Abstract
Cryobiology offers an important opportunity to assist in the management and study of wildlife, including endangered species. The benefits of developing “genome resource banks” for wildlife are profound, perhaps more so than for traditional uses in terms of livestock and human fertility. In addition to preserving heterozygosity and assisting in the genetic management of rare populations held in captivity, frozen repositories help insure wild populations against natural and human-induced catastrophes. Such banks also are an invaluable source of new knowledge (for basic and applied research) from thousands of species that have yet to be studied. However, it is crucial that genome resource banks for wildlife species be developed in a coordinated fashion that first benefits the conservation of biodiversity. Spurious collections will be of no advantage to genuine conservation. The Conservation Breeding Specialist Group (CBSG; of the International Union for the Conservation of Nature and Natural Resources—World Conservation Union’s Species Survival Commission) has promoted international dialogue on this topic. CBSG working groups have recognized that such repositories be developed according to specific, scientific guidelines consistent with an international standard that ensures practicality, high-quality ethics, and cost-effectiveness. Areas requiring priority attention also are reviewed, including the need for more basic research, advocacy, and support for developing organized repositories of biomaterials representing the world’s diverse biota.
Introduction
Most scientists, and the public alike, understand the need to protect and manage wildlife resources. Three reasons often are cited for conserving biodiversity, or the wealth of diverse species on the planet. The first and probably the most important reason is that biodiversity serves as Earth’s life support system, regulating local climate, devouring environmental pollutants, safeguarding watersheds, creating and preserving soils, and converting solar energy into the biochemical components that allow all life to proceed. Second, biodiversity provides a boundless economic resource for foods, chemicals, fiber, clothing, structural materials, energy, recreation, and medicines. For example, organisms produce the resources from which the majority of Western society’s pharmaceuticals are derived ( Raven 1994 ). Third, many would argue that humans have a moral responsibility to protect this wondrous web of life, a rather esoteric justification that is felt deeply across most cultures ( Maybury-Lewis 1992 ).
Few would argue that the highest priority for conserving biodiversity is to protect habitat—ecosystems in which diverse wild animals and plants coexist in a natural balance. Despite valiant efforts of governments, nongovernmental organizations, and individual people, we are failing to save sufficient habitat to maintain biodiversity or the genetic heterozygosity required to sustain healthy species and populations. Although there are approximately 8000 protected areas worldwide, 96% of the remaining biodiversity remains undefended ( Wright 1993 ). A contemporary rate of species extinction is unknown, although E. O. Wilson (1992) has predicted that as many as 50,000 species are being lost annually, a rate likely to continue for several more decades. Michael Soulé (1991) , a well-known conservation biologist, has proposed a multilevel protective classification system— a biospatial hierarchy that ranges from whole ecosystems (landscapes) to communities, species, populations, and eventually to ex situ germplasm and seed bank repositories.
The value of the latter, termed here “genome resource banks” (GRBs 1 ), extends far beyond simply ensuring the protection of the world’s unique animal and plant species in liquid nitrogen. If collected, stored, and used appropriately, such repositories have merit for enhanced species and population management as well as vast basic and applied research with economic and social impacts ( Wildt 1997 ; Wildt et al. 1997 ). Particularly exciting will be the continued melding of molecular technology with the availability of diverse biota, especially microorganisms representing an infinite variety of unknown genes. Who knows the eventual value and ultimate potential of these snapshots of bio- and genetic diversity?
Although most attention has been placed on species loss, the related challenge of preserving gene variation and the evolutionary potential within species, populations, and individuals should never be ignored. Thousands of species are rapidly losing genetic variation that, in turn, can cause genetic drift, demographic bottlenecks, founder effects, and inbreeding depression. For example, the wild population of Florida panthers ( Puma concolor coryi ) became so small that incestuous matings became common, resulting in cardiac defects, a high seroprevalence to serious pathogens, and numerous reproductive abnormalities, including severe teratospermia and cryptorchidism ( Roelke et al. 1993 ). GRBs can have relevance to resolving issues related to continued losses in heterozygosity. Therefore, for conservation biology at large, traditional ways of preserving biodiversity are being reconsidered, including exploring strategies such as the banking of biomaterials.
