PART II TECHNICAL DOCUMENTATION

STATUS AND PROSPECTS OF EXISTING CAPTIVE POPULATIONS

GENETIC ANALYSIS OF PRZEWALSKI'S HORSES IN CAPTIVITY

Oliver A. Ryder

The wild horse of the Asian steppe was first described to western scientists by Poliakov in 1881. The considerable interest that this significant discovery created ultimately resulted in the capture of Przewalski's horse foals, their transfer to captive environments, subsequent acclimatization, and successful captive breeding. Many zoologists were fascinated by this unique creature but, were it not for the dedicated efforts of the early owners and breeders of Przewalski's horses they would almost surely be extinct today.

All Przewalski's horses living today in captivity trace their ancestry to twelve animals brought out of Mongolia at the turn of the century and a single mare captured in 1947. Growth of the captive population was at first slow and the population numbers declined severely during World War II. The dedicated interest of zoologist Dr. Erna Mohr resulted in the publication of the first Pedigree Book of the Przewalski's Horse. International Symposia have been held to consider the biology of this endangered species beginning in 1959. Following the publication of the first Pedigree Book by Dr. Mohr in Prague, Czechoslovakia zoo has maintained the studbook under the direction of Dr. Jiri Volf. The initial studbook and the annual update of new information have set a standard for all subsequent studbooks.

A studbook is a historical record of births, deaths and parentage of individuals. Although many extremely useful tools for genetic analysis are available now it is still the case that the single most powerful tool for genetic analysis is studbook data. Thus the numerous volumes of the Pedigree Book of Equus przewalskii constitute one of the most sophisticated and useful records of long-term captive breeding.

Aspects of analysis of studbook data such as the calculation of demographic parameters suggest the growth potential of captive populations. Other calculations based upon pedigree data are reflections of the degree to which the genetic contributions of individuals brought into captivity have been assimilated into subsequent generations of captive individuals. The techniques and methodologies of pedigree analysis are a continuing focus of medical scientists and zoologists. For very few species of large mammals do pedigree data exist over ten or more generations. Consequently the application of new and sophisticated genetic analysis techniques provides additional insights concerning the extent to which captive populations may differ from the free-living populations from which they were derived.

DEMOGRAPHIC ANALYSIS

The growth of the captive population of Przewalski's horses has been thoroughly documented by Studbookkeeper Dr. Volk and elaboration has been provided by Bouman, Foose and others. The captive population is growing at a sufficient rate that significant new collections of Przewalski's horses have been established in Asia and Australia while, at the same time, substantial increases in the sizes of collections have taken place at institutions that have long histories of propagating the species. It is growing increasingly clear that Przewalski's horse individuals surplus to the requirements for adequate conservation of the species ex situ are now available in limited numbers and that in a few short years substantial numbers of individuals will be available for appropriate restoration and reintroduction projects. The relatively large numbers of individuals in the younger age classes of the world population of Equus przewalskii suggest that sufficient numbers of animals for establishing an appropriate programme of restoration of the species to its historic range in the People's Republic of Mongolia will be available (Figure 1). The broad base of the age distribution suggests that, under continued patterns of reproduction such as have been occurring recently, the population is capable of rapid expansion.

INBREEDING IN PRZEWALSKI'S HORSE

The early history of breeding Przewalski's horses in captivity was greatly influenced by the significance of domestic horsepower for human societies. Numerous governmental and non-governmental institutions were interested in the possibility of producing superior domestic horses and, considering this background, it is not at all surprising that numerous hybrids between Equus przewalskii and E. caballus were created. Fortunately, only one documented hybrid is depicted in the International Studbook. Similarly, selective inbreeding has long been recognized as an important tool for the agricultural breeder. It is not unexpected that the early breeding of Przewalski's horses included matings of close relatives. Furthermore, the pioneering work of modern population geneticists was unavilable to those first owners and breeders of Przewalski's horses. The propensity for normally outbreeding populations to suffer losses of vitality (i.e. fitness depression) as a result of consanguineous matings is well documented for mammalian populations. The negative effect may be only transitory and, for those populations able to have sufficient numbers of offspring surviving to produce numerous progeny themselves, the deleterious alleles may be purged. In populations founded by relatively few individuals, inbreeding is unavoidable and some genetically less-fit individuals are expected to succumb. In such small populations low to moderate levels of inbreeding may be necessary in order to avoid population extinction (e.g. Speke's gazelle at St. Louis Zoo, Missouri, USA ([Templeton and Read, in press]).

