PART 7
SCIENCE AND TECHNOLOGY: HOW CAN THEY HELP?



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BioDiversity PART 7 SCIENCE AND TECHNOLOGY: HOW CAN THEY HELP?

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BioDiversity Sterile culture is one propagation method for the ex situ preservation of plant diversity. Photo courtesy of John Einset.

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BioDiversity CHAPTER 30 CAN TECHNOLOGY AID SPECIES PRESERVATION? WILLIAM CONWAY Director, New York Zoological Society, Bronx, New York In the preservation of biological diversity, the use of technology is a last resort. When the preservation of ecosystems falters, their fragments may have to be cared for piece by piece. FOUR OBSTACLES TO SPECIES SURVIVAL A species of limited distribution faces at least four obstacles. First, there may not be sufficient habitat and the possibility of obtaining numerous large new nature preserves is remote. Even protecting some areas already designated as preserves is not proving possible, and no land whatsoever will be set aside for large numbers of species. Second, many of the preserved habitats will be in pieces too small and too subject to change to sustain unmanaged, genetically and demographically viable populations of the animals and plants they seek to protect (Soulé and Wilcox, 1980). Third, although the majority of wild species must persist outside of wildlife preserves, large land vertebrates and great aggregations that conflict with humans will be mostly confined to refuges and those outside will require continual monitoring, protection, and help. Finally, human populations will continue to grow for some time, inexorably reducing resources available to other species, while human land-use patterns, cultural attitudes, and economic practices will continue to shift and change (Myers, 1979). PROBLEM AND APPLICATION Despite the factors mentioned above, the loss of a wild population is not always the result of irreversible habitat change. It can come about for transient economic

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BioDiversity and cultural reasons, such as overhunting of the American bison, Arabian oryx, white rhinoceros, and American beaver. Or it may happen for correctable environmental reasons, such as introduced pesticides (the peregrine falcon and bald eagle) and introduced predators and competitors (the Hood island giant tortoise and Howe Island wood rail). And the loss may also last for unpredictably long periods, as with the Pere David’s deer, Mongolian wild horse, European wisent, perhaps the Siberian tiger, brown-eared pheasant, Mauritius pink pigeon, and Guam rail; it could even be forever. But populations of all these species and many more have been increased in one situation or another by intervention strategies (see Table 30–1). Where a wild population’s ability to survive is lost, especially where the threat and destruction may be temporary, e.g., for the American bison, peregrine falcon TABLE 30–1 Ex Situ Care and Biotechnology. Each Technique Has Been Utilized with the Species Listed Below It on a Long-Term or Experimental Basis Intervention Technique Species Short-term propagation and reintroduction Golden lion tamarin, cheetah, wolf, red wolf, American and European bison, Arabian oryx, onager, Andean condor, bald eagle, peregrine, Hawaiian goose, Lord Howe Island wood rail, Guam rail, European eagle owl, Guam kingfisher, Galapagos giant tortoise, Galapagos land iguana, Ash Meadows Amargosa pupfish Long-term propagation Lion-tailed macaque, Siberian tiger, Pere David’s deer, European bison, Przewalski horse, brown-eared pheasant, Edward’s pheasant, Bali myna, white-naped crane, addax, slender-horned gazelle, scimitar-horned oryx, gaur, Gérevy’s zebra, Puerto Rican horned toad, Chinese alligator, Mauritius pink pigeon, Madagascar radiated tortoise, Aruba Island rattlesnake Relocation, transplantation Koala, mongoose lemur, aye-aye, brown lemur, chimpanzee, gorilla, squirrel monkey, wooly monkey, spider monkey, common marmoset, black rhinoceros, white rhinoceros, red deer, white-tailed deer, mule deer, moose, Tule elk, bighorn sheep, musk-ox, pronghorn antelope, roan antelope, mountain goat, African elephant, more than 400 species of birds, many reptiles and amphibians Fostering, cross-fostering Peregrine, bald eagle, whooping crane, masked quail, polar bear (captive), many species of waterfowl, pigeons, cranes (in nature and captivity), and passerine birds in captivity Artificial incubation Gharial, Siamese crocodile, Chinese alligator, green turtle, ridley, hooded crane, whooping crane, white-naped crane, and many other birds, reptiles, amphibians, and fishes Artificial rearing Hundreds of species of most vertebrate groups Artificial insemination Alligator, ocellated turkey, brown-eared pheasant, whooping crane, squirrel monkey, yellow baboon, giant panda, guanaco, Speke’s gazelle, gemsbok, bighorn sheep Embryo transfer Gaur, bongo, eland, common zebra, Przewalski horse, cottontop marmoset, yellow baboon

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BioDiversity (see Cade, Chapter 32), bald eagle, Arabian oryx, or Ash Meadows Amargosa pupfish, we need new scientific comprehension and a responsive technology. We must be able to relocate, sustain, or store a threatened population, to start and stop its propagation, and to reintroduce or remove it. Because many populations of species in nature are becoming fragmented and isolated from each other, the emigration and immigration necessary for them to find unrelated mates of the right age and gender are becoming impossible. In such instances, intervention technologies will be necessary to effect the required movements. In such small populations, localized catastrophes, disease, sex and age imbalances, and even inbreeding can threaten viability (Schonewald-Cox et al., 1983). In response, technology may make it possible to remove or insert individuals into populations or even embryos or zygotes with needed characteristics into individuals (see Dresser, Chapter 34). Where conservation biologists identify threatened but critical coevolutionary links, especially those between keystone species essential to ecosystem stability and diversity, sustaining these links for a time by scientific management of predators, competitors, even of environmental chemistry, microclimate, and with reintroductions, may be our only option for preservation. LIMITATIONS OF SCIENCE AND TECHNOLOGY But if such technological treatments and repairs are possible, why can not science and technology simply save biodiversity? Perhaps, as H.L.Mencken is reported to have said in a different context: “For every complex problem there is a simple answer and it is wrong.” Most losses of biological diversity, to say nothing of lost ecological services, are quite beyond human ability to repair. Too many very intricately interdependent species are being lost too rapidly with too many unpredictable consequences for others. Besides, sustaining species in a freezer, in a captive population, or in small fragmented refuges provides little to the Earth in the way of basic ecological services. However, intensive care and biotechnology can preserve some diversity that would otherwise be lost. But the greatest dimension of such preservation is depressingly slight compared with that which can be or could have been sustained in adequately designed and protected nature preserves and by understanding accommodation outside preserves. NUMBERS VERSUS TECHNOLOGY It is the numbers, whether they be those of the great variety of creatures requiring help or those representing the scarcity of biologists and dollars to help them, that discourage prospects for sustaining a sizeable proportion of living creatures solely through technology—despite our most ardent wishes or most arrogant imaginings. There are perhaps 400,000 species of plants. In Chapter 31, Ashton discusses their preservation in gardens and arboreta. But there may be 30 million kinds of invertebrates, mostly insects. Despite their importance, the overwhelming number of