GRBs for Wildlife Conservation
Living wild animals are conserved in situ (in nature) or ex situ (in intensively managed programs in captivity, usually zoos). The latter should be highlighted because of the focus of zoos on genetic and demographic management of high-priority species. For example, more than 90 animal species of diverse phenotypes and geographic origin are bred in captivity in North American zoos strictly to maximize genetic diversity. This diversity is achieved through a “Species Survival Plan” approach under the umbrella of the American Zoo and Aquarium Association. Zoos holding a particular species agree to collaborate by exchanging valuable individuals to keep the captive population genetically vigorous. Selection for breeding and animal exchange are based on the principles of small population biology and an overall relatedness factor known as mean kinship ( Ballou and Lacy 1995 ). This model is important because it demonstrates that moving living animals between breeding institutions, although arduous and expensive, can be successful in maintaining genetic diversity and avoiding inbreeding.
Another form of in situ and ex situ conservation is the long-term collection, storage, and use of biomaterials, that is, germplasm, embryos, tissues, blood products, and DNA. This is not a new concept. The US National Academy of Sciences declared in 1978 that “what is done for domestic species (i.e., sperm and embryo freezing) should be done for all species” ( NRC 1978 ). The US Agency for International Development, the US Congress' Office of Technology Assessment, and the National Science Foundation have made similar calls for action ( Wildt et al. 1993 ). The reason is simple—such collections could have a profound value for conservation, for basic and applied research, and as a biologic resource for human society.
Advantages of GRBs
Advantage 1: Preserved Extant Genetic Diversity
The existenc e of a GRB provides “insurance” for existing genetic diversity within a species. Thousands of species survive in nature but often in fragmented habitats, similar to the situation faced by animals living in geographically disparate zoos. These small populations are susceptible to inbreeding depression, acute environmental catastrophes, and disease epidemics. Most of the Earth’s biodiversity exists in locations sensitive to epizootics and drastic shifts in social and political structure. The organized and systematic sampling of biomaterials from free-living and zoo-maintained species would help preserve existing genetic diversity in a viable archive. Once collected, these materials could be maintained in perpetuity as an insurance policy for the web of life.
Advantage 2: Invaluable Genetic Resource
An ideal GRB is composed of an array of biologicals (beyond germplasm and embryos), including blood products, tissue, and DNA. These materials have innumerable uses in phylogenetic, systematic, and disease studies related to conservation biology. For example, DNA can be used to quantify the amount of genetic diversity in wild and captive populations, thereby providing information to drive management decisions. Biomaterials can be used to understand the processes underlying patterns of diversity, such as gene flow, selection, and mating. Blood samples can be screened for clinical chemistries, thus providing data on species norms that will be helpful for resolving future health problems. Similar data collected longitudinally can help identify the onset and causes of disease epidemics that, in turn, can speed remedial actions. Finally, these stored materials can have applications to the well-being of humans and domestic animals. For example, we have used stored sperm from wild species to study the cellular significance of teratospermia, a prevalent human condition ( Wildt 1994 ). Others have formulated new concepts about immunodeficiency diseases from studying the stored blood and tissue of wild felids (e.g., O’Brien 1995 ). The number of such examples would increase significantly if the academic-biomedical world had access to wildlife biomaterials.
Advantage 3: Economics
A GRB provides as yet unknown opportunities for improving quality of life, especially in developing countries. Wildlife species contain genes that confer special adaptations to strengthen reproductive capacity or survival for a unique environmental niche. For example, a species’ genotype may make it particularly resistant to parasites or to a particular disease. These genes are of great value, especially if incorporated into local livestock to create hardier, more productive animals. In some cases, the process may be more simple than relying on sophisticated gene therapy. For example, the Smithsonian Institution has a collaborative project in progress in Thailand testing the value of cryopreserved sperm fromnative wild cattle ( Bos guarus and Bos janvanicus ) for boosting the genetic vigor and productivity of common indigenous, domesticated cows. Hybrid offspring produced by artificial insemination are large, genetically vigorous calves that have natural disease resistance ( Figure 1 ). In this case, the stewardship of such a GRB is motivated by financial incentives (enhanced animal agriculture). However, at the same time, the repository is protecting and ensuring dwindling numbers of native wild cattle while illustrating their economic value to local communities. Of course, there is the need to monitor closely the exploitation of wildlife for human benefit with appropriate checks and balances. The acceptable criterion is always an affirmative answer to the following question: Does the use of this biomaterial to help humans have equal or greater benefit for protecting and conserving the donor species? The Thailand example illustrates how such projects (in this case, the use of frozen spermatozoa) could benefit both the rare species (wild cattle) as well as society.
