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ILAR Journal V32(1) 1990
State of the Art

State of the Art

Semen Collection, Evaluation, and Cryopreservation in Exotic Animal Species: Maximizing Reproductive Potential
Barbara S. Durrant

Barbara S. Durrant is head of the Reproductive Physiology Division, Center for Reproduction of Endangered Species, Zoological Society of San Diego.

Introduction
In 1980, Thomas Lovejoy of the World Wildlife Fund stated that reduction in the biological diversity of the planet is the most basic issue of our time and that slowing this process of "biotic impoverishment" is a great challenge to the ingenuity of biologists. Extinction of a species represents the loss of a resource that has evolved through thousands, perhaps millions, of years of mutation and natural selection. The genetic diversity that now exists in captive exotic animals will steadily dwindle through random processes without continuous input from wild populations. The National Research Council (1978) has stressed the importance of conserving this genetic material and emphasized that if immediate action is not taken, much of this vital resource will be depleted in the near future.

It is incumbent upon American zoos, as stewards of a large proportion of the earth's captive species, to maintain them in sufficient numbers to ensure that genetic variability is adequate for long-term genetic health. Notter and Foose (1985) have calculated that nearly 50,000 individuals would be required to maintain 99 percent of a species' genetic diversity for 1,000 generations. Because of severe space limitations in zoos and animal parks, this ideal genetic diversity will not likely be attainable for any of the species we are attempting to save from extinction. Perhaps a more realistic goal is to preserve 90 percent of a species' diversity for 1,000 generations with less than 5,000 individuals. Even that greatly reduced number of individuals is impossible to maintain in captivity.

However, long-term germ plasm storage can minimize holding space requirements and increase the number of species that can be conserved through captive breeding. The judicious assimilation of genetic material from frozen reserves into extant breeding populations will ensure continuance of genetically diverse species in captivity.

Importation of wild-caught individuals of endangered species is now greatly restricted. When cryopreservation protocols have been perfected, it will be feasible for researchers to collect germ plasm "in the field" to sustain captive breeding programs. Incorporation of germ plasm from individuals remaining in the wild into captive populations will increase the genetic diversity of captive groups without the losses that would occur by removing free-ranging animals from the wild.

Using frozen gametes and zygotes for artificially enhanced reproduction offers greater potential for genetic contribution from underrepresented captive individuals to extant and future generations. Overrepresented individuals can contribute germ plasm to future generations when their genes may again benefit the population. Because the majority of captive species are within a few generations of genetic input from the wild, file most immediate need is to preserve the genetic diversity that currently exists in captive populations.

The efficacious use of cryopreserved germ plasm requires the development of artificial insemination (Al), in vitro fertilization (IVF), and embryo transfer (ET) techniques for each endangered species.

The potential value of cryopreserved gem plasm for basic and applied research has yet to be realized in wildlife species (Ryder and Benirschke, 1984). Once sufficient germ cells and embryos have been preserved to assure the vigorous continuation of a species, excess genetic material in these forms can be made available for biological research to benefit the species. Frozen germ plasm will provide researchers with materials to conduct fundamental studies in disease transmission, genetics, sperm-egg interaction, and early embryonic development.

Eaglesome et al. 0980) have compiled considerable data concerning infection of the gametes and early embryos of domestic species by viral, bacterial, and protozoan agents. Knowledge of the vulnerability or resistance of semen and embryos to various disease agents is of great importance when formulating import-export regulations concerning the transfer of genetic material across state and national borders. This issue is a matter of considerable concern to exotic animal breeders as well as commercial livestock industries. Expansion of these studies to include exotics will depend upon the availability of germ plasm from cap-five wild species.

Heterologous in vitro fertilization can be used to evaluate the karyotype of sperm cells (Rudak et al., 1978). The technique could potentially facilitate identification of lethal or undesirable chromosomal aberrations and, by removing affected males' semen from the breeding population, the elimination of the defect from the population.

Embryo sexing by staining or karyotypic analysis prior to ET will allow the maintenance of the most favorable sex ratios in captive breeding groups. For species that are normally housed in single male groups (i.e., Przewalski's horses [Equus przewalskii] and lion-tailed macaques [Macaca silenus]), problems associated with housing excess males could be alleviated by embryo sexing.

Intraspecies in vitro fertilization studies may elucidate mechanisms of fertilization or early embryonic development that can be enhanced for greater reproductive success or inhibited for contraception.

Our future dependence on cryopreserved germ plasm to maintain genetic diversity obligates the curators of "frozen zoos" to store only the highest quality samples. Although the successful management and propagation of endangered species depends on the long-term storage and discriminating use of cryopreserved germ plasm, the technology to properly preserve it is not yet in place. To achieve the goal of functional germ plasm depositories, extensive research must be undertaken to optimize cryopreservation techniques for each species. Although individual research facilities and zoological institutions are beginning to establish and stock private germ plasm banks, no large-scale centrally organized or coordinated program exists for sampling, evaluating, preserving, and using available sources of germ plasm (Council for Agricultural Science and Technology, 1984).