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BioDiversity specialized invertebrates makes it logistically impossible for technology to contribute to the preservation of a significant representation for restoration programs. There are only about 41,000 vertebrates. Of these, 19,000 or so are fishes, about 9,000 are birds, 6,000 are reptiles, 3,100 are amphibians, and 4,300 are mammals (E.O.Wilson, personal communication, 1987). For those that have been or will be totally displaced from nature, we have only a few specialized propagation centers and the world’s zoos. Zoos currently house about 540,000 mammals, birds, reptiles, and amphibians—an almost trivial number in relation to original wild populations but significant in the impact on human interest. The number is roughly equal to 1% of the domestic cats in American households, 10% of the cats and dogs euthanized annually in the United States, or about 25% of the deer taken by U.S. sportsmen each year. Zoos are popular but have little room. The spaces for animals in the world’s zoos could all fit comfortably within the District of Columbia. Even if half these spaces were suitable for propagation of vanishing animals, the individual numbers of each species necessary to keep viable populations would make it impractical for zoos to sustain more than 900 species very long and probably far fewer in conventional breeding programs (Conway, 1986). But it will not always be necessary to sustain a population for a long time or to do so conventionally. Zoos, revised and improved, can come to have a special role in species preservation, for they represent a unique devotion of local human resources to the care of foreign wildlife. In the past few years, the world’s zoos have bred more than 19% of all the living mammals and more than 9% of the birds. Thus far, criteria of genetic uniqueness as well as the practicality of care have guided long-term propagation programs. In the future, more attention must be given to ecological criteria with an eye toward the future needs of restoration programs, to species that naturally occur at low density, to the great predators and large ungulates, and to the primates. A foundation for such help rests in growing international collaboration and, in the United States, in unequalled programs of coordinated animal data gathering in the International Species Inventory System and species management in the Species Survival Plan of the American Association of Zoological Parks and Aquariums. Zoos are breeding orangutans and Chinese alligators, Bali mynas, pink pigeons, and Puerto Rican horned frogs, addax, slender-horned gazelles, wattled cranes, and black lemurs. They have pioneered rare embryo transfers between animal species, artificial rearing techniques, cross-fostering between species, and necessary long-term contraceptives for population management—a host of fundamental technological tools essential to the prospects of helping species in extremis. Even so, extensive scientific and technological advances would be necessary to appreciably expand the space available for the care of species losing their homes. But where cold storage of sperm and embryos is possible, a herd of wild cattle or antelope can be cared for in a space no larger than a soda straw, moved without risk of trauma, and stored indefinitely—if we are satisfied to have our wild cattle and antelopes in soda straws. Yet, we must consider the alternatives and their time scales. Unfortunately, practically all that we know of animal reproductive physiology has been worked out with a few domestic and laboratory species. The technology

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BioDiversity of sperm and embryo storage in use with domestic cattle is the product of 20 years of research, millions of dollars, and thousands of specimens—a critical matrix for investigation and discovery. A vast amount of research would have to be undertaken before a technology of embryo and sperm storage and transfer as reliable as that in use with cattle could be available for wild species without domestic analogs. But where is the economic incentive for such research? Where are the animals? The apparently simple techniques of artificial insemination, for example, have been successful with scarcely 20 wild species of mammals. Nonetheless, development of the scientific understanding necessary to long-term propagation is a technological fulcrum for many intensive species care programs. In Chapter 33, Seal discusses some of the challenges. TECHNOLOGY IS EXPENSIVE Unhappily, high cost is characteristic of high-tech applications, and whereas the capability and the money to apply advanced technologies to preservation is located mostly in wealthy northern countries, the largest problems of species loss are in poor tropical countries. Money used for high-tech intervention strategies obviously can not be used to preserve habitat. For less-developed countries, habitat preservation is the only realistic strategy, unless help comes from outside. Whatever the help, no available amounts of money can ensure the protection of many species in nature, even vertebrates, such as the addax and scimitar-horned oryx from the Sahel or Guam’s kingfisher and rail. Ex situ care and biotechnology are their only hope. Besides, support from different sources is usually restricted to different purposes. Except in local education, research, and propagation programs in zoos, for example, municipal funds are usually unavailable to international species preservation. In such differentiation of source, competition for funds between preservation options can be diminished. After all, ex situ care and technology are used only after it is evident that conventional conservation efforts could fail. BUYING TIME Can technology be used to ensure continuing evolution? Both intensive management and habitat reduction reduce the chances of directional habitat-responsive evolution. And in small unmanaged populations, genetic drift is much more powerful than natural selection. But before worrying about whether species must continue to evolve to survive, please reflect upon the time scale of concern. The profoundly immediate problem is to save as many species as possible through the next 150 years. It seems inevitable that most large land vertebrates and many plants eventually will survive only as wards of humans, scientifically managed or cared for, even reestablished, at some point. Because of an overall decline of diversity, those species that persist through the feeble efforts of science and technology will become proportionately more important. Saving 200 of 2,000 mammal species seems more important than saving 200 of 4,300. Furthermore, it is the larger forms, among animals if not plants, that will most likely profit from these intervention strategies;

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BioDiversity creatures that are not only spiritual symbols for the ongoing promotion of conservation but evolutionary conservative; more irreplaceable than the merely irreplaceable. Preservation science and technology must become an active branch of conservation biology, because future habitat restoration, if any, will depend upon its progress. Thus, technology is not a panacea for the disease of extinction. It is a palliative—a topical treatment with which to buy time, to preserve options for a few populations and species judged of special value. In the final analysis, it is no more important than the species it sustains, which would otherwise be lost forever, and no less. REFERENCES Conway, W. 1986. The practical difficulties and financial implications of endangered species breeding programmes. Int. Zoo Yearb. 24/25:210–219. Myers, N. 1979. The Sinking Ark. Pergamon Press, Oxford. 307 pp. Schonewald-Cox, C.M., S.Chambers, B.MacBryde, and W.L.Thomas, eds. 1983. Genetics and Conservation. Benjamin/Cummings, London. 722 pp. Soulé, M.E., and B.A.Wilcox, eds. 1980. Conservation Biology: An Evolutionary-Ecological Perspective. Sinauer Associates, Sunderland, Mass. 395 pp.

