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 Zoology, 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 cryopreservation 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 biologists 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 embryos 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 semen, 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 Temperature Storage of Biological Resources” Society for Low Temperature Biology 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 freezing, 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 Conservation Biology 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 embryo 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 vertebrate 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 physiology. Even if researchers and practitioners want only simple information such as the anatomy 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