The U.S. Congress Office of Technology Assessment, in its 1987 report, "Technologies to Maintain Biological Diversity," describes a series of steps through which useful technology emerges: first, basic research provides an understanding of the nature of a biological system. As a result of these basic studies, researchers define requirements and develop techniques to manage a species or its genetic resources. Next, researchers must translate techniques into technologies, and finally, technologies must be applied to site-specific circumstances. The difficulty in transferring basic research to applied research is regarded as a primary impediment to the development of useful technologies.

The dearth of consistently successful AI or ET programs for exotic animals illustrates the difficulty of extrapolating successful protocols from domestic species or humans to endangered species and the need for carefully designed studies in male and female reproductive and gamete physiologies.

It has been stated that the most efficient method for preserving gene pools is semen cryopreservation (Gee, 1984). Collecting semen is less problematic than collecting ova or embryos; thus, more samples can be made available for cryopreservation studies. In addition, semen thawing and AI require less training and equipment than do embryo thawing and ET. Therefore, Al is feasible for more zoological institution personnel. For example, American cattle breeders performed 200,000 ETs in 1985 primarily using fresh embryos collected and transferred by reproductive physiologists or veterinarians. In contrast, almost all of the 10.5 million dairy calves produced by AI that year were conceived with frozen semen inseminated by farm personnel and trained technicians (U.S. Congress, Office of Technology Assessment, 1987). Because most zoos do not employ reproductive physiologists or veterinarians trained in embryo transfer techniques, AI offers the opportunity for more zoos to cooperate in programs designed to maximize the genetic diversity of captive species.

Semen Collection
The intractability of most exotic species severely limits our ability to collect semen without chemical restraint. Hence, electro-ejaculation (EE) has become the standard collection technique in zoos and primate centers (Figure 1). The physiological mechanisms of EE are well understood (Martin, 1978), and the prerequisite tranquilization or immobilization appears to be the most significant health concern associated with the procedure.

Adherence to a regimented protocol of stimulation for each EE procedure has been suggested (Wildt et al., 1983). However, because response to EE can vary greatly between males of the same species and even between successive attempts with the same male, standard EE practice calls for modification of the protocol during each procedure based on the animal's response (Merilan et al., 1981). Voltage and number of stimulations required for erection, ejaculation, or both is affected by the animal's plane of anesthesia. Generally, the deeper the anesthetic plane, the more stimulation is required (Gould et al., 1978). Some tranquilizers and anesthetics are contraindicated for EE (Meltzer et al., 1988). Even identical doses of anesthetics are not always similarly effective, probably due to physiological differences between males, including those differences resulting from the stress of capture and restraint. The small body of literature describing the influence of anesthesia on EE must be greatly expanded to provide guidelines for exotic species.

Semen collection with an artificial vagina (AV) offers the advantage of frequent sampling without the stress of chemical or physical restraint (Figure 2; Durrant et al., 1985). The contact required to train a male to service an AV excludes all but the most tractable animals. AV semen collection in chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) has been reported (Fussell et al., 1973; Gould et al., 1985 ). In San Diego, a hand-raised cheetah (Acinonyx jubatus) contributed 200 semen samples to a cryopreservation program over 4 years using an AV, and a second cheetah contributed 30 samples over 9 months (Durrant et al., 1989). Three mother-raised cheetahs were sufficiently nonaggressive to permit routine collection with an AV.

An alternative opportunity for noninvasive semen collection is the use of ejaculates obtained by masturbation (Gould et al., 1985; Martin et al., 1978). At San Diego a number of primate species, including the drill baboon (Papio leucophaeus) and the lion-tailed macaque, have provided ejaculates for detailed evaluation and cryopreservation. Rewarding successful masturbatory behavior (preferably in an off-exhibit area) may be, for certain males, a most efficient method of obtaining large numbers of samples for cryopreservation, artificial reproduction studies, or both.

Semen collection in avian species is not widely practiced in zoos despite the relative ease of capture and physical restraint Imprinted birds have been trained to deposit semen on or in appropriate receptacles (Boyd and Schwartz, 1983). Manual "massage" collection has been described for various species (Figure 3), including cranes ( Grus canadensis : Gee, 1983), budgerigars ( Melopsittacus undulatus; Samour et al., 1986), and pheasants (Lophophorus lhuysii; Spiller et al., 1976). Although a relatively uncommon technique in birds, EE has been reported in parrots (Harrison and Wasmund, 1983) and ducks. The latter have been shown to respond to EE with larger volume and higher concentration ejaculates than with the massage technique (Watanabe, 1957).

There are very few published reports of semen collection in reptiles. Cloacal massage and Al of resultant semen were reported in snakes by Mendgen et al. in 1980. EE yielded semen samples heavily contaminated with urates. A combination of EE and manual penile massage was successful in producing ejaculates in unanesthetized Galapagos tortoises (Geochelone elephantopus) and Aldabra tortoises (G. gigantea) (Durrant, unpublished data, 1982). Larsen and Cardeilhac 1984) did not find EE useful in alligators (Alligator mississippicrisis), but were able to collect sperm by penile groove cannulation and postmortem epididymal extraction.

Postmortem extraction of sperm can be developed as an important source of germ plasm. Sperm from a pygmy chimpanzee (P. paniscus), lion-tailed macaque, and paras monkey (Cercopithecus patas) frozen as long as 12 hours after death have shown acceptable postthaw motility and ability to penetrate hamster ova in vitro (Durrant, 1987).