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BioDiversity CHAPTER 31 CONSERVATION OF BIOLOGICAL DIVERSITY IN BOTANICAL GARDENS PETER S.ASHTON Director, Arnold Arboretum, Harvard University, Cambridge, Massachusetts Conservation is already, and very appropriately, recognized as being a major activity for botanical gardens in both their research and educational programs. In this field, arboreta and botanical gardens have a particular and important potential, which I discuss in this chapter. In nature, plants frequently exist in small populations. Examples include many rare endemics, such as those of mountain peaks and many in the Mediterranean dry sclerophyll scrublands, especially in the Cape Province of South Africa and in Southwest Australia, and those of certain rain forests. Over the relatively short time we realistically have had to work as conservation managers, extremely small stands have been found to persist in nature. Higher plants, being sedentary, are often highly site-specific. This facilitates the development of logical plans for demarcating minimal areas for in situ conservation based on ecological knowledge and principles of island biogeography. On the whole, the most favorable sites are a few environmentally heterogeneous reserves of sufficient size to minimize edge effects (e.g., changes in species composition at the periphery caused by in- and out-migrations from adjacent unprotected lands). Ideally, these would be loosely connected by small stepping stones or corridors to allow for the exchange of genes (Diamond, 1975). Identification and immediate protection of sites of high conservation value must be our highest priority in the absence of even the grossest information upon which to base plans, including basic inventory as well as distributional and ecological data on many of the richest biota. This underlines the vital necessity of increasing inventory and ecological information as a prerequisite to developing any logical plan for conservation. In practice, of course, the luxury of regional planning often does not exist. The conservationist only succeeds in

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BioDiversity raising awareness when the plant is reduced to endangerment in one or a few isolated localities or, at best, is offered a patchwork of lands for which the farmer and the planner have failed to find other uses. As development proceeds and natural habitats become increasingly fragmented, extinction accelerates (Wilcox and Murphy, 1985). The most endangered floras are those of the arable lands; the current distribution of preserves takes little account of this. Even when the luxury of time for planning does exist and centers of species richness and endemism can be identified and conserved, many locally endemic plant species refuse to follow the rules and occur in isolated areas where, overall, conservation priorities are low. Even under ideal circumstances, though, decreases in and fragmentation of natural areas is certain to lead, as predicted by the theory of island biogeography, to substantial increases in extinction rates, though it is uncertain whether these rates would be on the scale calculated for large animals (e.g., Schonewald-Cox, 1983; Soulé et al., 1979; Simberloff and Abele, 1984). This is therefore a case for some form of selective program of ex situ conservation, that is, conservation through cultivation. Too little is known of the ecology of any plant species, let alone those that are rare and endangered, to consider the transfer of species from one natural community to another. But in addition to their immobility, plants have many practical advantages over animals for ex situ conservation. Plants are generally easy to propagate asexually through a variety of methods, including division of the rootstock, cuttings, and tissue culture. For conservation of heritable character traits, experience with a variety of crop plants has shown that gene cloning is a realistic possibility. Propagants, vegetative or seed, can be collected with minimal disturbance to the wild population and are cheap and easy to transplant. In comparison to animals, management of ex situ plant populations is relatively simple and inexpensive. Plants do not require caging, and in practice, genotypes can often be maintained for long periods, though probably not permanently, through propagation or forced rejuvenation (e.g., see Rackham, 1976, on the effect of pollarding on trees recorded over half a millennium). Furthermore, plants need less constant care than animals. The bisexuality of the majority of higher plants implies broadly that minimum population sizes for maintenance of heterosis can be half those of populations comprising two sexes. In addition, their modular construction allows considerable phenotypic plasticity. Their habitat requirements can generally be reasonably accommodated ex situ provided that competition is excluded. They do not manifest demanding behavioral traits and are rarely dangerous to humans. Added to these attributes, plants are both attractive and unobtrusive. They are more often scented than smelly, and they are generally perceived by humanity as benign. In short, they are welcomed adornments to the human environment. Is is certainly true, then, that the rapidly increasing demand for ex situ conservation, occasioned by the inexorable destruction of natural habitats, presents botanical gardens with both a challenge and an opportunity, the likes of which have not arisen for more than a century.

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BioDiversity METHODS OF EX SITU CONSERVATION In comparison to conservation of animals, flowering plants have a disadvantage in the extraordinary diversity of their reproductive systems and, notably, their sexual differentiation. These differences are considered to be major determinants of genetic patterns within populations. The kind of reproductive system influences the minimal viable population sizes needed for conservation (Wilcox and Murphy, 1985) both in situ and ex situ and controlled pollination strategies for stock regeneration. In brief, self-pollinating and vegetatively reproducing species will vary more genetically between than within breeding populations (Allard, 1960). But among outbreeders, especially dioecious species, the reverse will hold true, though differences in gene frequency between reproductively isolated populations will increase over time. In self-pollinating species, representative samples of a wide range of breeding populations should be sampled, but individual samples need be represented by comparatively few individuals. For outbreeders, individual populations should be well sampled, but fewer representatives of different populations will generally be necessary. Methods of ex situ conservation now available can conveniently be classified according to the part of the plant that is conserved—the whole organism, seed, tissues, or genetic material in culture. When kept in ex situ living collections, whole plants have educational value and can be displayed, and for species that take a long time to reach reproductive maturity, mature specimens on hand are advantageous for research. A relative disadvantage of whole plants is their higher maintenance costs in comparison to other means of ex situ plant conservation. For example, there are high requirements for space, especially for trees. Conversely, annuals require frequent, controlled pollination and reestablishment, unless they are inbreeders or methods of vegetative propagation are available. Whole plants conserved in gardens and plantations often readily hybridize with related taxa. Controlled pollination is therefore obligatory for regeneration from seed among outbreeders. When grown in single-species plantations, whole plants are more susceptible to communicable diseases than when scattered, as in nature, in a matrix of other species. On the other hand, plants will as a rule prosper outside their natural range in the absence of coevolved pathogens. This explains why crop plants, particularly long-lived tropical species such as Hevea rubber, have flourished best in plantations outside their region of origin. Clearly, this biological reality causes political problems, which must be faced and overcome if ex situ conservation is to succeed and its subjects are to be exploited to benefit humans. It also identifies a conflict between the needs for conservation per se and the need for display or education when demonstration of indigenous flora has high priority. In contrast to masses in plantations, however, specimen plants will not incur this danger. With present technology, the preferred method of ex situ conservation is through storage as seed. The principal advantage of seed banks is their economy of space and the larger sample sizes that then become possible and, in countries with high labor costs, their low labor demands. The principal practical disadvantage is their