Avoidance of close inbreeding is generally observed in natural populations of feral horses and other equids. Consequently, to more closely simulate the genetical relationships presumed to have been exhibited by free-living populations of Przewalski's horses as well as to minimize the loss of genetic variability, inbreeding levels should not dramatically increase. Analysis of the world trend in levels of inbreeding as measured by the average coefficients of inbreeding of foals born each year is presented in Figure 2. As the world population grows and with periodic animal transfers among major geographical centres of breeding, e.g. Europe, North America, British Isles, as suggested by Bouman (1979) the worldwide increase in coefficients of inbreeding has been arrested. Since the inception of the Species Survival Plan for the Asian Wild Horse under the auspices of the American Association of Zoological Parks and Aquariums a previous trend toward increasingly higher values of coefficients of inbreeding in North American collections has been reversed (Figure 2).

Fig. 1 All institutions - population age structure for species: Przewalski Horse

Fig. 2 Przewalski horse inbreeding coefficients: yearly trend

Coefficients of inbreeding for all Przewalski's horses listed in the International Studbook issued in Prague on 1 January 1984 are presented in Appendix 1.

SELECTIVE BREEDING OF PRZEWALSKI'S HORSES

The milieu in which much of the captive breeding of Przewalski's horses has occurred included both conscious and unconscious selection. Unconscious selection began when the first foals began to die because they could not, in their wildness, adapt to their surrogate mothers and the journey to the west. Even currently, as in-experienced stallions run into fences and break their necks, unconscious (or unwilling) selection occurs. Conscious selection takes other forms. Some selection of stallions by phenotype has undisputedly occurred. On a worldwide basis there has been a 20-year trend of relative reduction on the genetic contribution of the E. caballus mare (who was the dam of #56, Halle 1) to the gene pool of captive E. przewalskii (Figure 3). During the same period of time there has been a dramatic increase in the relative genetic contribution of the last wild-caught mare, #231 Orlica III (Figure 3). Under common circumstances and for species whose populations are quite small (less than 100) conscious selection should be severely restricted. For other, larger, populations, and for the special case of introgression (in which genes from one species are introduced into another species) some selection against individuals with extreme attributes (“outliers”) or selection for reduction in the overall genetic contribution of the introgressing individual(s) is acceptable (Frankham et al., in press). This is, in fact, the currently occurring worldwide trend for captive E. przewalskii.

The extent to which the selection against the domestic mare has reduced the genetic variation derived from #58 Halle C, the single genetically effective offspring of two wild-caught individuals (#11, Bijsk 1 and #12, Bijsk 2), is a subject of current investigation (MacClure et al., in press; Gilpin et al., in preparation). It should be noted that studies of blood group and electrophoretic variation in Przewalski's horses have indicated that lower average levels of heterozygosity are present in those horses descended from #56 Halle 1 without the incorporation of genetic input from #285, Askania 3, Bars, than are observed among either the descendants of #56 Halle 1 after the introduction of genetic contribution from Bars to the captive gene pool or among the horses lacking genetic input from the domestic mare (Ryder et al. Equus, in press; Ryder et al. 1982).

POPULATION MANAGEMENT FOR GENE POOL PRESERVATION AND RESTORATION

There is ample documentation establishing the phenotypic diversity of the Przewalski's horses imported from Mongolia (Mohr 1959). Genetic analysis of proteins and DNA from recent generations of Przewalski's horses has readily demonstrated that substantial variability remains within the captive gene pool. It is this reservoir of genetic variation that represents the potential source of adaptive variation upon which natural selective forces will act as individuals are restored to free-living conditions. Consequently, an important aspect of the total effort to preserve Przewalski's horses and restore them to appropriate habitats is the maintenance of genetic variation within the captive gene pool in order that the restoration efforts have the greatest chances for success. The dependence of an established free-living population of Przewalski's horses upon the captive population will not cease until many generations of breeding in nature have occurred. Even at that time it is highly probable that some periodic exchanges of individuals will be considered desirable. Genetic bottlenecks must be anticipated as a result of strong selective forces acting on the reintroduced population as has been demonstrated for the reintroduction of the Arabian oryx (Stanley-Price et al., in press).