Semen Evaluation
Male fertility is a symphony of physiology, endocrinology, and behavior. It is a complex continuum that begins with spermatogenesis and proceeds to sperm maturation in the epididymis, ejaculation, sperm transport through the female reproductive tract, and finally, penetration of the ovum. Each step in this scheme (greatly oversimplified) is the subject of intense scientific investigation aimed at defining the myriad biochemical processes leading to normal fertility and the perturbations that result in subfertility or sterility. The biological basis of male infertility is still not well understood even in the most extensively studied species.

The ultimate test of male fertility is, of course, conception. The fertilizing capacity of sperm can be evaluated by examining ova for evidence of fertilization following natural breeding or artificial insemination. Unfortunately, for the majority of exotic mammals, the time of ovulation is not known, and artificial insemination techniques have not been perfected. Removal of ova from females for microscopic examination is not a procedure that could be justified for routine fertility assessment. An alternative strategy would be to inseminate ova in vitro. However, the ova of endangered species are not readily available for such studies. These difficulties also plague researchers working with human subjects and have led to attempts to develop laboratory tests of semen quality that are correlated with fertility.

Traditional parameters of semen quality include volume and sperm motility, concentration, and morphology. As single predictors of fertility, none of these measurements are sufficiently sensitive. When these parameters are combined, the accuracy of fertility assessment is enhanced but still incomplete (as none of these criteria can predict the ability of sperm to perform effectively in the female reproductive tract).

Truly comprehensive semen analysis involves evaluation of the ability of sperm to

· reach the site of fertilization,
· undergo capacitation and the acrosome reaction,
· penetrate the zona pellucida, and
· fuse with the ooplasm and decondense (Bedford, 1981).

In vitro tests have been developed for each of the above parameters, and their correlation with fertility has been the much disputed topic of many research reports.

Motility, once considered an adequate predictor of fertility, is not consistently correlated with in vitro or in vivo fertilizing capacity (Anderson et al., 1980; Hall, 1981). Recognition that motility of sperm in culture medium on a glass microscope slide may not represent its ability to traverse the female reproductive tract prompted development of the postcoital test (Tredway et al., 1978). Quantification of sperm numbers and motility in human cervical mucus following insemination gives a more accurate measure of the functional motility of sperm. For exotic animals, recovery of cervical mucus following breeding is impractical or impossible. The collection of estrous cervical mucus for in vitro testing is likewise unfeasible. Gaddum-Rosse and associates (1980) offered an alternative by recording the progress of human sperm through estrous bovine cervical mucus (BCM). Keel and coworkers (1987) found that, for humans, BCM penetration was superior to the postcoital test in its ability to evaluate the functional motility of sperm and predict pregnancy. The BCM penetration test is simple to perform with commercially available mucus preparations, and its usefulness in semen evaluation of exotic animals warrants investigation.

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Its greatest advantages are elimination of the subjective nature of routine semen evaluation and the addition of detailed motion analysis unquantifiable by visual examination. Reports correlating CASA parameters with fertility are scarce. However, Mathur and coworkers (1986) reported a significant difference in the swimming speed and linearity of sperm from fertile versus infertile men as calculated by CASA. These differences could not be detected by manual analysis. Amann (1989) reported a significant multiple correlation between six swimming parameters and a competitive fertility index in bulls. Because the sperm of each species varies in size and morphology, CASA programs written for human or bovine semen are not immediately applicable to most exotics. The reprogramming necessary to analyze each sperm type likely to be encountered in a zoo physiology laboratory is, at least at this time, prohibitive. In addition, the extensive analysis necessary to correlate CASA parameters with fertility will be difficult to carry out with the limited number of samples available for exotic species. Tests that evaluate sperm's ability to reach the site of fertilization must be considered to be only one facet of comprehensive semen analysis. The ability of sperm to capacitate, acrosome react, and penetrate the zona pellucida and the ovum must also be assessed. The mammalian zona pellucida is rarely if ever penetrated by sperm of other species. Thus, to assess the ability of sperm to negotiate the zona, homologous ova are required in the assay system. Postmortem collection of ova with intact zonae may provide an occasional opportunity to perform homologous zona penetration assays. Perhaps a more feasible system would be to determine if sperm of the target species were capable of penetrating zonae of closely related species from which they could be routinely collected and frozen or salt stored (Yanagimachi et al., 1979).

Ruling out in vivo fertilization assays and penetration of homologous ova in vitro for logistical reasons, heterologous ova must be substituted for tests of fertilizing capacity. In 1972, Yanagimachi reported the penetration of zona-free hamster ova by guinea pig sperm. Since then, the unique ability of hamster ova to be penetrated by sperm of other species has been extensively explored as a means to determine the fertilizing capacity of sperm. This heterologous sperm penetration assay (SPA; Figure 4) has been developed for widespread use in the prediction of IVF success in humans (Yanagimachi et al., 1976). When fertile and infertile men were compared by the SPA and by traditional spermiogram, Rogers (1985) found far fewer false positives and false negatives with the SPA. Experiments by van Kooij and coworkers (1986) demonstrated the ability of the SPA to differentiate infertile men with normal spermiograms from fertile men. The heterologous nature of the assay dictates caution when interpreting results, as mechanisms of oolemma fusion and decondensation may differ from homologous fertilization in vivo. However, there is general agreement that the SPA is a valid indicator of sperm's ability to capacitate, acrosome react, fuse with the oolemma, and decondense (Prosad, 1984).