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BioDiversity matchmaker for participating zoos who wish to swap bloodlines. As zoo animal sperm and embryo banks become a reality, it is hoped that frozen embryo and semen samples can also be cataloged in the ISIS computer as an aid to zoos interested in transporting germplasm rather than animals themselves for the introduction of new bloodlines. In consideration of the above concerns, this chapter discusses reproductive technology as it applies to the long-term preservation of animal germplasm and maintenance of biological diversity. Most ongoing wild animal research is directed toward the improvement of genetic and species diversity. Scientists have realized that the development of advanced reproductive technology, such as embryo transfer, gamete cryopreservation, and artificial insemination, may represent the real key to the future for many species who are currently threatened by extinction. They have also realized that this technology will do a great deal to improve and maintain the genetic diversity within captive populations. However, much of the application of this technology to wild species is still in its infancy. EMBRYO TRANSFER Embryo transfer is a technique by which fertilized ova and early embryos are recovered from the reproductive tract of a donor female, the genetic mother, and are transferred into the tract of a recipient female, the foster mother, in whom the embryos develop into full-term fetuses and live young. The first successful transfer of mammalian embryos was performed in the rabbit by Heape in 1891. His observations stimulated relatively little further research until about 1950. Since then, there has been an explosion of research in this area. Numerous published reviews and textbooks describe both the methods and the fundamental principles on which the technique of embryo transfer rests [see reviews by Betteridge (1977), Mapletoft (1984), Seidel (1981), and Sreenan (1983), and texts by Adams (1982), Cole and Cupps (1977), and Daniel (1978)]. Although the specifics of the methods depend upon the species used, the general principles are the same whether performed in laboratory animals such as mice and rabbits, in large domestic animals such as horses and cattle, or in wild species such as baboons and antelope. Embryo transfer in two species, mice and cattle, has become absolutely routine. It is not an exaggeration to state that tens of thousands of living mice and cattle have been produced by embryo transfer. In general terms, the transfer of embryos of other species has been modeled on techniques devised for these two species. Although there are fewer live young, probably thousands of live rabbits, pigs, and horses have been produced by embryo transfer. The application of embryo transfer to wild species is a relatively recent event. Its history can be highlighted as follows: 1975 The first successful nonhuman primate surgical embryo transfer in a baboon (Papio cynocephalus) (Kraemer et al., 1976). 1976 The first successful wildlife surgical interspecies embryo transfer between mouflon (wild sheep; Ovis musimon) and domestic sheep (Ovis aries) (Bunch et al., 1977). 1981 Second successful surgical transfer of an embryo from a wild species into a domestic species—gaur (Bos gaurus) to Holstein (Bos taurus) (Stover et al., 1981).

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BioDiversity 1983 First successful nonsurgical embryo transfer performed with an eland antelope (Tragelaphus oryx) (Dresser et al., 1984a). 1983 First successful nonsurgical embryo transfer with the eland antelope (Tragelaphus oryx) involving a previously frozen embryo (Kramer et al., 1983). 1984 First successful primate interspecies embryo transfer—macaque (Macaca fascicularis) to rhesus (M. mulatta)—following in vitro fertilization (Balmaceda et al., 1986). 1984 First frozen embryo transfers in nonlaboratory species of primates accomplished in the common marmoset (Callithrix jacchus) (Hearn and Summers, 1986). 1984 First successful nonsurgical interspecies embryo transfer between two different species of wild animals—bongo (Tragelaphus euryceros) to eland (Tragelaphus oryx). These bongo antelope embryos were brought from Los Angeles to Cincinnati and transferred fresh, 12 hours after collection (Dresser et al., 1984b). Embryos from a wild species had never before been transported long distances. 1984 Nonsurgical interspecies embryo transfer from Grant’s zebra (Equs burchelli) to horse (E. caballus) (Bennett and Foster, 1985; Foster and Bennett, 1984). 1984 First long-term frozen embryo transfer in a wild species: an eland (Tragelaphus oryx) embryo previously frozen for 1.5 years successfully transferred nonsurgically to an eland surrogate (Dresser, 1986). 1984 Interspecies embryo transfer from Przewalski’s horse (Equs przewalski) to New Forest pony (Equs caballus) (Kydd et al., 1985). 1985 First successful embryo transfer in Dall sheep (Ovis dalli) (K.Mehren, Metro Toronto Zoo, personal communication, 1986). 1987 First nonsurgical embryo transfer between guar (Bos gaurus), an endangered species, and Bos taurus, a domestic Holstein, by Dresser and colleagues. Synchronization of the donor and recipient animals in embryo transfer can be accomplished through precisely timed injections of prostaglandins, such as Lutalyse and Estrumate. These hormone analogs serve to stimulate the ovaries to begin a new cycle. Superovulation of the donor is accomplished through the injection of fertility hormones such as follicle-stimulating hormone (FSH). Superovulation has been fairly successful with the ungulates, but optimal drugs and dosages have yet to be refined for most other species. As many as 31 embryos have been collected from one FSH-stimulated eland cow (Dresser, 1983). On the other hand, fertility drugs seem to have little or no effect on the equids (such as zebras and Przewalski’s horse) (Hearn and Summers, 1986). Clearly, the hormone regimen that produces the optimal superovulation response within a given species seems to be fairly individualized and much work is needed in this particular area. In felines, the superovulation of donors and the synchronization of donors and recipients is complicated by the fact that most cats are induced or reflex ovulators, meaning that they ordinarily do not ovulate without the stimulation of copulation. Human chorionic gonadotropin (HCG) has been administered to domestic cats to cause ovulation to occur (sometimes in conjunction with stimulation by a vasectomized male) (Bowen, 1977). Researchers in several institutions have been working on embryo transfer in domestic cats with limited success (Dresser et al., 1987; Goodrowe et al., 1986; Kraemer et al., 1979), but little work has been done thus far on wild cats (Bowen et al., 1982; Reed et al., 1981). It is hoped that domestic cats may be able to serve as surrogates for incubating embryos from small endangered

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BioDiversity Gaur calf born to Holstein surrogate on 5/25/87 at Kings Island Wild Animal Habitat. This was the first nonsurgical interspecies embryo transfer between an endangered species and a domestic species. Photo by Dr. Betsy L.Dresser. wild cats such as the black-footed cat (Felis nigripes). A great deal more work needs to be done in this area to determine the best regimens for stimulating cat’s ovaries to produce more than the usual number of follicles and to cause the ovulation of these follicles and, thus, to obtain embryos. The development of embryo transfer techniques is essential if genetic diversity within captive populations is to be maximized. The ability to introduce new bloodlines into a captive population through the transfer of nonlocal embryos into surrogates, would be far preferable to the transport of adult animals for breeding purposes, or to the depletion of wild herds to add new breeding stock to captive populations. In addition, it is a goal of many zoo researchers to develop interspecies embryo transfers to the point at which embryos can be collected from endangered species and transferred to surrogates of a more common species, thereby greatly increasing the reproductive potential of the donor species. Other important benefits result from embryo transfer. For example, it has been found that disease transmission between different populations can be dramatically reduced by embryo transfer (see Hare, 1985, for review). This happens because the intact embryo collected from a diseased mother is almost always free of the microbial or viral disease agent and does not transmit the disease to the foster mother. Alternatively, the surrogate mother may confer passive immunity to her