Fig. 3 Przewalski horse founder blood representation

In order to minimize the loss of gene pool resources for captive Przewalski's horses it is desirable to incorporate a relatively greater proportion of males born in one generation as sires of the next generation. For example three living stallions had sired 133 foals as of 1 January 1982 whereas some stallions with comodious pedigrees are underrepresented or not represented at all in subsequent generations. A listing of the reproductive contributions of all stallions having sired offspring listed in the studbook from its inception until 1 January 1982 appears in Appendix II.

Rather than placing single stallions in many small zoos with inadequate exercise space for Przewalski's horses, several new male-only groupings have been created in zoos in the British Isles and the United States. One new stallion group at Woburn Abbey re-establishes the species at one of the historical sites of early captive breeding success. Such groupings probably more closely reflect the natural groupings of adolescent males occurring in free-living populations of other equids. Care must be taken in the creation of all-male groups. Factors such as the relatedness of the colts, the age differential among individuals, and animal density must be taken into consideration. Even so, fighting and occasional injury may occur.

PRZEWALSKI'S HORSE AND THE EVOLUTION OF MONODACTYL EQUIDS

Following Poliakov's identification of the Mongolian wild horse as a species distinct from domestic horses in 1881, controversy surrounded the systematic status of the wild horse of Mongolia. The discovery that the diploid chromosome number of E. przewalskii differs from that of domestic horses provided new insights into the systematic differentiation of the two taxa (Benirschke and Malouf 1966). More recently, blood grouping analysis, electrophoretic studies and comparative studies of DNA have added further information concerning the genetic differentiation of Przewalski's and domestic horses (Ryder et al. in press; Ryder et al. 1979; Ryder et al. 1981; Ryder et al. 1982). A comparison of mitochondrial DNA restriction maps of E. przewalskii (representing three separate female founders) and E. caballus suggests the extent of divergence of their mitochondrial genomes is not indicative of a recent close genetic relationship. These data rather suggest that it was well before humans domesticated wild horses that Przewalski's horses and the wild ancestors of domestic horses had diverged from a common ancestor (George and Ryder 1983; George and Ryder, submitted for publication).

ACKNOWLEDGEMENTS

The contributions of The Canyon Colorado Equid Sanctuary, Wagon Mound, New Mexico (USA) in the form of computer-generated studbook analyses (represented by Figures 1, 2 and 3 as well as Appendix I) are gratefully acknowledged. This work was supported by NIH grant GM23073, the Zoological Society of San Diego, and the Ceasar Kleberg Foundation.

REFERENCES

Bouman J. 1979 A possible stallion exchange strategy in order to decrease inbreeding in the Przewalski horse. In: Genetics and Hereditary Diseases of the Przewalski Horse. J. Bouman (ed). Foundation Pres. Prot. Przewalski Horse. Rotterdam. pp. 119– 124.

Buisman A.K. 1982 and van Weeren R. Breeding and management of Przewalski horses in captivity. In: Breeding Przewalski Horses in Captivity for Release in the Wild. Bouman and Bouman (eds). Foundation Pres. Prot. Przewalski Horse. Rotterdam.

Foose T.J. 1980 Demographic management of endangered species in captivity. Intl. Zoo Yrbk. 20: 154–165.

Frankham R., Hemmer H., Ryder O.A., Cothran E.G., Soule M.E., Murray N.D. and Snyder M. Selection in captive populations. (In press, Zoo Biology).

George M. 1983 and Ryder O.A. Evolutionary relationships among the Equidae inferred by mitochondrial DNA mapping. Abstract presented at the 63rd Ann. Mtg. of the American Society of Mammalogists.

George M. and Ryder O.A. Mitochondrial DNA evolution in Equus. (Submitted).

MacClure J.W., VandeBerg J.L., Read B. and Ryder O.A. Pedigree analysis by computer simulation. (In press, Zoo Biology).