The sperm of numerous exotic species--including dolphin (Tursiops truncatus; Fleming et al., 1981), marmoset (Callithrix jacchus; Moore, 1981), bat (Lambert, 1981), cynomolgus monkey (M. fascicularis; Hoffman and Curtis, 1984), rhesus macaque (M. mulatta; Boatman and Bavister, 1984), budgerigar (Samour et al., 1986), patas monkey, lion-tailed macaque, and pygmy chimpanzee (Durrant, 1987), tiger (Post et al., 1987), and cheetah (Durrant et al., 1989)---are capable of penetrating hamster ova with varying success. However, no attempt has been made to correlate the SPA with fertility in any of these species.

The SPA may find its greatest usefulness in the evaluation of various cryopreservation protocols. Combined with other semen analysis parameters, the loss or retention of ability to penetrate hamster ova could be used to discriminate between freezing methods and aid in the development of optimal cryopreservation protocols for endangered species.

The acrosome reaction is considered an end point of successful sperm capacitation. Human sperm must be acrosome reacted prior to penetration of zona-free hamster ova (Yanagimachi, 1984). Thus, the SPA may be used to formulate in vitro capacitation techniques. The ability to capacitate sperm in vitro is essential for IVF studies or for Al using immature sperm collected from the epididymis.

Other less complex assays of the acrosome reaction are available. For many species, light microscopic examination is not sufficient to visualize the unstained acrosome. Because electron microscopy is expensive and labor intensive, several acrosome stains have been developed flint can be evaluated with light microscopy. The divalent cation ionophore A23187 is often used to induce the acrosome reaction in vitro to confirm that the stain technique being tested can differentiate reacted from unreacted sperm (Green, 1978). A triple stain designed by Talbot and Chacon (1981) is frequently used to assess spontaneous (normal) acrosome reactions and those occurring as the result of sperm degeneration. Although this technique requires only a light microscope for acrosomal status evaluation, it is time consuming. A faster method uses chlortetracycline fluorescent stain (Ward and Storey, 1984), which causes reacted sperm to fluoresce in the region of the acrosome. The disadvantage of this technique and other fluorescent stains is the requirement for a microscope equipped with epifluorescence optics. The potential toxicity of fluorescent stains can be assessed by incorporating a supravital stain into the assay.

Exclusion of a supravital stain is a measure of the structural integrity of the sperm plasma membrane (Figure 5>; Schrader et al., 1986) and is a well-studied adjunct to additional semen evaluation (Eliasson and Treichl, 1971; LasIey et al., 1942). Supravital stains are not equally applicable to sperm of ail species (Varner et al., 1987), and the suitability of each stain must be determined for each species. Because supravital stains are easily applied to semen samples in the field for later examination under a light microscope, this simple technique may be a valuable addition to semen analysis in exotic species.

A relatively new technique that measures the functional or physiological integrity of the sperm plasma membrane is the hypoosmotic swelling test (HOS; Jeyendran et al., 1984). The test, which is easily read with a light microscope, assesses the ability of the membrane to transport water. If the membrane is functional, the sperm tail will coil within the swollen membrane. These authors reported a significant correlation between the percentage of swollen tails and percentage of human sperm penetrating in the SPA. This straightforward technique--in combination with other evaluation techniques--may be a useful adjunct to semen evaluation in exotic species. It has been shown to correlate well with sperm motility in cheetahs and pheasants (Durrant, unpublished data; 1989).

No single semen evaluation parameter is likely to be proven predictive of fertility. However, combinations of the above techniques, such as a fluorescent acrosome stain, a supravital stain, and the HOS test, may give reasonably accurate profiles of potential fertility.

The direct comparison of various analyses using the same samples (or at least the same males) will streamline evaluation of semen of each species by eliminating redundant tests and demonstrating which are most highly correlated with fertility. For example, the SPA, HOS, and triple acrosome stain were all applied to aliquots of the same ejaculates from men of known fertility or infertility (van Kooij et al., 1986). Only the SPA was significantly different between fertile men and infertile men with normal spermiograms. Multivariate discriminant analysis has been used to select semen parameters capable of predicting fertility. One study with human subjects found semen volume, sperm motility, the log of sperm concentration, the SPA, and the HOS to be significant discriminating factors, which, when taken collectively, predicted the fertility of these men with 77.6 percent accuracy (Wang et al., 1988).