A gaur hybrid calf produced in Thailand by artificial insemination with thawed spermatozoa stored in a local genome resource bank.
A gaur hybrid calf produced in Thailand by artificial insemination with thawed spermatozoa stored in a local genome resource bank.
Advantage 4: Easy Movement of Genetic Material
If captive wildlife populations are truly to serve as “hedges” against extinction, then intensive genetic management is essential, especially to avoid inbreeding depression. In the Species Survival Plan paradigm, genetic diversity is currently managed through (1) the sharing and movement of animals among cooperating institutions for breeding, and (2) the occasional supplementation of new genes by animals extracted from the wild. As long as adequate genetic diversity exists in the captive population, option 2 is unnecessary (and, in most cases, modern zoos rarely support their living collections through wild animal importations). Nonetheless, the occasional infusion of new wild genes could benefit most intensive management programs.
Moving germplasm from a GRB and then using assisted breeding (e.g., artificial insemination) could meet intensive management objectives without requiring the translocation of stress-susceptible wild animals. Zoos simply exchange germplasm rather than animals. The GRB could be bolstered by the occasional collection of sperm from random freeliving and anesthetized males that are producing surplus germplasm. Wildlife anesthesia is so advanced that this is a safe and effective option. Furthermore, these snapshot collections of sperm from wild, free-living populations would avoid any future need to import more animals into zoos. The current wild populations remain intact where their very presence (especially large, charismatic vertebrates) serves to protect native habitat. In theory, it also may be possible to infuse new genes into isolated wild populations via a GRB and assisted breeding. For example, wild females could be captured and held short term for ovulation induction and artificial insemination before being released back into nature. This route may be viable for remediation in cases of severe habitat fragmentation, at least to sustain populations until corridors are established between isolates.
Advantage 5: Increased Efficiency in Captive Breeding
A computer match of two animals as “ideal genetic mates” and their translocation to a common site for natural breeding is no guarantee of success. On the contrary, a significant obstacle to contemporary zoo genetic management is behavioral incompatibility between designated mates. Thus, even a sophisticated selection process combined with the most modern zoo environs cannot entice some individuals to copulate with each other due to sex partner preferences. Under these conditions, assisted breeding, such as artificial insemination, is the logical option. A GRB containing frozen semen from the appropriate males facilitates the assisted breeding process. Without the frozen repository, artificial insemination requires delicate timing to ensure that the sperm are collected near the time of ovulation. Two coordinated teams of biologists (one for the male, one for the female) usually are the norm. The process is complicated further if the sperm donor is located at another institution, thereby requiring the tricky long-distance shipping of fresh spermatozoa. Thus, access to viable frozen germplasm is much preferred.
Advantage 6: Reduced Genetic Problems
A GRB containing sperm or embryos extends the generation interval of the donor indefinitely. As long as viable germplasm or embryos remain in the repository, the genes do not die with the individual. Rather, they can be rederived and infused into the population at any time in the future, theoretically a century or more from now. This approach of reintroducing “original” genes over time decelerates natural losses in diversity as a result of genetic drift.
Advantage 7: Fewer Space Problems
A GRB can help wildlife managers resolve one of their greatest contemporary challenges—too little zoo space available to protect too many species, subspecies, and populations. Thousands of species deserve captive management attention, but zoos using conventional approaches can adequately manage fewer than 100 total species for conservation ( Conway 1986 ). This situation occurs because a minimum number of animals (and space) is required per species to allow maintaining adequate amounts of genetic heterozygosity. The problem is not resolved by building more or larger zoos. Rather, the issue is how to make current facilities more efficient. A GRB can help reduce space needs, as in the case of males and the possibility of germplasm storage in liquid nitrogen rather than the reproductive systems of living individuals. Thus, species’ zoo space requirements could be minimized, allowing newly available room to be redirected to other species in crisis.
