Blood typing studies of 12 Przewalski horses : Equus przewalskii:at San Diego Zoo and Wild Animal...
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224 H U S B A N D R Y
The following two articles report on similar studies carried out in Britain and the United States on the genetics o f Przewalski horses. The nomenclature employed by the two groups o f workers dgers in certain respects. The British group has followed the practices o f the Newmarket Equine Research Station which are generally in accordance with European usage. The blood group terminology is that agreed by recent international convention. The American laboratory has not adopted the new international convention for blood grouping as it does not cover all thefactors used in the study.
Both laboratories have described new alleles but, at this stage, it is not possible to corzfirm whether they are recopiring the same alleles, as sera have not yet been exchanged between the two groups.
In the circumstances the Editors did not consider it pracfical to attempt to standardise the nomenclature but the alternative designations given below should help readers to make direct coniparisons between the two articles, where this is possible.
A glossary o f general ternis used in the following three articles appears on pp. 238-239.
Editor B I O C H E M I C A L MARKERS IN HORSES
British convention alternative
Catalase F-S M Carbonic anhydrase I Albumin S, F-S, F B, A-B, A
M or D
BLOOD FACTORS IN HORSES
International Systetn convention A Aa
D Da Db Dc Dd De Df
P Pa Pb
Q Qa Qb QC
alternative A, A H
D El E, E' J a Y
Q SI I Sr2
Blood typing studies of 12 Przewalski horses Equus przewalskii
at San Diego Zoo and Wild Animal Park A. TROMMERSHAUSEN-SMITH,' 0. A. RYDER,' & Y. SUZUKI' lDepartment o f Reproduction, Serology Laboratory, University o f Calfirnia, School o f Veterinary Medicine, Davis, California 95616, and lResearch Department, San Diego zoological society, San Diego, Cal$vnia 92112, USA
Comparative genetic studies of domestic horses Equus caballus and Przewalski horses E. przewalskii may help in understanding the evolutionary relationship between these present day Eguus species. It is known that the chromosome num- ber of E. przewalrkii is 2n=66 and that of E.
caballus 2n= 64. Despite this difference in chromo- some number, the two species cross to form fertile hybrids (2n= 65). Comparative chromoso- mal studies with banding techniques indicate considerable similarity between the two equine species (Ryder et al., 1978). The reduction in
H U S B A N D R Y
Henrietta Bogatka Jeanhold Bosaga Belkina BeIzar Bolinda Bogdo Roland Bektari Borkas Roxina
(Severinx Berta) (Rolandx Bonnette) (Bertoldx Bonnie Jean) (Jeanhold x Bogatka) (Jeanhold x Belaya) (Jeanhold x Belaya) (Roland x Bonnette) (Roland x Bonnette) (Rertold x Rolanda) (Jeanhold x Belaya) (Jeanhold x Bolinda) (Severin x Roxanne)
SEX STUDBOOK NO.
? 268 3 so4 6 469 0 639 0 603 6 638 P 406 6 473 d 320 P 678 6 685 0 480
Table I. Przewalski horses from San Diego Zoo and Wild Animal Park used in blood typing study.
chromosome number from 66 in E. przewalskii to 64 in E. cubufftrs is thought to be the result of a Robertsonian fusion between two acrocentric chromosomes of E. przewalskii.
Blood type comparisons between Przewalski horses and domestic horses can complement karyotype studies in understanding the relation- ship of these two species. In the literature at the present time are only two references detailing blood types of six Przewalski horses (Podliachouk & Kaminski, 1971 ; Trommershausen-Smith et al., in press) (but see Fisher et al., pp. 228-235 this volume). Thls report extends our previous paper and details the genetic markers found in the blood of IZ Przewalski horses from the San Diego Zoo and San Diego Wild Animal Park, including seven animals not previously reported.
MATERIALS A N D METHODS Blood samples were obtained from animals at the San Diego Zoo and Wild Animal Park. Two blood samples, approximately 10 ml each, were collected in evacuated blood collecting tubes from each animal, one tube containing anticoagulant (ACD solution) and the other without.