Analysis of sperm morphology has long been part of traditional semen evaluation. Individual males with high proportions of severe sperm anomalies, such as detached heads, would be expected to be subfertile due to impaired motility (Figure 6). The relationships between less serious sperm defects and fertility are not clear. In one study of domestic stallions, the male with the highest number of abnormal sperm had the highest pregnancy rate (Voss et al., 1981). Two clouded leopard (Neofelis nebulosa) males of proven fertility had extremely high proportions of sperm abnormalities, as did six other males of unknown fertility (Wildt et al., 1986). Free-ranging and captive cheetahs have uniformly poor semen quality, including a large percentage of morphological abnormalities (Durrant et al., 1985; Wildt et al., 1983). All proven sires at the Zoological Society of San Diego exhibit these semen characteristics that, if observed in domestic felids, would signal potential infertility (Durrant et al., unpublished data, 1988).

Correlation of sperm or semen characteristics with fertility should not be mistaken for fertility prediction. The final step required to develop predictive semen analysis is to apply techniques shown to be correlated with fertility in one set of samples to different sets of samples from males of unknown fertility (Amann, 1989). This step has not been carried out for any species. Without large numbers of samples from known fertile and infertile males and the ability to use aliquots of the same ejaculate for evaluation and AI, it may not be possible to use semen evaluation parameters to predict fertility in most exotic animals.

Semen Cryopreservation
Semen cryopreservation is a complex and poorly understood technique that is consistently successful in a very limited number of species. Because the biochemical properties of semen vary widely between species, protocols must be redesigned for each new animal under investigation.

The remarkable success enjoyed by the dairy industry using frozen semen for AI is the result of many years of research and field trials with thousands of experimental animals. Additional factors are the relative resistance of bovine sperm to damage during the freezing and thawing process and intense selection of males for semen quality and high fertility.

From this history of cryopreservation protocol development in cattle, the basic experimental parameters may be derived. First, an appropriate extender or diluent must be formulated for each species. Second, the correct concentration of the proper cryoprotectant equilibrated at the right temperature for the optimum period of time must be added to that medium. Finally, freeze rates and thaw methods must be established. Comprehensive semen analysis before and after freezing is essential for assessing damage and discriminating between various cryopreservation protocols. Each of these steps represents a significant investment of time, money, and semen.

Numerous reports in the literature claim the successful freezing of semen of exotic species (see reviews by Graham et al., 1978, and Wildt, 1986). The end point of most of these early experiments was the demonstration of postthaw motility, however slight. (For purposes of this paper, the birth of offspring following AI with frozen semen will define successful semen freezing.)

Few studies have involved evaluation of frozen semen by any parameter other than motility. Haigh and coworkers (1986) compared three media with and without EDTA and estimated acrosomal damage of cryopreserved wapiti (Cervus elaphus) sperm with light microscopy in addition to motility at thaw. Motility differed in only one medium when EDTA was added, but acrosomal damage was decreased in two media when it was added, which perhaps indicates the superior discriminating power of acrosomal assessment. Howard and colleagues (1986) studied sperm motility, duration of motility, and acrosomal damage of African elephant (Loxodonta africana) sperm frozen in several freezing media and in two thawing media. The percentage of normal acrosomes was the most effective parameter for discriminating between the different freezing media. Cryopreservation of ferret (Mustela putorius) semen in three media, by two freezing methods and two thawing methods, was evaluated by postthaw motility and acrosomal integrity (Howard et al.,1989). One protocol was selected based on these studies, and a 70 percent pregnancy rate was achieved following AI. Sixteen cryopreservation protocols for cheetah semen compared extender composition and pH, freeze rates, and packaging methods (Durrant et al., 1989). Postthaw motilities did not differ between cryopreservation methods, and the SPA was not satisfactory as an indicator of retained fertilizing capacity in this species. These preliminary studies represent the beginning of a new field of study.

In 1972 the first pregnancies were established in an exotic species--the fox (Vulpes vulpes)--with frozen semen (Aamdal et al., 1972). A slowly emerging literature reports successful pregnancies in the following exotic animals: reindeer (Rangifer tarandus; Dott and Utsi, 1973), wolf (Canis lupus; Seager et al., 1975), crane (Sexton and Gee, 1978), red deer (C. elaphus; Kelly and Moore, 1981), wapiti (Haigh et al., 1984), addax (Addax nasomaculatus; Densmore et al., 1987), kestrel (Falco sparverius; Brock et al., 1984), falcon (F. peregrinus; Parks and Bardaswick, 1987), and budgerigars (Samour et al., 1988).

For many exotic species, semen freezing techniques probably will be similar to those developed for closely related domestic species. Taxonomically unique species, such as the cheetah, however, may require extensive research to elucidate appropriate protocols. For example, studies with antelope or gazelle semen will logically begin with cryopreservation techniques developed for domestic cattle, the semen of wild equids may freeze as well as domestic stallion semen using the same methods, and human semen freezing methods will serve as models for freezing primate sperm. Comprehensive evaluation of sperm through the application of multiple analyses both before and after freezing is essential to the development of freezing protocols, as well as the identification of appropriate evaluative procedures for sperm of each species.

Summary
The importance of semen cryopreservation to the future genetic health of captive exotic animals cannot be overstated. Unfortunately, limited availability of samples for analysis and funding shortages represent significant impediments to basic research in this field. However, by modifying semen freezing techniques developed for domestic animals and humans, progress should be relatively rapid. Cooperation among zoological institution personnel and university scientists will further enhance research efforts on behalf of endangered wildlife.