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BioDiversity offspring that develop from transferred embryos. This may occur either through the placental blood supply or via the colostrum, the first milk. That is, it has been found that cattle of a given breed may quickly succumb to local diseases when imported into a new location. However, live calves produced by transfer of embryos from a foreign breed into another (native or endemic) domestic breed will usually be as disease-resistant as this domestic breed. This accident of biology should have important consequences for the transplant of both domestic and wild species from one location to another. Interspecies embryo transfer has enjoyed limited success in wild animals, but much more research needs to be done in this area. Intergeneric, e.g., eland to cow (Dresser et al., 1982a) or water buffalo to cow (Drost, 1983), as opposed to interspecies, e.g., tiger to lion (Reed et al., 1981), embryo transfers have never been successful. It seems that most embryos can develop to the early blastocyst stage in the oviducts of unrelated species (Daniel, 1981), but further development requires a much closer relationship between donor and recipient and similarity in time and type of implantation, placenta formation, rate of ovum transport, length of gestation, birth weight, and both neonatal and maternal postpartum behavior. Several techniques have been tried to help overcome the surrogate mother’s immune response and prevent the rejection of the foreign embryo in an interspecies embryo transfer. Recently, a domestic horse mare gave birth to a donkey foal after she had been injected with donkey white blood cells (Antczak, 1985). Prior to this treatment, all other donkey embryo transfers into domestic horses had failed. It is not yet clear why the procedure was successful. Although various attempts have been made to collect embryos from nonhuman primates at various zoos and primate centers over the years, very few of these procedures have been reported in the scientific literature. For a review of the existing literature on superovulation and ova collection attempts in the more common nonhuman primates, see Bavister et al. (1985), Clayton and Kuehl (1984), Hodgen (1983), and Kraemer et al. (1979). There has been dramatic progress in embryo micromanipulation in little over 6 years, especially as it applies to domestic animals. The research of Willadsen (1979, 1980) and Willadsen et al. (1981) is most notable in this regard. In the most important application of micromanipulation to exotic animals, an embryo is microsurgically bisected into two or more pieces, thereby producing genetically identical twins or triplets. This technique, although not yet successful with exotic species, could help to quickly increase the numbers of endangered or rare animals. Another result of micromanipulation has been the production of chimeras, which are embryos that are a product of combining two embryos at a relatively early stage of development. It is possible to prevent the surrogate uterus from recognizing the foreign embryo by combining different blastomeres. The younger cells tend to form the trophoblast that gives rise to the placenta, and the older cells tend to form the inner cell mass that will form the fetus. Chimeric embryos can be constructed so that the cells that constitute the trophoblast belong to the surrogate species and the cells of the inner cell mass belong to the donor species. A reupholstered embryo

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BioDiversity such as this can effectively trick the surrogate’s uterus into thinking it is carrying a native embryo (Vietmeyer, 1984). Interspecies embryo transfer and inner cell mass transfer should not be confused with hybridization of species. Offspring contain only genetic material of the original species. Many people now recognize that the method of chimera production might be utilized to rescue endangered species. It might be possible to construct chimeras consisting of an embryo from an endangered species plus an embryo of a common, but related species. The common species might then carry the fetus of the endangered species to term. In summary, then, embryo transfer has become a widely used technique to produce live young animals, especially of domestic species. Although it is as yet still a novel procedure in wild species, continued research will inevitably make transfer of wild animal embryos as successful as the transfer of domestic animal embryos. CRYOPRESERVATION OF EMBRYOS The first successful freezing of mammalian embryos was reported in 1972 (Whittingham et al., 1972; Wilmut, 1972). Since those reports, more than 300 articles and 100 abstracts have been published on this one subject. There have also been three full meetings (Ciba Foundation, 1977; Muhlbock, 1976; Zeilmaker, 1981) and numerous symposia devoted exclusively to the freezing of mammalian embryos. Numerous reviews have also been published (Lehn-Jensen, 1981; Leibo, 1977, 1981; Maurer, 1978; Rall et al., 1982; Renard, 1982). Freezing of mouse, rabbit, and bovine embryos has now become a routine procedure. Altogether, embryos of 11 mammalian species have now been successfully frozen. Again, success means that live young have been born from frozen embryos. To date, the species that have been successfully preserved include the mouse (Whittingham et al., 1972), cow (Wilmut and Rowson, 1973), rabbit (Bank and Maurer, 1974), sheep (Whittingham et al., 1972), rat (Whittingham, 1975), goat (Bilton and Moore, 1976), horse (Yamamoto et al., 1982), human (Trounson and Mohr, 1983), baboon (Pope et al., 1984), antelope (Dresser et al., 1984a), and cat (Dresser et al., 1987). The freezing of mouse embryos has become so routine that banks of tens of hundreds of thousands of mouse embryos have been frozen to preserve valuable genetic stocks for extended times. For some species, most notably the mouse, rabbit, and cow, preservation by freezing has reached such a high level of sophistication and reliability that approximately 80 to 90% of frozen embryos will develop in vitro when thawed and cultured. Moreover, the procedures to freeze embryos have become increasingly simplified. Regardless of the methods used, it can be reasonably estimated that thousands, if not tens of thousands, of live young of domestic species have been produced from frozen-thawed embryos. Again, because of extremely limited experimental material, only a few attempts have been made to transfer previously frozen wild animal embryos into recipients.

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BioDiversity The procedure has yielded some limited success (Cherfas, 1984; Dresser et al., 1984a; Hearn and Summers, 1986; Kramer et al., 1983). Cryopreservation will undoubtedly prove to be an important adjunct to reproductive research in nondomestic animals. Geneticists Thomas Foote and Ulysses Seal have determined that a population of 250 properly managed animals of a particular species can theoretically preserve 95% of the original genetic diversity of the group after 50 generations (400 years) (Myers, 1984). The world’s zoos, however, have limited facilities and often cannot accommodate large numbers of animals of each species they maintain. Success in the area of cryopreservation will allow zoo professionals to overcome the limited space in zoos and wild animal preserves by maintaining the bulk of the desired genetic diversity in liquid nitrogen freezers. Embryos containing new bloodlines could be recovered in the wild and brought back to zoos in the frozen state to improve the bloodlines of captive populations without depleting the wild herds. U.S. government restrictions currently prohibit the importation of embryos from other countries, but scientists have been actively lobbying for a change in these restrictions. It appears that a change may be possible in the future. ARTIFICIAL INSEMINATION Artificial insemination is the introduction of semen into the vagina or cervix by artificial means. This procedure was supposedly used by the Arabs in ancient times, but the first documented success in the modern world occurred in 1784 with the artificial insemination of a dog (Betteridge, 1981). In the 1930s, artificial insemination of livestock was used extensively in Russia. Arthur Walton demonstrated its potential as an effective method to transport genes in the 1920s and 1930s by shipping fresh rabbit, sheep, and bull semen from England to other European countries (Betteridge, 1981). The ability to successfully freeze semen resulted from the discovery of the cryoprotective action of glycerol by Polge, Smith, and Parkes in 1949 (Betteridge, 1981). Artificial insemination is very common in the agricultural industry today. Foote (1981) estimated that close to 90 million head of cattle were produced worldwide in 1977 by artificial insemination with previously frozen semen samples. Artificial insemination has had limited success in wild animals thus far, especially with certain species of mammals and birds. Success has been attained for the following species: Nondomestic Mammals Addax1 Brown brocket deer Ferret Rhesus monkey Guanaco Reindeer Fox1 Baboon Llama Red deer Wolf1 Squirrel monkey Blackbuck1 Speke’s gazelle Persian leopard Chimpanzee1 Bighorn sheep Giant panda1 Puma Gorilla1 1Frozen semen.