Mohr E. 1959 Das Urwildpferd. A. Ziemsen Verlag. Wittenberg. Lutherstadt.

Ryder O.A., Bowling A.T., Brisbin P.C., Carroll P.M., Gadi I.K., Hansen S.K. and Wedemeyer E.A. Genetics of Equus przewalskii, Poliakov (1881): Analysis of genetic variability in breeding lines, comparison of equid DNAs, and a brief description of a cooperative breeding program in North America. (In press, Equus 2: [Berlin]).

Ryder O.A., 1981 Brisbin P.C., Bowling A.T. and Wedemeyer E.A. Monitoring genetic variation in endangered species. In: Evolution Today. Scudder and Reveal (eds). pp. 417–424.

Ryder O.A., 1982 Fisher R.A., Putt W. and Whitehouse D. Genetic differences among subgroups of a captively-bred endangered species: The case of the Mongolian wild horse, E. przewalskii. Ann. Proc. Am. Assoc. Zool. Parks Aquar. pp. 91–102.

Ryder O.A., 1979 Trommershausen-Smith A.T., Hansen S.K., Suzuki Y., Sparkes M.C., Sparkes R.S., Clegg J.B., Oosterhuis J.O., Nelson L.S., Robinson P.T., Meier J. and Benirschke K. Biochemical variation in Przewalski's horses of the Munich line in the United States. In: Genetics and Heredity Diseases of the Przewalski Horse. J. Bouman (ed). Foundation Pres. Prot. Przewalski Horse. Rotterdam. pp. 41–60.

Templeton A.R. and Read B. Factors eliminating inbreeding depression in a captive herd of Speke's gazelle (Gazella spekei). (In press, Zoo Biology).

Volf J. 1960 Pedigree Book of the Przewalski Horse. Zoological Garden, Prague, Czechoslovakia.

et seq.

APPENDIX I

CANYON COLORADO EQUID SANCTUARY
FOR SPECIES: Przewalski Horse
MATRIX/KEY (STUDBOOK NO.) CROSS REFERENCE TABLE

APPENDIX II

GENETICALLY EFFECTIVE PRZEWALSKI'S HORSE STALLIONS: NUMBER OF OFFSPRING PER STALLION

EXPERIENCE IN PRZEWALSKI HORSE MANAGEMENT IN ASKANIA-NOVA

Yu. Musienko, N. Krylov, N. Lobanov and V. Klimov

In 1987 the Askania-Nova zoo, one of the oldest in the USSR, will mark its 100th anniversary. It was established in 1885–1887 in the steppes of Tavria on the Black Sea northern shores. In historic times these steppes gave refuge to numerous herds of wild horses - tarpans eliminated by man last century.

When N. Przewalski discovered a new species of wild horses that were seen by few people and doubted by many others, it was decided in Russia to catch and transmit them to Europe. By doing so, the existence of true wild horses could be proved.

At the end of the 19th century an expedition was sent to Mongolia and in 1900 the Askania-Nova zoo was the first in Europe to accommodate wild horses. Mares Staraya-I and Staraya-II and a stallion Vaska initiated captive reproduction of these horses and first foals were born. During 40 years 37 foals were produced in the zoo to form an old Askania line. The horses propagated normally till the Second World War. They formed a basis of Munich (Hellabrunn) line, thus preserving blood of the Askania line in current Przewalski horses. The Second World War that brought about vast devastations to Europe and the USSR put an end to existence of the line, the horses in Askania-Nova were destroyed.

In 1948 a stallion Orlik-Robert was delivered from Germany and in 1957 the zoo admitted a mare Orlitsa-III caught in the wild in Mongolia. These animals became founders of a new Askania line.

LINE STRUCTURE, DEMOGRAPHY

The horses of this line began to reproduce since 1960 and 100 animals have been produced over 25 years. Some of them (50 percent) form a nucleus of the line, the others have been distributed among the USSR and other countries' zoos. As related to the foals produced per year, the Askania-Nova zoo is acknowledged to be a leader among the world's zoos. The average surplus accounts for 12–15 percent per annum exceeding other zoos' indices by 5–7 percent. Practically all the sexually mature mares are productive. In some years fertility reached 100 percent, averaging 60 percent on the whole. Among fifty horses available in Askania-Nova only ten are efficient. The zoo does its utmost to improve these indices.