References

Aamdal, .1, K. Anderson, and I. A. Fougner. 1972. Insemination with frozen semen in the blue fox. Proc. 7th Int. Congr. Anim. Reprod. Artif. Insem. 2:1713-1716.

Amann, IL P. 1989. Can the fertility potential of a seminal sample be predicted accurately? J. Androl. 10(2):89--98.

Anderson, E. W., B. W. Pickett, and J. L Voss. 1980. The relationship between motility and fertility of frozen stallion semen. Proc. 9th Int. Congr. Anim. Reprod. Artif. Insem. 3:371.

Bedford, J. M. 1981. Why mammalian gametes do not mix. Nature 291:286.

Boatman, D. E., and B. D. Bavister. 1984. Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. J. Reprod. Fertil. 71:357-366.

Boyd, L. L., and C. H. Schwartz. 1983. Training imprinted semen donors. Pp. 24-31 in Falcon Propagation: A Manual on Captive Breeding, I. D. Weaver and T. J. Cade, eds. Ithaca, New York: Peregrine Fund.

Brock, M. K., D. M. Bird, and G, A. Amah. 1984. Cryogenic preservation of spermatozoa of the American kestrel. Int. Zoo Yearb. 23:67-71.

Council for Agricultural Science and Technology. 1984. Animal Germ Plasm Preservation and Utilization in Agriculture. Council for Agricultural Science and Technology Report No. 101. Washington, D.C.: U.S. Government Priming Office.

Densmore, M. A., M.J. Bowen, S.J. Magyar, M. S. Amoss, R. M. Robinson, P. G. Harms, and D. C. Kraemer. 1987. Artificial insemination with frozen, thawed semen and pregnancy diagnosis in addax (Addax nasomaculatus). Zoo Biol. 6:21-29.

Dott, H., and M. N. P. Utsi. 1973. Artificial insemination of reindeer, Rangifer tarandus. I. Zool. London 170:505-508.

Durrant, B. 1987. Penetration of hamster ova by non-human primate spermatozoa. J Androl. 8:27P.

Durrant, B., T. Schuerman, and S. Millard. 1985. Non-invasive semen collection in the cheetah (Acinonyx jubatus). Proc. Am. Assoc. Zool. Parks Aquar. 1985:564-567.

Durrant, B. S., I. K. Yamada, and S. E. Millard. 1989. Development of a semen cryopreservation protocol for the cheetah. Cryobiology 26(6):542-543.

Eaglesome, M. D., W. D. D. Hare, and E. Singh. 1980. Embryo transfer. A discussion of its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents. Can. Vet. J. 21:106.

Eliasson, E., and L. Treichl. 1971. Supravital staining of human spermatozoa. Fertil. Steril. 22:134-137.

Fleming, A. D., R. Yanagimachi, and H. Yanagimachi. 1981. Spermatozoa of the Atlantic bottlenose dolphin, Tursiops truncatus. J. Reprod. Fertil. 63:509-514.

Fussell, E. N., L. F. Franklin, and R. C. Frantz. 1973. Collection of chimpanzee semen with an artificial vagina. Lab. Anim. Sci. 23:252-255.

Gaddum-Rosse, P., R. I. Blandau, and W. I. Lee. 1980. Sperm penetration into cervical mucus in vitro. I. Comparative studies. Fertil. Steril. 33:636-643.

Gee, G. F. 1983. Crane reproductive physiology and conservation. Zoo Biol. 2:199-213.

Gee, G. F. 1984. Value of frozen tissue collections for gene pool preservation. Pp. 14-16 in Collection of Frozen Tissues: Value, Management, Field and Laboratory Procedures, and Directory of Existing Collections, H. C. Dessaner and M. S. Hafner, eds. Lawrence, Kansas: Association of Systematics Collections.

Graham, E. F., M. K. L Schmehl, B. K. Evensen, and D. S. Nelson. 1978. Semen preservation in non-domestic mammals. Symp. Zool. Soc. London 43:153-173.

Green, D. P. L. 1978. The induction of the acrosome reaction in guinea pig sperm by the divalent metal cation ionophore A23187. J. Cell Sci. 32:137-151.

Gould, ICG., H. Warner, and D. E. Martin. 1978. Rectal probe electro-ejaculation of primates. I. Med. Primatol. 7:213-222.

Gould, K.G., D. E. Martin, and H. Warner. 1985. Improved method for artificial insemination in the great apes. Am. J. Primatol. 8:61-67.

Haigh, J. C., M. P. Shadbolt, and G. J. Glover. 1984. Artificial insemination of wapiti. Proc. Am. Assoc. Zoo Vet. 1984:174.

Haigh, J. C., A. D. Barth, and P. A. Bowman. 1986. An evaluation of extenders for wapiti, Cervus clapbus, semen. J. Zoo Anita. Med. 17:129-132.

Hall, J. L. 1981. Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil. Steril. 35:457-463.

Harrison, G. J., and D. Wasmund. 1983. Preliminary studies of electro-stimulation to facilitate manual semen collection in psittacines. Proc. Ann. Meet. Assoc. Avian Vet. 1983:207-213.