Thus, the potential of GRBs for wildlife study and conservation, as well as the general life sciences, is enormous. However, we must consider whether the technology to translate the possibilities into reality is available. In terms of DNA and molecular/cellular approaches, illustrations are provided in advantages 2 and 3 above. In the context of the value of germplasm, there now are many examples of propagating endangered species by assisted breeding with cryopreserved sperm. The laboratories of the National Zoological Park and its Conservation & Research Center of the Smithsonian Institution have developed protocols for routinely producing offspring in the cheetah ( Acinonyx jubatus ), Eld’s deer ( Cervus eldi ), and scimitar-horned oryx ( Oryx dammah ), all using cryopreserved sperm. In the black-footed ferret ( Mustela nigripes ), one of North America’s most endangered species, artificial insemination with fresh or frozen sperm is commonly used to produce offspring from the most genetically valuable individuals. These animals are then used as breeding stock or for direct reintroduction onto the plains of four states in the American West ( Figure 2 ).
Young, genetically valuable black-footed ferret produced by assisted breeding, just before reintroduction into Arizona (photograph by Lisa Ware).
Young, genetically valuable black-footed ferret produced by assisted breeding, just before reintroduction into Arizona (photograph by Lisa Ware).
These successes did not arise easily. In fact, it has been argued that there is a general misperception, even in the scientific community, that assisted breeding technologies are easily adapted among wildlife species ( Wildt and Wemmer 1999 ). In truth, reproductive techniques that work for one species have little relevance to another (i.e., artificial insemination technology successful with cattle fails when applied to the cheetah). The reason should be obvious inasmuch as reproductive mechanisms can be (and usually are) highly diverse, even among evolutionarily related species ( Wildt and Wemmer 1999 ). Thus, for GRBs to be routinely effective using cryopreserved germplasm requires substantial parallel efforts directed toward understanding the basic biology of every species of interest. Finally, it is also important that most successes have centered on the use of frozen spermatozoa. Much more research is required to develop methods of effectively cryopreserving oocytes and gonadal tissue.
Nonetheless, the examples of consistently producing cheetahs, Eld’s deer, scimitar-horned oryx, and black-footed ferrets by artificial insemination illustrate the potential value of cryopreserved germplasm for helping to manage wildlife species. The issue then becomes: How do we proceed to develop such repositories?
Organizing GRBs for Wildlife
Despite the first calls for wildlife GRBs more than 2 decades ago, little progress has been made for several reasons. First, discussing the collection, storage, and shared use of biomaterials, especially those of rare species that involve complex proprietary issues, will always provoke controversy about how to proceed. Additionally, from the beginning, there has been misdirected thinking that such repositories should be established in a top-down fashion (i.e., huge international or national repositories, radiating outward to the scientific community and eventually down to individual species). Given the sheer expense and biopolitical challenges of creating such infrastructure, it seems logical to search for alternative approaches.
A different type of dialogue emerged about a decade ago through the Conservation Breeding Specialist Group (CBSG1) of the International Union for the Conservation of Nature and Natural Resources (IUCN 1 )-World Conservation Union’s Species Survival Commission. CBSG, one of more than 100 IUCN-sanctioned specialist groups, is a “neutral” catalyst, specializing in tackling novel and often controversial issues influencing wildlife conservation. Through its worldwide network of more than 700 members, CBSG has facilitated awareness and debate on this issue in a host of international venues. CBSG workshops provided a social framework for multiple stakeholders (most with diverse viewpoints) to reach cooperative consensus. The formation of strong alliances, partnerships, and “buy-in” from group work is one of the most positive characteristics of current strategic planning for conservation. CBSG’s objective with respect to GRBs has been to provoke international awareness and debate. To date, such workshops have been held in North America, Africa, and Australia. CBSG members have formulated a resolution statement on the value of such repositories that has been submitted to the IUCN-World Conservation Union ( Wildt 1997 ). Among the highlights of this statement are the following:
It is “recognized that the efficiency and efficacy of intensive conservation efforts can be increased many fold by applying recent advances in reproductive technology. These included assisted breeding and the low temperature storage of viable animal germplasm, namely spermatozoa, oocytes and embryos. Germplasm banks: (1) offer a high degree of security against the loss of diversity and, therefore, entire species from unforeseen catastrophes; (2) minimize depression effects of genetic drift and inbreeding; and (3) provide a powerful method for managing the exchange of genetic diversity among populations. Ancillary conservation benefits include banks for basic and applied research including repositories of serum, DNA and cultured cell lines from germplasm donors that permit studies on disease status, detection of microbial antibodies, pedigree determination, taxonomic status, geographical differentiation of populations and cellular physiology.” Therefore, it is recommended that “the development of genome resource banks is a valuable component of integrated conservation programs.”