Demographic figures are given in Table 1. As one can see, the population's reproductive potential is increasing.

Analysing the line structure, mention should be made of two

Prague-line mares involved in reproduction (Gasana and Vada) as well as two females and a male imported from the USA in 1982. The latter represent American branches of the Munich line. The line geneology clearly indicates three families - offspring of Orlitsa-III, Vada and Gasana. As for the stallions, the horses relate to Orlik-Robert and his offspring and Gordyi of the Prague line. One more line is likely to be formed, involving a stallion Sigor from the USA into reproduction. Therefore, the new Askania line includes blood of all the wild founders of the Przewalski horse captive population. This fact favours genetic variability of the line and reproductive processes.

MANAGEMENT, BREEDING

Now the Przewalski horses in Askania-Nova occupy a portion of reserve feather grass steppe divided into large enclosures. Depending on the time of the year they are kept in either isolated or common enclosures varing from 45 to 1550 ha. All-year round ranging promotes better development and reproduction of horses. In relation to morphology, exterior, coloration, hair coat and behaviour, the Askania-line horses are closer to a standard of wild horses that has been repeatedly indicated by other specialists. They are more successful in reproduction as compared with horses from other zoos. The inbreeding level is very high in captive population on the whole, while being much less in Askania-Nova. Inbreeding coefficient of the line makes 0.25 on the average though it surpasses 0.30 in some animals. It happens due to long-term consanguineous mating because a stallion Pegas has been a major sire for a long period of time. To remedy the situation, in 1982 the Askania-Nova zoo exchanged horses with the US Bronx and San Diego zoos. Askania-Nova received two mares Lisa and Bollette and a stallion Sigor that made it possible to propagate horses in several directions. Since 1983 the animals have been mated and managed in four groups according to the prepared programme on interbreeding:

1. stallion Pegas and American mares
2. stallion Sigor and three Askania-line mares
3. stallion Parad and eight Askania-line mares with young stock of 1–2 years of age
4. group of bachelors - genetic reserve of the population.

Group 2 produced three foals in 1984 and two foals were born to the American mares. The international exchange of horses has favoured the animals' reproduction and the line genetic structure that will make a high level of genetic variability possible. Now the Askania-Nova zoo owns representatives of two pure lines and can breed horses in several directions and provide the total population with animals having new gene combinations. In the future efforts will be taken to enhance the quality and numbers of the line by: stimulation of reproductive processes, increasing the level of the line genetic variability, isolation of the Prague-line horses and their separate management, pure breeding, line breeding, formation of new lines and breeding groups with qualitatively new gene variants.

POSITIVE AND NEGATIVE FEATURES OF BREEDING OF PRZEWALSKI HORSES AND WHAT DO WE EXPECT FROM THEIR RESERVES

Jirí Volf

The breeding of the Przewalski horse (Equus przewalskii Poliakov, 1881) in captivity was started in 1899–1904. At that time six transports from Middle Asia brought a total of 54 individuals to Europe. A low reproduction rate and extensive scattering of the breed prevented additions and could not prevent a natural decrease at the beginning. In the twenties the number of wild horses kept in captivity varied within the range of 20–30 animals. In the thirties this number gradually increased exceeding several times 40 individuals. During World War II the number again temporarily decreased; another ten years passed before it was possible to substitute the losses. Despite this, the future of the species was not secure due mainly to the fact that during this period the number of freely living wild horses decreased catastrophically.

In September 1959, the 1st International Symposium on the Protection of the Przewalski Horse took place and the Prague Zoo, at the time the most important breeder, was assigned the task of compiling an all-world card-index on this animal.

An accurate central card-index makes it possible to publish annually the international Pedigree Book of the Przewalski horse. This book increases the direct interest of breeders aimed at the reproduction of animals, surveys the results of all-world breeding, facilitates the exchange of animals and supplementation of breeding herds and due to its wide distribution propagates the protection of this endangered species. When I started to publish the Pedigree Book in 1960, 59 Przewalski horses lived at 17 breeding stations; by 1 January 1985, as many as 620 individuals were living at 100 zoological gardens, scientific institutes and private breeders. This means that during twenty-five years the number of Przewalski horses kept in captivity increased more than ten times.