Hoffman, M. L., and G. L. Curtis. 1984. Prevention of monkey sperm penetration of zona-free hamster ova by sperm antibody obtained from vasectomized cynomolgus monkeys. Fertil. Steril. 42:108-111.

Howard, J. G., M. Bush, V. de Vos, M. C. Schiewe, V. G. Purcel, and D. E. Wildt. 1986. Influence of cryoprotective diluent on post-thaw viability and acrosomal integrity of spermatozoa of the African elephant (Loxodonta africana). I. Reprod. Fertil. 78:295-306.

Howard, J. G., S. L. Hurlbut, C. Morton, F. Morton, M. Bush, and D. E. Wildt. 1989. Pregnancies in the domestic ferret after laparoscopic artificial insemination with frozen-thawed spermatozoa. J. Androl. 10:.528

Jeyendran, R. S., H. H. Van der Ven, M. Perez-Pelaez, B. G. Crabo, and L. J. D. Zaneveld. 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil. 70:219-228.

Keel, B. A., R. W. Kelly, B. W. Webster, K. L. Zumbach, and D. K. Roberts. 1987. Application of a bovine cervical mucus penetration test. Arch. Androl. 19:33-41.

Kelly, R. W., and G. Moore. 1981. Artificial Insemination of Red Deer. New Zealand Mining and Agriculture Annual Report, 1980/1981.

Lambert, H. 1981. Temperature dependence of capacitation in bat sperm monitored by zona-free hamster ova. Gamete Res. 4:525-533.

Larsen, R. E., and P. T. Cardeilhac. 1984. Artificial insemination, semen handling, and assessment of reproductive status in alligators. Proc. Am. Assoc. Zoo Vet. 1984:159.

Lasley, J. E., G. T. EasIey, and F. F. McKenzie, 1942. A staining method for the differentiation of live and dead spermatozoa. Anat. Rec. 82:167.

Lovejoy, T. E. 1980. Foreword. Pp. ix-x in Conservation Biology: An Evolutionary-Ecological Perspective, M. E. Soule and B. A. Wilcox, eds. Sunderland, Massachusetts: Sinauer Associated.

Martin, I. C. A. 1978. The principles and practice of EE in mammals. Symp. Zool. Soc. London 43:127-152.

Martin, D. E., C. E. Graham, and K. C. Gould. 1978. Successful artificial insemination in the chimpanzee. Symp. Zool. Soc. London 43:249-260.

Mathur, S., M. Carlton, J. Ziegler, P. Rust, and H. Willlamson. 1986. A computerized sperm motion analysis. Fertil. Steril. 46(3):484-488.

Meltzer, D. G. A., M. Van Vuuren, and M. S. Bomman. 1988. The suppression of electro-ejaculation in the Chacma baboon (Papia ursinus) by azaperone, J. S. Afr. Vet. Med. Assoc. 59(I):53.

Mendgen, G. A., C. G. Platz, R. Hubbard, and H. Quinn. 1980. Semen collection, freezing and artificial insemination in snakes. Pp.71-78 in Reproductive Biology and Diseases of Captive Reptiles, L B. Murphy and T. Collins, eds. Lawrence, Kansas: Society for the Study of Amphibians and Reptiles.

Merilan, C. P., B. W. Read, and W. L Boever. 1981. Semen collection procedures for captive wild animals. Int. Zoo Yearb. 21:241-244.

Moore, H. D. M. 1981. An assessment of the fertilizing ability of spermatozoa in the epididymis of the marmoset monkey (Callithrix jacchus). Int. J. Androl. 6:310--318.

National Research Council. 1978. Conservation of Germ Plasm Resources: An Imperative. Washington, D.C.: National Academy of Sciences.

Notter, D. R., and T. J. Foose, 1985. Concepts and Strategies to Maintain Domestic and Wild Animal Germ Plasm. Commissioned paper, Office of Technology Assessment. Washington, D.C.: U.S. Government Printing Office.

Parks, J. E., and V. Hardaswick. 1987. Fertility and hatchability of falcon eggs after insemination with frozen peregrine falcon semen. J. Raptor Res. 21 (2):70-72.

Post, G. S., H. C. Hensleigh, A. E Byers, U. S. Seal, T. J. Kreeger, N. J. Reindl, and R. L. Tilson. 1987. Penetration of zona-free hamster ova by Siberian tiger sperm. Zoo Biol. 6:183--187.

Prasad, M. R. N. 1984. The in vitro sperm penetration test: A review. Int. J. Androl. 7:5-22.

Rogers, B. L 1985. The sperm penetration assay: Its usefulness reviewed. Fertil. Steril. 43(6):821-840.

Rudak, E., P. A. Jacobs, and R. Yanagimachi. 1978. Direct analysis of the chromosome constitution of human spermatozoa. Nature 274:911-912.

Ryder, O. A., and K. Benirschke, 1984. The value of frozen tissue collections for zoological parks. Pp. 6-9 in Collections of Frozen Tissues: Value, Management, Field and Laboratory Procedures, and Directory of Existing Collections, H. C. Dessauer and M. S. Hafner, eds. Lawrence, Kansas: Association of Systematics Collections.

Samour, J, H. D. Moore, and C. A. Smith. 1986. Avian spermatozoa penetrate zona-free hamster oocytes in vitro. J. Exp. Zool. 239:295-298.