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BioDiversity Birds Reptiles Cranes (several species) Waterfowl (ducks) Tortoises1 Albino cockatiel Pheasants   Raptors     1Frozen semen. Much more semen has been collected from wild species than has actually been evaluated and used. The first successful artificial insemination of a wild species with previously frozen semen occurred in 1973 with the wolf (Seager, 1981). This was followed by the successful insemination of a gorilla (Douglass and Gould, 1981). There has been a great deal of time and effort spent trying to artificially inseminate wild-caught felidae (cats); most attempts have resulted in failure (Dresser et al., 1982b). The London Zoo finally produced a puma in 1980 through surgical artificial insemination with a fresh semen sample (Moore et al., 1981). This was followed in 1981 by a successful nonsurgical artificial insemination of a Persian leopard with fresh semen at the Cincinnati Zoo (Dresser et al., 1982b). There are three methods of semen collection: manual stimulation of the male reproductive tract, use of an artificial vagina, and electroejaculation (Cherfas, 1984). Electroejaculation was invented by two French workers, Jonet and Cassou, and is by far the most common collection mode for wild animals. Electroejaculation works by inserting a lubricated probe into the rectum of an anesthetized animal. This conveys mild pulsating electrical stimuli to the nerves of the reproductive tract, resulting in ejaculation. There is some question about the fertility of sperm collected through electroejaculation, but there is also a question about how viable a semen sample must be to be effective. For example, the successful gorilla insemination at the Memphis Zoo in 1981 was accomplished with a previously frozen sample whose motility was 10% and judged to be poor at the time of insemination (Douglass and Gould, 1981). A great many problems are associated with artificial insemination as it applies to wild animals. First of all, it necessitates the use of anesthesia, which is always a risk, for both semen collection from the male and insemination of the female. In addition, as mentioned above for electroejaculation, the fertility of semen obtained from artificial collection techniques is sometimes questionable when compared to that produced in a natural ejaculation. Sperm usually begin to die as soon as they are collected. Even if a fresh sample is used for insemination, it is likely to have undergone a certain amount of sperm loss. SEMEN CRYOPRESERVATION The freezing process is somewhat detrimental to sperm, and it is very unlikely for sperm to come out of a thaw as motile as they were going into the freeze (Cochran et al., 1985). Much work needs to be done in the area of semen cryopreservation for exotic animals. To date, sperm from at least 200 different species has been frozen, but very little of it has actually been thawed and tested (Seager, 1981). The ultimate test is production of offspring. From semen that has been tested, cryobiologists have found that sperm from each species needs to be extended and frozen under slightly different conditions to produce the optimal results.

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BioDiversity Extenders used to preserve the collected semen basically consist of a buffered solution that contains a cryoprotectant (e.g., glycerol), antibiotics, and either egg yolk or milk. Many variations have been tried in the basic recipe for semen extender. The agricultural industry has found that the optimal extender for a given species seems to be very species-specific and that is turning out to be true with wild animals also. Perhaps some clues for semen preservation can be found in the natural world from studying certain female reptiles who have the potential for keeping sperm viable within their bodies for up to 6 years after mating (Cherfas, 1984). Other problems with artificial insemination include difficulty in predicting the optimal time for inseminating the female and the fact that artificial insemination cannot occur as frequently as a female would have been inseminated naturally for the duration of her estrous cycle. On the positive side, artificial insemination can be a great boon toward improving the genetic diversity of a captive population of animals. As with embryo transfer, the risk and expense of transporting semen is far less than that of transporting a male animal for breeding purposes. Artificial insemination could also be used to overcome quarantine restrictions and the risks of disease. Often, the strict agricultural legislation has made transport of zoo animals more difficult and costly than is perhaps necessary. FUTURE PROSPECTS Since preservation of genetic material from a species is one of the keys to ensuring diversity, the development of reproductive technology for exotic species, such as cryopreservation of gametes, embryo transfer, and artificial insemination, should be emphasized and supported. Ex situ animal conservation programs that are dependent upon the long-term preservation of genetic variation should apply this technology as it becomes available because of the increasing realization that captive breeding programs are essential to prevent many species from becoming extinct. Loss of genetic diversity could also limit the potential of a population to adapt to new environments when reintroduced to the wild. A large amount of basic research is urgently needed before application of new technology will be routine for maintaining captive populations. It is hoped that the urgency will be recognized by many more scientists worldwide than at present, and that ex situ conservation programs will become the nuclei of genetic material for dwindling populations of wild animals. Extinction for some may be softened by the frozen zoo concept, which may turn out to be the single most important reproductive technology developed for exotic animals during this decade. Its effects will reach centuries into the future for many species. REFERENCES AAZPA (American Association of Zoological Parks and Aquariums). 1983. Species Survival Plan Handbook Publication. Oglebay Park, Wheeling, W. Va. 24 pp. Adams, C.E., ed. 1982. Mammalian Egg Transfer. C. R. C. Press Inc., Boca Raton, Fla.

