Semen Banking—Is It Now Feasible for Captive Endangered Species?

Semen banking—is it now feasible for captive endangered species?

W. V. Holt and H. D. M. Moore

This article proposes that serious consideration should now be given to the establishment of banks of genetic material from rare and endangered species. In the light of advances in the technology associated with semen assessment and preservation, the limitations that have so far discouraged the practice of semen freezing and banking can now be minimized and circumvented. The authors review these developments and suggest that semen preservation should be recon- sidered as a practical means of preserving the genetic diversity currently contained within captive stocks of wild animals.

The World Conservation Strategy published by and freezing was not sufficiently advanced to the International Union for Conservation of permit such a venture but considerable progress Nature and Natural Resources (IUCN) in 1980 has been made since then. Given the current identified the preservation of genetic diversity as rate of scientific development and innovation, an important priority, both for conservation and the view expressed by Foose et al. (1985), that the future development of scientific and indust- another two centuries of research will be re- rial innovation. In order to implement this prin- quired before germplasm banking is a realistic ciple we need to make the best use of new possibility, is unduly pessimistic. technological progress where appropriate. The last decade has seen many developments in the International co-operation in the maintenance technology associated with animal reproduction. by zoos of detailed studbooks, such as those for Ironically, much of the stimulus for this work has the Arabian oryx and Przewalski's horse, means arisen through the need for human infertility that the relative contribution of particular foun- treatments and greater agricultural efficiency. It der or parent animals to the current gene pool is appropriate, therefore, to use the fruits of this can be calculated. Certain individuals may con- research in conservation, countering the effects tribute excessively, minimally or not at all; these of world-wide population pressures. effects, which limit the gene pool for subsequent generations, need to be countered by planned The purpose of this review is to outline ways in breeding programmes (for examples, see Ches- which new advances in artificial insemination ser et al, 1980; Foose, 1980). These typically technology and genetics could now be applied involve the expensive and stressful transport of to achieve this end with mammalian species; we animals, but artificial insemination and embryo recognize that conservation of other phyla is transfer technology can be used both to avoid equally important, but these will not be discussed this procedure and to amplify the international here. In particular this article proposes that seri- distribution of valuable genetic resources. Furth- ous consideration should now be given to the ermore, recent developments in molecular biol- idea of setting up an international repository of ogy should facilitate the genetic analysis of popu- frozen semen from endangered species; ten lations and assist with the unequivocal identifica- years ago the technology for semen assessment tion of parent-offspring relationships. 172 Oiyx Vol 22 No 3, July 1988

Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 25 Sep 2021 at 23:16:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0030605300027800 Artificial insemination in endangered Table 1. Mammalian species that have been successfully species bred by artificial insemination Ten years ago artificial insemination and semen Primates Baboon freezing in cattle breeding were already estab- Chimpanzee Pan troglo- Fritz, 1986; lished procedures, and some advocates of their dytes Martin etal., application in conservation were rather naively Gorilla Gorilla g. 1978. gorilla Douglass, optimistic about applying the techniques to other 1981 species. Hence, some zoos started to collect and Carnivores Cheetah Acinoyx jubatus freeze semen from a wide range of species, with- Puma Felis concolor Moore etal., out due regard to the problems of using the Snow Panthera 1981 semen for insemination. These are underlined leopard uncia when it is considered that, after more than 30 Fox Unspecified Aamdal etal., 1 Q7R years of research farmers still do not use frozen Ungulates Addax Addax Densmore ram or boar semen for breeding because the nasoma- etal., 1987 culatus success rate is uneconomically low. Blackbuck Antilope Holt etal., cervicapra 1988 Despite these problems, artificial insemination Speke's Gazella Boever eta/., has been successfully performed in a number of gazelle spekei 1980 Red deer Cervus Krzywinski and species (Table 1), and the secret of success seems elaphus Jaczewski, to lie in the careful monitoring of semen quality 1978 and timing of inseminations. Since the economic Wapiti Cervus Haigheta/., elaphus 1984 aspects of artificial insemination are of minor Others Giant panda Ailuropoda Moore etal, importance when the aim is propagation of a melanoleuca 1984 rare species, the conservationist is not con- strained to use minimal sperm numbers in insemination doses, and considerable care can for penetrating the tough outer layers that sur- be taken in introducing the spermatozoa into round the ovum, it has always seemed reason- the female reproductive tract. Catheters to de- able to regard sperm motility as a good indicator liver semen samples can be guided through the of fertility. Unfortunately, the commonest cervix, or directly into the uterine horns, under method of rating sperm motility has always been visual control of the operator using fibreoptic to examine a drop of semen through the micro- endoscopes developed for surgical procedures. scope, making some subjective estimate of These procedures lead to improved pregnancy motility based on a simple scoring system. This rates, compensating for the damaging effects of technique is open to many pitfalls, and has pro- freezing upon sperm longevity and vigour. vided little hard evidence for relationships between motility and fertility. Recent research has shown, however, that Advances in semen assessment sperm motility can be measured objectively Faced with a fresh or frozen semen sample, it is using either time-lapse photography or newer necessary to evaluate its fertilization potential in computerized techniques to analyse images. order to avoid spending large sums on preserv- Measurements of sperm swimming speed (Milli- ing semen that stands minimal chance of ever gan et a/., 1980; Aitken et a/., 1982; Holt et a/., achieving a pregnancy. Clearly, attempting to 1985) established that, for human sperm, swim- predict fertility without performing direct trials is ming speed was a very valuable guide to fertility a major problem, but techniques to achieve this in vitro and in vivo. This now forms the basis of end are currently under development. a diagnostic test for fertility, and there is every reason to believe that it could be usefully applied Since sperm movement is one of the most obvi- to semen from the great apes. So far it has not ous characteristics of a semen sample, and is been possible to validate this technique for ungu- essential both for sperm colonization of the cer- lates, although data gathered in the Institute of vix and oviduct prior to fertilization as well as Zoology, Zoological Society of London, show Semen banking for captive endangered species 173

(a) Arabian oryx (b) Pere David's deer

Fresh sperm diluted in BWW Fresh sperm diluted in BWW 18 14 10 6 2

i_ Stored in egg yolk/citrate for 24 h at 5°C 18

Diluted in egg yolk/citrate (not cooled) 14 14 - 10 10 - 6 2 1 6 _> - 5 2 Stored in egg yolk/citrate for 48 h at 5°C 18 14 10 6 Diluted in egg yolk/citrate (cooled to 5°C) 2 26 22 22 Frozen /thawed 18 18 14 14 10 10 6 6 2 2 15 30 45 60 75 90 105 120 135 15 30 45 60 75 90 105 Swimming speed (Mm/sec) Figure 1. Frequency histograms showing how the swimming speeds of spermatozoa from (a) Arabian oryx and (b) Pere David's deer responded to the various stages of processing involved in semen freezing. Typically, these entail dilution in a cryoprotective medium containing egg yolk and glycerol, slow cooling to approximately 5°C, then freezing in liquid nitrogen vapour. After thawing, Pere David's deer spermatozoa, which showed little change in speed during processing, recovered full motility. In this example, the poor survival of Arabian oryx spermatozoa was indicated by their serious decline in swimming speed during processing. Frozen/thawed sperm were non-motile. (BWW refers to a physiologically balanced salt solution.) 174 Oryx Vol 22 No 3, July 1988 '

Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 25 Sep 2021 at 23:16:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0030605300027800 solved readily using the sophisticated computer that a universally applicable technique may soon techniques available today. be developed. Functional tests of sperm quality provide another valuable means of assessing their likely fertility. Design of semen freezing procedure Two types of approach have been evolved On their own merit, the advances discussed that reproduce in the laboratory the natural above now justify establishing banks of frozen events leading up to fertilization. Since the semen from endangered species, since the ability chances of spermatozoa reaching the fallopian exists to assay semen quality objectively at every tube, and hence the egg, depend largely upon stage, from selecting suitable donors to checking their vigour and longevity, it has been found that post-thaw, sperm survival and viability. Another monitoring their progress through materials such complementary advance contributes strongly to as cervical mucus, bovine serum albumin or gel the justification of this proposal; this is the rela- filtration media (Sephadex) provides a good in- tively recent technical development of cryomi- dication of their fertility. A recent example of the croscopy. Sephadex technique was provided in a study of water buffalo semen (Heuer et a/., 1987), where The ability to monitor resistance to freezing, from a 7 per cent difference in fertility between either species or individuals with particularly samples collected in June and November was labile spermatozoa, means that banks of poor reflected in a superior (nearly double) ability of semen need not, and should not, be maintained. November samples to traverse the gel. However, selectivity on this basis is fundamen- tally undesirable and steps to tailor the semen The second type of functional test is directed processing schedules for these animals need to towards assessing the ability of spermatozoa to be taken. This is where cryomicroscopy can undergo the preliminary events of fertilization, help. then to fuse with an egg. Yanagimachi et al. The cryomicroscope consists essentially of a (1976) reported that human spermatozoa were sophisticated temperature control system, able to penetrate the membrane of the hamster mounted around the specimen chamber of a egg, which had had its tough outer coating (zona light microscope. With this system, semen pellucida) removed; since then there have been samples can be observed as they undergo cool- many reports showing that the hamster egg is ing, freezing and thawing at controlled rates, and unusual in not blocking the penetration of sper- the whole sequence recorded on videotape or matozoa from other species. Tiger, dolphin, mar- film. Techniques of sperm motility measurement moset, wallaby, Pere David's deer (Figure 2) and can therefore be used to analyse the response even budgerigar spermatozoa have penetrated to cooling and freezing. The small number of the zona-free hamster egg, although further de- spermatozoa required for each freezing trial velopment does not occur, and the penetration means that several different cooling rates, or di- mechanism for mammals closely resembles the luting media, can be quickly evaluated to identify natural process. This test provides a direct optimal procedures for any individual sample; the method of investigating the fertilization potential whole procedure thus defined can then be repro- of spermatozoa and, despite the obvious draw- duced for bulk samples using a purpose-built backs of using sperm and ova of different species freezing unit. At present, cryomicroscopes are in artificial media, it appears to work. So far, the expensive both to purchase and to maintain, main difficulty of applying this test with exotic and to our knowledge there are none dedicated species has been the need to develop suitable to the preservation of gametes from endangered culture media for inducing fertilization to occur; species. They are, however, being used for com- this reflects the plethora of morphological and mercially directed research, where, for example, metabolic differences between spermatozoa their potential for the development of fish-farm- from different species. Happily, however, pro- ing technology such as the freezing of sperm, gress is being made towards meeting these re- eggs, embryos and unicellular organisms as feed quirements and there is good reason to believe has been clearly recognized. Semen banking for captive endangered species 175

Insemination timing certain hormones such as prostaglandins and To perform artificial inseminations successfully progesterone to bring a female into oestrus requires not only effective means of preserving bypasses the need for monitoring and allows the and handling semen, but also knowledge of the time of insemination to be planned ahead. This female reproductive cycle. Egg development is has an added advantage in farming practice not a continuous process like spermatogenesis, where the insemination times for all cows in a therefore unless spermatozoa are introduced to herd can be synchronized. With some knowl- the female reproductive tract at an appropriate edge of the natural oestrous cycle, this treatment time, i.e. several hours either side of ovulation, can, in principle, be used for controlling the time the insemination will almost certainly be unsuc- of insemination in most ungulates. It has already cessful. Fortunately, many female mammals are been used successfully for addax (Densmore et only sexually receptive for a relatively short al., 1987), scimitar-horned oryx and Pere David's period (oestrus) before ovulation, and the simple deer. Suitability of the procedure does need to expedient of observing receptivity to a vasec- be confirmed for each new species tried, how- tomized male is often sufficient for determining ever, to establish that prostaglandins do not in- the time of oestrus. In our own experience with terfere with egg development and ovulation. artificial insemination in the blackbuck Antilope Outside the range of ungulates the application cervicapra it was possible to determine oestrus of this method is less uniform, as some species in about 50 per cent of the females observed. do not respond to prostaglandin treatment. However, the cyclic production of steroid hormones during the oestrous cycle can be moni- Genetic aspects of semen banking tored readily by urine analysis using radio- immunoassay (see Hodges and Eastman, 1984), Since the purely technical problems that previ- or techniques based on bioluminescence. ously discouraged zoos from establishing semen banks for endangered species can now be largely Continuous monitoring of the reproductive cycle circumvented, there is currently every reason is a tedious and inefficient process; the use of for pursuing this policy. At least two more funda- 176 Oryx Vol 22 No 3, July 1988

Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 25 Sep 2021 at 23:16:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0030605300027800 mental questions need to be addressed, how- Studies of genetic variation have been per- ever; how do we select animals as semen donors, formed biochemically for many years using the and what proportion of the current gene pool slight molecular differences between proteins should be conserved? as markers of lineage; thus surveys have, for example, been performed upon the genetic The answers to these questions will differ for variability between geographically separate each species, being dependent partly upon exist- populations of animals such as reindeer, ing population size and partly upon inter-gener- cheetah, Przewalski's horse and many other ation intervals. For example, if semen storage species (for review see Powell, 1983). from Arabian oryx is to be considered then the Techniques of this nature, together with the pedigrees of individuals in the current world herd newer methods in molecular biology, will pro- needs to be considered. The records for this vide the information for making judgements re- species, which have been well kept for many garding the relative genotypic value of semen years (Mace, 1986), indicate that certain males donors. This link between genetics and semen have sired few offspring while others have pro- preservation is essential, given the ultimate aim duced a disproportionately large number. of maintaining genetic diversity. Should we aim to store the semen from the least successful males, so that their genetic contribution is not lost and can eventually be rein- Conclusions troduced into the bloodline? Conversely, should we concentrate on the most successful animals? The ultimate goal of conservation, captive breeding and semen banking is clear; to preserve Recent advances in genetics should help with the vigour and viability of species so that they some of these problems. Firstly, it is necessary do not decline and drift, eventually becoming to know that stud book records are correct, or unable to sustain themselves. Ideally, it will be where stud books do not exist, to possess the possible to reintroduce species into their native capability for assessing the proportion of the cur- habitat; some projects like Operation Oryx have rent gene pool represented by a particular indi- already been undertaken, but the need for gen- vidual. New DNA fingerprinting techniques are etic management is unlikely to disappear. now being used to confirm paternity in humans. Planned breeding programmes will not fulfil the Some of these probes are effective with other need to preserve sufficient genetic diversity for species, particularly the great apes, as well as optimal survival of endangered species, and sheep, mice and even sparrows (Burke and there is a definite requirement for the application Bradford, 1987; Wetton et a/., 1987). Although of technology in this area. It should be remem- the use of these probes with unusual species bered that frozen semen retains its viability for needs validation, they would be useful for pro- many decades, which provides an opportunity viding unequivocal identification of individuals. to plan for artificial inseminations several gener- Tracing of maternal lineages by analysis of ations ahead, thus regaining some of the genetic mitochondrial DNA has also been proposed (for traits lost during the intervening period. review see Powell, 1983); this approach is jus- The separation of X and Y chromosome-bearing tified by the observation that only maternally spermatozoa is not yet possible, but the develop- derived mitochondria are passed from one gen- ment of such technology is likely within the next eration to the next. Study of mitochondrial DNA decade as the influence of sex chromosomes also has the advantage that it can be used to upon the cell surface chemical architecture be- probe evolutionary distance between different comes clearer. Such an innovation would enable populations of similar species (Brown et a/., the managers of the future to choose the sex of 1979); in a recent review Benirschke (1983) cited offspring, perhaps in accordance with a pre- a study of two subspecies of white rhinoceros, determined breeding programme. It is not too where comparison of fragments obtained from early to give some consideration to the implica- mitochondrial DNA allowed the divergence to tions of this research, which could be used to be estimated at two million years ago. the benefit of endangered species. Semen banking for captive endangered species 177

Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 25 Sep 2021 at 23:16:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0030605300027800 This review has not dealt with the preservation Fritz, J. 1986. Resocialization of asocial chimpanzees. In of embryos, and embryo collection and transfer, Primates: the Road to Self-Sustaining Populations (ed. all of which are feasible in species like the cow, K. Benirschke), pp. 351-359. Springer-Verlag, New York. sheep and horse, and which are, in theory at Haigh, J.C., Shadbolt, M.P. and Glover, G.J. 1984. Artificial least, applicable to endangered species. On a insemination of wapiti. Proc. Am. Ass. Zoo Vet. p. 174. purely practical basis the widespread application Heuer, C, Tahir, M.N. and Amjad, H. 1987. Effect of season of these procedures is unlikely, in view of the on fertility of frozen buffalo semen. Animal Reprod. 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