The samples with no anticoagulant served as a source of serum used in starch-gel electrophoretic tests that defic phenotypic digerences for domestic horses in the following systems: albumin (Al), prc-albumin (Pr), post-albumin (Pa), transferri (Tf) and esterase (Es). The samples collected in ACD solution served as a source of red blood cells (RBCs) for the serologic tests. In these tests the RBCs of each Przewalski
horse were typed using a minimum of 24 specifically different blood typing reagents developed for domestic horses that define groups in the genetic systems designated A, C, D, K, P, Q, T and U (Stormont & Suzuki, 1964; Sandberg, 1973 ; Suzuki, 1978). Haemolysates were also prepared from the RBCs for use in starch-gel electrophoretic tests that differentiate phenotypic differences in the carbonic anhydrase (CA), catalase (CAT), 6-phosphogluconate de- hydrogenase (PGD) and phosphoglucomutase (PGM) systems. References for these techniques are cited elsewhere (Trommershausen-Smith et a]., 1976).
R E S U L T S
The 12 horses of this study are identified with respect to name, parentage, sex and studbook number in Table I. The five animals included in the previous report were Jeanhold, Bosaga, Belkina, Belzar and Bolinda. Jeanhold, the chief stallion at the Wild Animal Park, is the sire of Bosaga, Belkina, Belzar, Bektari and Borkas. Roland, the chief sire at the S a n Diego Zoo, is the sire of Bogatka, Bolida and Bogdo. Blood types were determined on both parents for two off- spring, Bosaga and Borkas. The complete blood types of all 12 horses are shown in Table 2. A new international nomenclature for equine blood group factors was established in 1974 (International Society for Animal Blood Group Research, 1g74), but it is not used in this report because it does not cover all of the factors used in t h i s study.
226 H U S B A N D R Y
SYSTEMS OF ELECTROPHORETICALLY DETERMINED MARX6RS SYSTEMS OF SEROLOGICALLY DETERMINED RBC MARKERS*
Henrietta Bogatka Jeanhold Bosaga Belkina Uelzar B O l i n d a Bogdo Roland Bektari Borkas Roxina
A C D K P Q T U
AJHZ C E, - - QsR TW - A1 C DE/E, - PX/- QS TW U/- A1/HZ - DE/E, - +P QS TW - A,/HZ - DE/E, - iP QS TW U/- AJHZ - E, +P QS TVW- A1 C/- DE/E, - &P QaR TW - Ai/HZ C Ea - - QS TW U/- A1 C D E / E , - P X QS TW - A1 C Ea - PX/- QS TW - Ai - Ea - - QRS TVW- A I / A ~ C/- DE/E, - - QRS TW U/- Ai/HZ - JEs - - RS TVW-
A1 Tf Pa Es PI CACAT PGDPGM
B E - F F G I s M M F F B E-F F G-I Prz M M F F-S B F F G P r z M M F P S B F F GI Prz M M ** ** B F F G Prz M M ** ** B E-F F G I Prz M M F F-S B D*-FF G PrzS M M ** ** B D*-FF GI Prz M M F F B E-F F & G I Prz M M F F B E-F F G Prz M M F F-S B F F G P r z S M M F S A-BE F-S G-H Prz M M ** **
* Factors are phenogrouped according to types found in domestic hoives and genotypes are given, if deducible, from parentage or progeny data. * * Not tested. Table a. Blood types of the IZ Przewalski horses shown in Table I.
RBC Antigenic Markers: In the A system (a four- factor system) all 12 horses were positive for A,, and six of the 12 also possessed H and Z , but none possessed A'. In domestic breeds, these four factors determine a complex series of at least eight alleles (Suzuki, 1978). This group of Przewalski horses suggests the presence of two of these alleles, aA1 and a HZ. The aAl allele is the most common A-system allele found in domestic horses, but the a H Z allele has only rarely been encountered.
In the C system (a one-factor system), seven of the IZ horses were C-positive. Blood factor C has an incidence of greater than 85% in nearly all domestic breeds which we have studied.
In the D system (a closed, six-factor system), all 12 animals had factor E, and, in addition, six animals had factors D and E'. One animal had factor J. None was positive for two other tested D system factors, namely E, and Y. In domestic horses, Ea is commonly found in all breeds. Factor D, rarely found in light horse breeds, is always associated with E' in a DE' phenogroup and such appears to be the case in the Przewalski horses. Factor J can be associated with pheno- groups JE2, JE' or JYEa but in the Przewalskis seems to be as the phenotype JE,. These 12 horses suggest the presence of at least three phenogroups (E,, JE, and DE') out of those known for the D system in domestic horses.
In the K system (a one-factor system) all 12 horses were negative, but t h i s was not unusual in view of the fact that K has a low incidence in most domestic breeds.
In the P system (a three-factor system), three animals reacted with our anti-P and anti-X reagents, suggesting the presence of a P-system allele P p x . Some animals also showed weak, atypical reactions with our P-system reagents. No animals reacted with anti-P'.