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BioDiversity Antczak, D.F. 1985. Cute cross-species birth. Sci. News 128:8. Balmaceda, J., T.Heitman, A.Garcia, C.Pauerstein, and R.Pool. 1986. Embryo cryopreservation in cynamologus monkey. Fertil. Steril. 45(3):403–406. Bank, H., and R.R.Maurer. 1974. Survival of frozen rabbit embryos. Exp. Cell Res. 89:188–196. Barney, G.O. (Study Director). 1982. The Global 2000 Report to the President of the United States. Vol. 1: The Summary Report—Special Edition with the Environmental Projections and the Government’s Global Model. Council on Environmental Quality, U.S. Government Printing Office, Washington, D.C. 360 pp. Bavister, B.D., K.Collins, and S.Eisele. 1985. Non-surgical embryo transfer in the rhesus monkey. Theriogenology 23:177. Bennett, S.D., and W.R.Foster. 1985. Successful transfer of a zebra embryo to a domestic horse. Equine Vet. J. Suppl. 3:53–62. Betteridge, K.J. 1977. Embryo Transfer in Farm Animals. Monograph 16. Canada Department of Agriculture, Ottawa. 92 pp. Betteridge, K.J. 1981. An historical look at embryo transfer. J. Reprod. Fertil. 62:1–13. Bilton, R.J., and R.W.Moore. 1976. In vitro culture, storage, and transfer of goat embryos. Aust. J. Biol. Sci. 29:125–129. Bowen, M.J., C.C.Platz, Jr., C.D.Brown, and D.C.Kraemer. 1982. Successful artificial insemination and embryo collection in the African lion (Panthera leo). Am. Assoc. Zoo Vet. 57–59. Bowen, R.A. 1977. Fertilization in vitro of feline ova by spermatozoa from the ductus deferens. Biol. Reprod. 17:144–147. Bunch, T.D., W.C.Foote, and B.Whitaker. 1977. Interspecies ovum transfer to propagate wild sheep. J. Wildl. Manage. 41(4):726–730. Cherfas, J. 1984. Test-tube babies in the zoo. New Sci. 16–19. Ciba Foundation. 1977. The Freezing of Mammalian Embryos. Symposium 52. Elsevier Excerpta Medica, Amsterdam. 330 pp. Clayton, O., and T.J.Kuehl. 1984. The first successful in vitro fertilization and embryo transfer in a non-human primate. Theriogenology 21:228. (Abstract) Cochran, R.C., J.K.Judy, C.F.Parker, and D.M.Hallford. 1985. Prefreezing and post-thaw semen characteristics of five ram breeds collected by electroejaculation. Theriogenology 23:431–440. Cole, H.H., and P.T.Cupps, eds. 1977. Reproduction in Domestic Animals. Academic Press, New York. 665 pp. Croner, S. 1984. An Introduction to the World Conservation Strategy. Prepared for the International Union for Conservation of Nature and Natural Resources (IUCN) by its Commission on Education. IUCN, Gland, Switzerland; United Nations Environmental Program, Nairobi, Kenya. 28 pp. Daniel, J.C., Jr., ed. 1978. Methods in Mammalian Reproduction. Academic Press, New York. 566 pp. Daniel, J.C. 1981. Preserving the genome of endangered species. SWARA—E. Afr. Wildl. Soc. 4:16–18. Douglass, E.M., and K.G.Gould. 1981. Artificial insemination in lowland gorilla (Gorilla g. gorilla). Pp. 128–130 in Annual Proceedings—American Association of Zoo Veterinarians. Hills Division, Riviana Foods, Topeka, Kans. Dresser, B.L. 1983. Embryos of the African eland (Taurotragus oryx). Pp. 9–11 in Proceedings of the Owners and Managers Workshop of the IXth Annual Meeting of the International Embryo Transfer Society. International Embryo Transfer Society, Fort Collins, Colo. Dresser, B.L. 1986. Embryo transfer in exotic bovids. Int. Zoo Yearb. 24/25:138–142. Dresser, B.L., L.Kramer, C.E.Pope, R.D.Dahlhausen, and C.Blauser. 1982a. Superovulation of African eland (Taurotragus oryx) and interspecies embryo transfer to holstein cattle. Theriogenology 17:86. (Abstract) Dresser, B.L., L.Kramer, B.Reece, and P.T.Russell. 1982b. Induction of ovulation and successful artificial insemination in a Persian leopard (Panthera pardus saxicolor). J. Zoo Biol. 1(1):55–57. Dresser, B.L., L.Kramer, R.D.Dahlhausen, C.E.Pope, and R.D.Baker. 1984a. Cryopreservation followed by successful transfer of African eland antelope (Tragelaphus oryx) embryos. Pp. 191–193 in Proceedings of the 10th International Congress on Animal Reproduction and Artificial Insemination. Standing Committee of the International Congress of Animal Reproduction and Artificial Insemination, University of Illinois at Urbana-Champaign, Ill. Dresser, B.L., C.E.Pope, L.Kramer, G.Kuehn, R.D.Dahlhausen, E.J.Maruska, B.Reece, and W.D.Thomas. 1984b. Successful transcontinental and interspecies embryo transfer from bongo an-

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BioDiversity telope (Tragelaphus euryceros) at the Los Angeles Zoo to eland (Tragelaphus oryx) and bongo at the Cincinnati Zoo. Pp. 166–168 in Annual Proceedings of the American Association of Zoological Parks and Aquariums (AAZPA). American Association of Zoological Parks and Aquariums, Wheeling, W. Va. Dresser, B.L., C.S.Sehlhorst, G.Keller, L.W.Kramer, and R.W.Reece. 1987. Artificial insemination and embryo transfer in the Felidae. Pp. 287–293 in Tigers of the World: The Biology, Biopolitics, Management and Conservation of an Endangered Species. Noyes Publications, Park Ridge, N.J. Drost, M. 1983. Reciprocal embryo transfer between water buffaloes and cattle. Pp. 63–71 in Proceedings of the Annual Meetings of the Society for Theriogenology. Society for Theriogenology, Hastings, Nebr. Foote, R.H. 1981. The artificial insemination industry. Pp. 13–39 in B.G.Brackett, G.E.Seidel, and S.M.Seidel, eds. New Technologies in Animal Breeding. Academic Press, New York. Foster, W.R., and S.D.Bennett. 1984. Non-surgical embryo transfer between a grant zebra and domestic quarterhorse: A tool for conservation. P. 181 in Annual Proceedings of the American Association of Zoo Veterinarians. Hills Division, Riviana Foods, Topeka, Kans. Goodrowe, K.L., J.G.Howard, and D.E.Wildt. 1986. Embryo recovery and quality in the domestic cat: Natural versus induced estrus. Theriogenology 25:156. (Abstract) Gorman, J. 1980. Brave new zoo. Discover 1:46–49. Hare, W.C.D. 1985. Diseases Transmissable by Semen and Embryo Transfer. Office Internationale des Epizost, Paris. 36 pp. Heape, W. 1891. Preliminary note on the transplantation and growth of mammalian ova within a uterine foster-mother. Proc. R. Soc. London 48:457–458. Hearn, J.P., and P.M.Summers. 1986. Experimental manipulation of embryo implantation in the marmoset monkey and exotic equids. Theriogenology 25:3–11. Hodgen, G.D. 1983. Surrogate embryo transfer combined with estrogen-progesterone therapy in monkeys. J. Am. Med. Assoc. 250:2167–2171. Kraemer, D.C., G.T.Moore, and M.A.Kramen. 1976. Baboon infant produced by embryo transfer. Science 192:1246–1247. Kraemer, D.C., B.L.Flow, M.D.Schriver, G.M.Kinney, and J.W.Pennycook. 1979. Embryo transfer in the nonhuman primate, feline and canine. Theriogenology 11:51–62. Kramer, L., B.L.Dresser, C.E.Pope, R.D.Dahlhausen, and R.D.Baker. 1983. The nonsurgical transfer of frozen-thawed eland (Tragelaphus oryx) embryos. Pp. 104–105 in Annual Proceedings—American Association of Zoo Veterinarians. Hills Division, Riviana Foods, Topeka, Kans. Kydd, J., M.S.Boyle, W.R.Allen, A.Shephard, and P.M.Summers. 1985. Transfer of exotic equine embryos to domestic horses and donkeys. Equine Vet. J. Suppl. 3:80–83. Lehn-Jensen, H. 1981. Deep freezing of cow embryos, a review. Nord. Vet. Med. 32:523–532. Leibo, S.P. 1977. Preservation of mammalian cells and embryos by freezing. Pp. 311–344 in D. Simatos, D.M.Strong, and J.M.Turc, eds. Cryoimmunologie: Colloque, Dijon, 17–18 Juin 1976. Organis Editions de l’Institut National de la Sant et de la Recherche Medicale, Paris. Leibo, S.P. 1981. Physiological basis of the freezing of mammalian embryos. Pp. 353–379 in M.E. Gershwin and B.Merchant, eds. Immunologic Defects in Laboratory Animals, Vol. 2. Plenum, New York. Letvin, N.L., and N.W.King. 1984. Human disease: Acquired immune deficiency syndrome. Animal model: Acquired immune deficiency syndrome-like disease in non-human primates. Comp. Pathol. Bull. 16:5–6. Lushbaugh, C.C., G.L.Humason, and N.K.Clapp. 1984. Spontaneous colorectal adenocarcinoma in cotton-topped tamarins. Comp. Pathol. Bull. 15:2, 4. Mapletoft, R.F. 1984. Embryo transfer technology for the enhancement of animal reproduction. Biotechnology 2:149–160. Mauer, R.R. 1978. Freezing mammalian embryos. A review of the techniques. Theriogenology 9:45–68. Moore, H.D.M., R.C.Bonney, and D.M.Jones. 1981. Induction of oestrus and successful artificial insemination in the cougar, Felis concolor. Pp. 141–142 in Annual Proceedings—American Association of Zoo Veterinarians. Hills Division, Riviana Foods, Topeka, Kans. Muhlbock, O., ed. 1976. Basic Aspects of Freeze Preservation of Mouse Strains. Gustav Fisher, Stuttgart, Federal Republic of Germany. Myers, N. 1984. Cats in crisis! Int. Wildl. 14 (Nov-Dec):42–52.