Samour, J. H., J. A. Markham, and H. D. M. Moore. 1988. Semen cryopreservation and artificial insemination in budgerigars (Melopsittacus undulatus). J. Zool. London 216:169-176.

Schrader, S. M., S. F. Platek, L. J. D. Zaneveld, M. Perez-Pelaez, and R. S. Jeyendran. 1986. Sperm viability: A comparison of analytical methods. Andrologia 18(5):530-538.

Seager, S. W. J., C. C. Platz, and W. Hodge. 1975. Successful pregnancy using frozen semen in the wolf. Ins. Zoo Yearb. 15:140-143.

Sexton, T. J., and G. F. Gee. 1978. A comparative study on the cryogenic preservation of semen from the sandhill crane and the domestic fowl Symp. Zool. Soc. London 43:89-95.

Spiller, N., J. Grahame, and D. R. Wise. 1976. Experiments on the artificial insemination of pheasants. World Pheas. Assoc. J. 2:89-96.

Talbot, P., and R. S. Chacon. 1981. A triple-stain for evaluating normal acrosome reactions of human sperm. J. Exp. Zool. 215:201-208.

Tredway, D., G. Buchanan, and T. Drake, 1978. Comparison of the fractional postcoital test and semen analysis. Am. J. Obstet. Gynecol. 130:647-652.

U.S. Congress, Office of Technology Assessment. 1987. Technologies to Maintain Biological Diversity, OTA-F-330. Washington, D.C.: U.S. Government Printing Office.

Van Kooij, R. L, M. Balema, A. Roatti, and A. Campana. 1986. Oocyte penetration and acrosome reactions of human sperm. II Correlation with other seminal parameters. Andrologia 18(5):503-508.

Vamer, D.D., C. R. Ward, B. T. Storey, and R. M. Kenney. 1987. Induction and characterization of acrosome reaction in equine spermatozoa. Am. J. Vet.

Res. 48(9):1383-1389.

Voss, J. L., B. W. Picken, and E. L Squires. 1981. Stallion spermatozoal morphology and motility and their relationship to fertility. J. Am. Vet. Med. Assoc. 1780):287-289.

Wang, C., S. Y. W. Chan, M. Ng, W. W. K. So, W. L Tsoi, T. Lo, and A. Leung. 1988. Diagnostic value of sperm function tests and routine semen analyses in fertile and infertile men. J. Androl. 9(6):384-389.

Ward, C. R., and B. T. Storey. 1984. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev. Biol. 104:287-296.

Watanabe, M. 1957. An improved technique of the artificial insemination of ducks. L Fac. Fish Anim. Husb. Hiroshima Univ. 1(3):363-368.

Wildt, D. E. 1986. Spermatozoa: Collection, evaluation, metabolism, freezing, and artificial insemination. Pp. 171-193 in Comparative Primate Biology, vol. 3:Reproduction and Development. New York: Alan R. Liss.

Wildt, D. E., M. Bush, L G. Howard, S. J. O'Brien, D. Meltzer, A. Van Dyk, H. Ebedes, and D. J. Brand. 1983. Unique seminal quality in the South African cheetah and a comparative evaluation in the domestic cat. Biol. Reprod. 29:1019-1025.

Wildt, D. E., L G. Howard, L L. Hall, and M. Bush. 1986. Reproductive physiology of the clouded leopard: I: Electroejaculates contain high proportions of pleiomorphic spermatozoa throughout the year. Biol. Reprod. 34:937-947.

Yanagimachi, R. 1972. Penetration of guinea pig spermatozoa into hamster eggs in vitro. J. Reprod. Fertil. 28:477-480.

Yanagimachi, R. 1984. Zona-free hamster eggs: Their use in assessing fertilizing capacity and examining chromosomes of human spermatozoa. Gamete Res. 10:187-232.

Yanagimachi, R., H. Yanagimachi, and B. J. Rogers. 1976. The use of zona-free animal ova as a test system for the assessment of the fertilizing capacity of human spermatozoa. Biol. Reprod.. 15:471-476.

Yanagimachi, R., A. Lopora, C. P. Odom, R. A. Bronsun, C. A. Maki, and G. Nicolson. 1979. Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of salt stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31 (5):562-574.

Figure 1 Semen collection by electroejaculation of tranquilized (top) cheetah (Acinonyx jubatus) and (bottom) cimitar-horned oryx (Oryx dammah).

Figure 2 Collection of semen from (top) cheetah (A. jubatus) and (bottom) tapir (Tapirus pinpinchaque) using an artificial vagina.

Figure 3 Collection of semen from a pheasant (Tragopan temmincki) using the massage technique.

Figure 4 Penetration of cheetah (A. jubatus) sperm into hamster (Mesocricetus auratus) ova in lite heterologous sperm penetration assay. Three decondensing sperm heads are seen within the ooplasm (400 x).

Figure 5 Supravital staining of hamster (M. auratus) sperm. Live sperm has excluded the stain; dead sperm is stained (100 x).

Figure 6 Sperm of the greater kudu (Tragelaphus strepsiceros) illustrating common morphological abnormalities (detached heads and coiled tail; 100 x).