In the Q system (a four-factor system) n i n e of the horses possessed blood factor Q. Two posses- sed a Q subtype, Q,. At least four horses also possessed factor S, and three factor R, but definitive absorption tests for factors R and S were not performed on all animals because of the small amount of blood available. For example, in Table 2, Borkas is shown to possess factor R, which appears to be lacking in his parents Jeanhold and Bolinda. Undoubtedly, if critical absorption tests for R were done for the parents, either or both would be found to possess factor R.
In the T system (a three-factor system) all of the horses were T-positive. but this is not unusual in view of the fact that most domestic horses are T-positive. With respect to the two other factors, V and W, four of the horses were V-positive and all were W-positive. This is in marked contrast with domestic horse breeds in which W is only infrequently found.
H U S B A N D I1 Y 227
Electrophoretic markefss: In the A1 system (a two allele system, for all practical purposes) 11 horses were of phenotype B and one was A-B. In many breeds of domcstic horses, the allele for 13 has a higher frequency than that for A, although in other breeds the frequencies are approxi- mately equal. No evidence ofthe rare A1 I type was seen.
In the Tf system (a multi-allele system), zones typical of those coded for by the common allele Tf were encountered. However, at least two other atypical band patterns, definitely dis- tinguishable from previously reportcd Tf alleles in horses, are also present. One pattern, designated Tf E by us, although not to our knowledge previously reported, has also been found by this laboratory in Quarter horses and Standardbreds. The second unusual Tf pattern has never been seen by us in other horse breeds and is designated as atypical Tf D or Tf D*.
In the Pa system (a two-allele system) 11 Przewalski horses were of Pa type F and one was PaF-S. The PuF allele is also that most frequently encountered in domestic horses. In the Es system (a six-allele system) there were
three phenotypes (Es G, Es G H and Es G-I) indicating the presence of three of the six recognised Es alleles. However, the presence of allele EsG in all IZ Przewalski horses is in con- trast with domestic horses in which allele ErC is not that frequent. Allele EsH is extremely rare and in &IS laboratory has been seen only in a few Morgans, Hackney ponies, Quarter horses and Peruvian pasos.
In the Pr system (a multi-allele system), 11 of the 12 Przewalski horses possessed a three-band pattern which we have yet to encounter in domestic horses. We have used the symbol Prz in Table z to indicate the three-zone pattern which is apparently unique to Przewalski horses. One horse (Henrietta) did not have the Pr Prz pattern, but had only the single zone characteristic of Pr S of domestic horses and two horses had both the distinctive Przewalski pattern and Pr S.
In the CA system (a multi-allele system) there was evidence for only one type, namely that coded for by allele CAM. CAM is the most commonly encountered allele in domestic horses.
In the CAT system (a two-allele, three-pheno- type system), probably only the CAT M pheno-
type was present. Early samples were reported as CAT F in two animals, but we believe that these analyses were incorrect andthatonly CAT M has been found. In domestic horses the M phenotype is the heterozygous expression of alleles CATS and CATP. Since it is unlikely that all these horses were heterozygous, it may be that the Przewalski horses have a CAT allele not previously recognised.
Eight of the 12 horses were tested for PGD and PGM. No polymorphism was evident in PGD (a threcallele system) as all were PGD F. In PGM (a three-allele system) both PGMF and PGMS alleles were found.
Natural antibodies: In a check for natural allo- antibodies to equine RBCs in the sera of Przewal- ski horses, a haemolytic anti-C of low titer (r/4) was found in several C-negative animals. Anti-C is also commonly found in low titer in C- negative domestic horses. The finding of an alloantibody to equine RBCs is important with respect to possible transfusions administered to critically ill Przewalski horses. Random selection of a donor not cross-matched by haemolytic testing to a recipient might indeed lead to serious complications of transfusion.
D I S C U S S I O N
Seventeen blood type loci were tested in IZ Equus przewalskii for genetic variation using methods known to detect genetic differences in E. caballrrs. Overall, the picture obtained from comparisons of Przewalslu horse blood types and those of domestic horses indicated a close relation- ship. In view of their known genetic similarity from chromosome banding studies, this observa- tion is not too startling. Some of the more striking findings from these studies were the relatively high frequency of variants considered rare in domestic horses such as Tf E, red cell factor W and red cell allele u H Z , as well as the discovery of hitherto unknown factorsTf D* and Pr Prz.