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BioDiversity Pope, C.E., V.Z.Pope, and L.R.Beck. 1984. Live birth following cryopreservation and transfer of a baboon embryo. Fertil. Steril. 42:143–145. Rall, W.F., D.S.Reid, and C.Polge. 1982. Physical and temporal factors involved in the death of embryos that contain ice. Pp. 303–305 in F.Franks, ed. Biophysics of Water. Proceedings of a working conference held at Cirton College, Cambridge, June 29–July 3, 1981. John Wiley and Sons, New York. Reed, G., B.Dresser, B.Reece, L.Kramer, P.Russell, K.Pindell, and P.Berringer. 1981. Superovulation and artificial insemination of Bengal tigers (Panthera tigris) and an interspecies embryo transfer to the African lion (Panthera leo). Pp. 136–138 in Annual Proceedings—American Association of Zoo Veterinarians. Hills Division, Riviana Foods, Topeka, Kans. Renard, J.P., Y.Menezo, M.C.Pheron. 1982. Effect of lipid supplement on the viability of D7–D8 Ovine blastocysts after freezing and thawing (Abstract). In Embryo Transfer in Mammals, 2nd International Congress, Annecy. Foundation Marcel Merieux, Lyon, France. Seager, S.W.J. 1981. A review of artificial methods of breeding in captive wild species. Dodo, Jersey Wildl. Preserv. Trust, 18:79–83. Seidel, G.E., Jr. 1981. Superovulation and embryo transfer in cattle. Science 211:351–358. Snyder, R.L., G.Tyler, and J.Summers. 1982. Chronic hepatitis and hepatocellular carcinoma associated with woodchuck hepatitis virus. Am. J. Pathol. 107:422–425. Sreenan, J.M. 1983. Embryo transfer procedure and its use as a research technique. Vet. Rec. 112:494–500. Storrs, E.E., C.H.Binford, and G.Migaki. 1980. Experimental lepromatous leprosy in nine-banded armadillos (Dasypus novemcinctus Linn). Am. J. Pathol. 92:813–816. Stover, J., J.Evans, and E.P.Dolensek. 1981. Interspecies embryo transfer from the gaur to domestic holstein. Pp. 122–124 in Annual Proceedings—American Association of Zoo Veterinarians. Hills Division, Riviana Foods, Topeka, Kans. Stuhlman, R.A. 1979. Diabetes mellitus. Animal model: Spontaneous diabetes mellitus in Mystromys albicaudatus. Am. J. Pathol. 94:685–688. Trounson, A.O., and L.Mohr. 1983. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305:707–709. Vietmeyer, N. 1984. From horses come zebras. Int. Wildl. 14(Nov-Dec):12–13. Whittingham, D.G. 1975. Survival of rat embryos after freezing and thawing. J. Reprod. Fertil. 43:575–578. Whittingham, D.G., S.P.Leibo, and P.Mazur. 1972. Survival of mouse embryos frozen to −196° and −269°C. Science 178:411–414. Willadsen, S.M. 1979. A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins. Nature 277:298–300. Willadsen, S.M. 1980. The viability of early cleavage stages containing half of the normal number of blastomeres in the sheep. J. Reprod. Fertil. 59:357–362. Willadsen, S.M., C.Polge, L.E.A.Rowson, and R.M.Moor. 1974. Preservation of sheep embryos and liquid nitrogen. Cryobiology 11:560. (Abstract) Willadsen, S.M., H.Lehn-Jensen, C.D.Fehilly, and R.Newcomb. 1981. The production of monozygotic twins on preselected parentage by micromanipulation of nonsurgically collected cow embyros. Theriogenology 15:23–30. Wilmut, I. 1972. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. 11:1071–1079. Wilmut, I., and L.E.A.Rowson. 1973. Experiments on the low-temperature preservation of the cow embryos. Vet. Rec. 92:686–690. Wolkomir, R. 1983. Draining the gene pool. Natl. Wildl. 21(Oct-Nov):24–28. Yamamoto, Y., N.Oguri, Y.Tsutsumi, and Y.Hachinohe. 1982. Experiments in the freezing and storage of equine embryos. J. Reprod. Fertil. Suppl. 32:399–403. Zeilmaker, G.H., ed. 1981. Frozen Storage of Laboratory Animals. Proceedings of a workshop at Harwell, U.K., May 6th–9th, 1980. Gustav Fisher, Stuttgart, Federal Republic of Germany. 193 pp.