, The Zoological Society of London and The British Museum of Natural History in London. The explicit aim of this project is to ensure that DNA and samples from as many threatened species as possible are stored and available for future study. The existence of such banks of frozen materials opens up the unintentional possibility that nuclear transfer technologies (cloning) could be used in future breeding programmes, once the technology is more successful and reliable. Several wild species have already been cloned, including banteng and mouflon, types of wild cattle and sheep respectively (for reviews, see (4, 15, 24). The low success rates of simpler technologies, caused largely by the general lack of background reproductive data, means that cloning is not a viable option with which to support conservation programmes. Many cloning attempts are required to obtain some success, and when the occasional success occurs the outcome is the production of small numbers of animals that have a high probability of future poor health. Nevertheless, we should probably be aware that this is a future option and take suitably aseptic precautions when sampling and storing the tissues. It would be a pity to contaminate viable cells with micro-organisms that might cause disease in any resultant offspring. Cloning has become routine for domestic livestock and now merits little media attention. Nonetheless, many biotechnologists have speculated on the potential value of cloning for wildlife preservation and, regardless of potential improvements in cloning technology, there are a few cautionary remarks to make. For example, why not clone the giant panda or the Iberian brown bear whose declining numbers are so dramatic as to suggest the need for rather extraordinary remediation measures? One excellent reason is quite logical. Where would the recipient oocytes come from in the case of a Giant panda? Various bear species have been proposed as recipients, but from an evolutionary point of view the Giant panda is not a closely related species. This would be equivalent to proposing that human cloning could be carried out using rabbit oocytes as recipients! Nuclear-cytoplasmic interactions are necessary for the

49 normal function of most mitochondrial genes (27), and cross species interactions may not permit these to take place. Such objections are often dismissed by technologists, as strategies such as the use of mixed mitochondrial populations within the cytoplasm might eventually lead to the elimination of all but the most appropriate mitochondrial genotypes (26). While this may be true in theory, it does not provide a credible background for the production of animals that will be fully fit in every respect and able to participate in future population development. Few other issues highlight the gulf in viewpoints between biotechnologists and conservation biologists better than this. Having said this, it is probably unwise to dismiss cloning technology as being valueless for conservation. There are some scenarios where it may be useful, one being the potential for conserving threatened amphibians. For example, amphibian populations all over the world are currently experiencing significant declines, possibly due to emerging diseases such as chytridiomycosis and ranaviruses (6). This phenomenon, popularly known as the “amphibian extinction crisis” signals the urgent need for the implementation of protective measures, and it is surprising that there is a dearth of studies on methods of assisted reproduction in these species. Some research on the collection and freezing of spermatozoa has recently been published (2, 3), but most of this work has commenced within the last five years and its value for conservation remains unclear. Application of the nuclear transfer technique to amphibians is worthy of exploration because it would avoid the need for developing semen collection and embryo transfer technologies; moreover, amphibian cloning has been studied since the 1960s (for review, see (7)). To date, no fertile amphibians have been produced by cloning from somatic cell nuclei although embryonic cell nuclei have been successful in this respect. Further research in this field can only be useful from both practical and scientific perspectives. If cloning were ever to be adopted as a useful conservation strategy considerable thought would be needed in designing appropriate implementation strategies. There is a widespread perception that it could be used to multiply the number of individuals in a population, thereby apparently solving the urgent conservation issue to hand. However, this would not serve the purpose of maintaining genetic diversity and supporting the viability of the population. In fact, the reverse would be true if all clones were generated from a small genetic pool. The way to avoid this scenario would be to clone using the widest possible genetic pool, using tissues and cells from as many individuals as possible from the extant population. This will not be possible in the future unless far sighted individuals collect the requisite number of samples well in advance of a population crash. Financial and logistic constraints will probably prevent such a sampling strategy from being implemented as the requirements for genetic research will be met by sampling from a smaller set of individuals. The message here is that any projects aimed at future breeding exercises should probably be setting up extensive cell and tissue banks right now. It will take a brave (and rich) conservation organization to set this up on what would almost certainly be a speculative basis.

CONCLUSIONS

Throughout this review I have tried to emphasize that technical capability alone, whether for the production of offspring by artificial breeding techniques or for the successful cryopreservation of gametes, does not necessarily mean that cryobiology and reproductive technology will make a lasting contribution to animal conservation. However, the converse, i.e. these techniques cannot make any contribution whatever, is equally untrue. A balanced view is the most realistic and useful, but unfortunately only a small number of conservation planners take this into account. The tragedy of this dichotomy of perspectives is that both sides of what seems to be a divided community have the best of intentions.

50 REFERENCES

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51 26. Smith LC, Bordignon V, Garcia JM & Meirelles FV (2000) Theriogenology 53 , 35-46. 27. Spikings EC, Alderson J & John JCS (2006) Human Reproduction Update 12 , 401-415. 28. Swanson WF (2003) ILAR Journal 44 , 307-316. 29. Taggart DA, Leigh CM, Steele VR, Breed WG, Temple-Smith PD & Phelan J (1996) Reproduction Fertility & Development 8, 673-679. 30. Taggart DA, Schultz D & Temple-Smith PD (1997) Australian 19 , 183-190. 31. Zomborszky Z, Nagy S, Nanassy L, Szabari M & Bodo S (2005) Animal Reproduction Science 90 , 185-190.

Accepted for publication 9/10/07

52