Equus przewalskii is a very rare species and only a few hundred animals exist in zoos throughout the world. It is not known whether any Przewal- ski horses survive in the wild. It is of prime concern to zoos to maintain this rare species, but the limited number of founder animals which have established the genetic base may be critical to the future of t h i s zoo species (Mohr, 1971).
218 H U S B A N D R Y
Of the Przewalski horses tested in t h i s study, some were genetically related animals originally obtained from stock at the Catskill Game Farm in New York and the rest were their descendants. In view of the high degree of relationship of these animals, it is surprising to find that the 12 animals were polymorphic at 13 of the 17 tested loci. Perhaps this indicates a broad spectrum of genetic polymorphism in the founder animals and may augur well for the future of zoo Przewalski horses.
CONCLUSION Blood typing studies of IZ Przewalski horses at the San Diego Zoo and wild Animal Park using methods for detecting genetic variation in 17 systems in domestic horses indicated a close genetic relationship between these chromosomally disparate but interfertile species. At least two genetic markers not previously found in domestic horses were evident. Polymorphism was found at 13 of 17 tested loci in this interrelated group of animals.
ACKNOWLEDGEMENTS The skillful assistance of Linda S. Nickel, who per- formed the electrophoretic tests, and B. G. Morris, who performed the blood typing tests, is gratefully acknowledged. W e are also grateful for the generous
cooperation of the veterinary staff of the Zoological Society of San Diego, particularly Drs L. S. Nelson, J. E. Oosterhuis and P. T. Robinson, for providing us with blood samples for these studies.
RESEARCH (1974): Meeting of the horse research section at the 14th ISABR conference. h i m . Blood Grps biochem. Genet. 5: 196. MOHR, E. (1971): The Asiatic wild hotre. London: J. A. Allen and Co. PODLIACHOUK, L. & KAMINSKI, M. (1971): Comparative investigations of Equidae. A study of blood groups and serum proteins in a sample ofEquus prrewnbkii PoliakoE Anim. Blood Grps biochem. Genet. 2: 239-242. RYDER, o., BPEL, N. & BBNIRSCHKE, K. (1978): Chromo- some banding studies of the Equidae. Cytogenet. Cell Genet. 20: 323-350. SANDBERG, K. (1973): The D blood group systcm of the horse. Anim. Blood Grps biochem. Genet. 4: 193-zo~. STORMONT, c. & SUZUKI, Y. (1964): Genetic systems of blood groups in horses. Genetics 50: 915-929. SUZUKI, Y. (1978): Bloodgroups in horses. Thesis. Tokyo University of Agriculture, Tokyo.
(1976): Use of blood-typing to confirin principles of coat-color genetics in horses. J . Hered. 67: 6-10.
BBNIRSCHKE, K. & %YDER, 0. A. (iu press). Blood type markers in 5 Przewalski horses. Equus.
INTERNATIONAL SOCIETY FOR ANIMAL BLOOD GROUP
TROMMERSHAUSBN-SMITH, A., SUZUKI, Y. & STORMONT, C.
TROMMERSHAUSBN-SMITH, A,, SUZUKI, Y., STOmONT, C.,
Manuscript siibmitted 13 December 1977.
Gene markers in 40 Przewalski horses Equus przewalskii
R. A. FISHERI, W. PUTTI, A. M. SCOTTa, C. M. HAWKEY3, P. D. BUTCHER3, D. G. ASHTON4 & P. BIRCHER6 fMRC Human Biochemical Genetics Unit, The Galton Laboratory, University College London, WoZJion House, 4 Stephenson Way, London, NWi zHE, PEquine Research Station, POB 5, Balaton Lodge, Snailwell Road, Newmarket CB8 7 0 W, 8Nujield Laboratories o f Comparative Medicine, The Zoological Society ofLondon, Regents Park, London, NWi 4RY , 4The Zoological Society ofLondon, Whipsnade Park, Dunstable, Bedfordshire LU6 zLF, and 6Manuell Zoological Park, Colden Common, Nr. Winchester, Hampshire, Great Britain
The Przewalski horse Equus przewalskii is now thought to be extinct in the wild. It is usually considered to be a defined species of the Equidae although closely related to the domestic horse Equus caballus. The animals now extant total some 300 and the numbers appear to be increasing, but
since they are all descended from only 13 animals, they are a very inbred population (Bouman, 1977).
Inbreeding often leads to loss of fitness and there are signs that this may have happened to some degree in certain Przewalski horse herds.