Furthermore, CBSG working groups have recognized that:
“…the establishment of genome resource banks must be matched by developing strategies for use as a genuine and practical conservation asset for supporting natural breeding. Furthermore genome resource banks should follow specific, scientifically developed guidelines consistent with an international standard, thus ensuring their use as a meaningful, practical, ethical and cost-effective conservation tool.”
To achieve the latter, CBSG has nurtured the idea of a GRB action plan, a written document that guides the various stakeholders of a particular species through the challenges of developing an effective banking program. Such written plans are a high priority because there are many complicated, interactive factors dictating the success (or failure) of such a program. Participants in writing action plans have been amazed at the variety of issues that require attention. A second reason for a written plan is that the purpose of such a repository is shared use of limited (endangered) resources. This sharing, in turn, provokes concerns about equability and proper biomaterials use. Finally, because there is a financial cost to such banks, it is likely that a detailed, written plan will be necessary to help secure long-range funding support.
Therefore, guidelines for writing a GRB action plan have been generated and include sections on the following: (1) justification for establishing a repository; (2) current knowledge of life history and natural reproduction; (3) current knowledge of assisted reproduction; (4) studbook and regional collection plan status; (5) status of the species in the wild; (6) accessibility of existing animal resources for contributing to the banking process; (7) type and amount of germplasm to preserve in relation to genetic management; (8) technical germplasm collection, storage, use, and ownership issues; and (9) resources and funding. The subsequent creation of prototype action plans has identified even more challenges, especially dealing with type and amount of germplasm to collect. For example, with a given male of a given genetic value, how many sperm need to be stored to meet management objectives, now and in the future? This and other such questions are not simple to answer.
The subject of the value of cryopreserved germplasm to genetic management has stimulated hours of philosophical and scholarly discussions. There have been at least three related consensus recommendations: (1) Develop at least two banks for each species, one designated as an “in perpetuity” repository (for use only when the species approaches extinction) and the other for routine management of living animals in the ex situ and in situ populations. (2) Whenever possible and appropriate, systematically sample the free-living population to capture all extant diversity. (3) Use frozen sperm and assisted breeding, if only occasionally, because simulation modeling has demonstrated the power of such repositories and their potential to greatly enhance the ability of managers to meet goals of maintaining maximum genetic diversity ( Johnston and Lacy 1995 ).
The type of freewheeling dialogue facilitated internationally by CBSG and described above has been a positive initial step. The first priority always should be avoiding random, spurious collections of biomaterials without a clear conservation or scientific goal. In short, CBSG activities have concluded that GRBs for wildlife are potentially valuable conservation tools, but only in the face of multiple stakeholders, extensive cooperation, strong science, and a written action plan.
Priorities for Achieving Effective GRBs for Wildlife
Advocacy and Support
There is a need for society at large, and especially political decision makers and the biomedical community, to understand the crucial need for genetic resource conservation. There is a first-order need to promote the value of GRBs for wildlife in political arenas to generate philosophical and financial support. Scientists in the biomedical world have diverse sources of available funding for human and laboratory animal research, but it is important to remember that there is no “National Institutes of Health for Endangered Species.” There is a paucity of biomedical studies actually directed at wildlife, even within the zoological community, due to a lack of competitive funding sources. The development of national or commercial resources will draw attention and the most talented researchers to this field. When there is funding incentive, science (and scientists) will emerge to find ways to address critical issues.
Emphasis on Basic Research in Wildlife Species
Most species on the planet have gone completely unstudied. Mittermeier and Mittermeier (1999) recently estimated the known existence of more than 40,000 vertebrate species. But how many of these have been studied systematically from a biomedical perspective? What proportion of these species have been studied adequately to allow reproductive technologies to be efficient? The answer is only a small fraction ( Wildt and Wemmer 1999 ). There is a fundamental need to characterize biodiversity (invertebrates as well as vertebrates) while continually prospecting for its value to society by searching for clues about how to use biomaterials to protect genetic integrity and every species. Meanwhile, an equally high priority will be the need to systematically study the reproductive physiology and endocrinology of each species to generate the information that will allow frozen germplasm to be used for the production of young.