Irrespective of this indisputable success some undesirable aspects accompanying the worldwide breeding of wild horses should be taken into consideration.

First of all is the domestication effect. This is manifested by morphological changes, of the extremities and the cranium in particular. We found that these changes could be proved as soon as the first generation of horses was born in captivity. This change affects above all the lower jawbone, where the Corpus mandibulae narrows and the jaw assumes the fine form of tweezers. The cause of this weakening of the lower jawbone is the relatively soft food ingested by the animals in captivity which does not adequately put to use the biting apparatus of the horses.

However, negative selection, used by some breeders mainly with stallions, also cannot be overlooked. As a consequence, the dark-nosed form has presently almost disappeared from breeding herds (this form was formerly described by Pallas as Equus equiferus typicus). Some of the hyppologists considered the so-called meal-nose as a specific art of wild horses. Up to now it has depended on every single breeder whether he prefers the lighter form of wild horses with striped extremities, which can be for instance found in the Prague breed, or the darker form with black extremities kept at the Munich collection.

One has also to be aware of the breeding of relatives, which has already been observed in Przewalski horses for a long time. All wild horses living in captivity at present descended from only ten imported individuals: one pair which came to Falz-Fein in 1899 and a mare in 1902, two pairs of the Hagenbeck transport in 1901 and a pair in 1902, and the last the mare “Orlica III”. It lived in the Scientific Research Institute in Askania Nova (Ukraine) until the end of 1974. It is a pity that for a long time she was crossed with domestic stallions and did not influence to a great extent the breeding of wild horses all over the world. Only three purebred descendents of her remain.

In my opinion, it is possible to suppress partly the above mentioned aspects. The most effective approach would be the release of a greater number of individuals to a reserve. As the number of Przewalski horses in captivity is continually rising this step can be fully justified.

The reserve should ensure good living conditions for the animals so that the many years of effort of the breeders are not be wasted.

In the reserve the horses must be completely safe from their natural enemies - beasts of prey - as well as from hunters. We can use wild asses - Kulans (Equus hemionus kulan) as an example here. They found a new home on the island Barsa-Kelmes in the Aral sea some time ago.

Further important factors, which must be observed when releasing wild horses to a reserve, include soil and climatic conditions. The animals have to rub their hoofs regularly, because otherwise these would grow to such dimensions that these individuals could become completely lame. Thus, a hard sandy ground is essential.

The continential climate with a greater variation of temperature and little rainfall is a natural area for wild horses. We know that moisture is in fact much more unfavourable for wild horses than the greatest possible frost. Therefore, the reserve should have an arid climate.

This also influences feeding conditions favourably. Thus, prairie grass favourably influences the development of the chewing muscles and the whole mandible.

In addition, from the point of view of genetics, the reservation has to be isolated from domestic horses. The danger of cross-breeding holds not only for domestic stallions, but also for domestic mares that cannot withstand the aggression of wild stallions, and also for domestic mares that are introduced to the herd by wild stallions.

If a suitable reserve is found all breeders should be informed. If some of them are willing to give a part of their herd for the new reserve, they should be given the opportunity to get personally acquainted with the reserve and give their possible comments and recommendations before shipping the horses - I think that about 5% of the world's wild horses should be introduced at first, that means about 25–35 animals.

I believe sincerely that the foundation of a special reserve of Przewalski horses will reflect the successful international cooperation in the preservation of this endangered species and, in addition, will secure its fruitful future.

I wish your session in Moscow full success.

EQUUS PRZEWALSKI - 1 January 1985

Location of animals, with numbers of males and females

STATUS OF EXISTING CAPTIVE POPULATIONS

M. Knowles

SUMMARY

The present picture of numbers of animals in different locations is given in the paper by the Studbook keeper Dr. J. Volf and the paper by Dr. O. Ryder on this subject. The only comment additionally needed is to indicate that there are sufficient animals in captivity for a release to be made without prejudice to the captive population. There is also a strong commitment from the captive breeding population to make animals available.