Cooperation and Sharing
Because developing countries hold most of the world’s biorichness, it is imperative that emerging regions benefit from the collection, storage, and use of biomaterials. Such assistance should come in the form of direct compensation to allow self-sufficiency in conservation as well as the banking process itself. This assistance involves industrialized regions providing training and all types of resources for program development within the range country and within the locales of interest. Responsibilities and expectations for biomaterials collection, storage, and use by developed versus developing countries (including proprietary issues) have been addressed previously ( Wildt 1997 ).
Genesis of Individual Banks
Advocacy for wildlife GRBs begs the question: Just where do we begin collecting, especially given the sheer number of life forms needing attention? In an ideal world, one might envision huge repositories, large buildings with many curators managing vast arrays of viable faunal (and floral) biomaterials. However, for now, it may be too ambitious to develop centralized repositories, largely for economic reasons. It may make more sense to link extant organizations. For example, museums and zoos could establish partnerships with academia or other organizations (e.g., the American Type Culture Collection) to expand existing collections to include wildlife specimens. The first step would be to coordinate production of GRB action plans (see above). A related idea is to sponsor remote repositories in developing countries that are connected to Western partners who provide funding, necessary equipment/supplies, and training in prospecting and curation. Thus, a grass roots type of approach that involves wildlife managers, biomedical scientists, and appropriate regulatory authorities with interest in particular taxa may be the most reasonable initial strategy from which successful models will emerge. Regardless, a high priority will be to set standards for databases, safe and effective monitoring, quality control, and long-term support of these specialized accessions.
Summary
Biodiversity is important for sustaining a healthy Earth, but it also is immensely valuable to the health and lifestyle of human society. Contemporary wisdom holds that biodiversity is best saved in nature through the protection and preservation of habitats. This indeed is an absolute truth and should always remain our highest priority. Interestingly, frozen repositories of biomaterials sometimes have provoked concern among conservation biologists who fear that (1) a GRB may be considered a substitute for living animals existing in nature, or (2) funds needed to establish and maintain highly technological banks detract from support of field research and protection. This article and others ( Wildt 1997 ; Wildt et al. 1997 ) argue that a GRB is only one tool in an arsenal of technologies needed to combat the ever-growing threat to biodiversity losses. Repositories of biomaterials are “insurance” and a valuable research resource that, if used appropriately, will strengthen in situ conservation programs. Furthermore, the origin of funding for GRBs is unlikely to come from sources that are competitive for field research or other conservation initiatives.
A partnership between the biomedical and wildlife communities would definitely benefit both. Science is key to conserving wildlife, whereas the wild animals (and plants) comprise valuable genes and knowledge that can have a positive impact on society. In effect, the variety of species on the planet offers the scientific community an incredible opportunity for exploring a host of fascinating mechanisms and possibilities in the life sciences. However, to meld these two worlds and to save the laboratory, as it were, requires that we first develop means to collect and store these biomaterials. The need now is for serious promotion of the utility of GRBs and related scientific partnerships and for development of necessary resources. More vocal public and policy advocacy is required, not only by those trained as “conservationists,” but also by those (especially colleagues in academia) sufficiently intrigued to take advantage of this unique opportunity. This approach requires the active participation of individuals and groups with a range of disciplines and interests to join the rapidly growing network of scientists, zoo managers, and wildlife authorities dedicated to exploring, preserving, and protecting biodiversity
Acknowledgments
The author thanks W.F. Rail, U.S. Seal, J. Ballou, and R. Lacy for their contributions into the theoretical value of GRBs for wildlife. The hybrid cattle project’s principal investigators are Drs. Barbara Wolfe and Budhan Pukazhenthi, and Dr. JoGayle Howard leads the black-footed ferret assisted breeding program. Portions of the concepts and results presented here are derived and extrapolated from two manuscripts of the author, Wildt 1997 and Wildt et al. 1997 .
References
Abbreviations used in this article: CBSG, Conservation Breeding Specialist Group; GRB, genome resource bank; IUCN, International Union for the Conservation of Nature and Natural Resources.
