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Identification and parentage verification of individual horses by blood typing tests
W@u'l¢l A SPECIAL, NON-REVIEWED SECTION
IDENTIFICATION A N D PARENTAGE VERIFICATION OF INDIVIDUAL H O R S E S BY BLOOD TYPING TESTS C l y d e J. S t o r m o n t
INTRODUCTION Horse blood typing as a means of identifying individual animals, verifying parentage, and solving problems of questionable parentage did not come into its own until the 1960s. T M Since then the amount of equine blood typing, performed primarily for the registries, has increased considerably. Following the recent lead of The Jockey Club (USA), more and more equine registries will be requiring a parentage check by blood typing on all horses to enter their Stud Books. From a forensic point of view this is as it should be, because when a well-established method is available to authenticate parentage assignments, and to serve as a bulwark of breed integrity, that methodology should be used to assure the accuracy of all pedigrees. In this article I shall briefly describe the kinds of blood typing tests performed in this laboratory. Then I shall turn to a discussion of the efficacy of the tests in detecting errors in parentage assignments, followed by a section on the burden of truth as it relates to parentage verification cases, concluding with a section on positive horse identification by blood typing.
MATERIALS AND METHODS Two blood samples are required from each horse, one collected in a yellow-stoppered Vacutainer tube, which contains ACD anticoagulant, and the other in a dry, red-stoppered Vacutainer tube. We recommend the use of Vacutainers that draw approximately 7 ml of blood. The blood sample drawn in ACD solution serves as a source of red blood cells (RBCs) that are tested not only for a variety ofserologically deffmed antigenic markers, but also are used in electrophoretic tests that define molecular differences in hemoglobin and 6-phosphogluconate dehydrogenase. The blood sample drawn in the dry tube serves as a source of serum for use in the electrophoretic tests. W e are often asked by veterinarians if they should spin the tubes down and decant the serum into separate tubes. The answer is no, because in order for the samples to remain uncontaminated the stoppers should not be removed. Author's address: Stormont Laboratories, Inc., 1237 E. Beamer SL, Suite D, Woodland, California 95695 USA. Acknowledgement: I am indebted to Dr. Yoshiko Suzuki for suggestions in the preparation of the manuscript and to Jonathan Dodd for his work in putting the manuscript and tables in final form. 176
The Serologically Defined Genetic Markers The membrane of the RBC is literally peppered with a variety of antigenic determinants, referred to as blood factors, which occur at multiple sites on the membrane. Some of the blood factors are genetically fixed, i.e., they occur on the RBCs of all members of the species under study. Others segregate genetically; they appear on the RBCs of some but not all members of the species under study, a classic example being blood factors A and B of the ABO system of human blood groups (after Stormont13). The number of segregating blood factors varies considTABLE 1 Symbols of Allelic Genes Which Encode Phenogroups in the Blood Group Systems of Horses Sy~ems
Alleles*
Ae¢, A~:e' Ac, A~,A co, AO...etc.
A
g , A a , A a ~ , A a d g , A b,
C
C', Ce
D
D- D adt D adln D bcm D ~ D cgm D c°rr° D eefgm Dehoimn,'DdelO,'Ddl., bOkl,'Ddfla, bdol...etc.
K
K',K k
P
'Daghm,
p-,p~,pc, pal,p, paca,p~, p~...etc.
Q
Q-, Qae, QabC, Qb, Qe ...etc.
T U
TW, Trw, TV, "I~ U-, Uu
* The number of alleles and their specific symbols are brought up-to-date at the workshops held at the biennial meetings of the International Society of Animal Blood Group Research. The T system s' is not as yet officially recognized by the ISABR.
TABLE 2 The Eight Genetic Systems of Electrophoretic Markers that are Routinely Tested for in the Author's Laboratory Systems
Symbol
Albumin Transferrin Esterase Postalbumin or Xk 6--phosphogluconate dehydrogenase Protease inhibitor* Vitamin D binding protein Hemoglobin
No. of Alleles References
Alb Tf Es Pa PGD
3 12 6 3 3
8,9 10,11 11 12,13 14
Pi Gc Hb
8 2 4
11,12,15,16 17 18,19
*This system was originally called the prealbumin (Pr) system prior to the discovery that the zones of prealbumin proteins are actually a l protease inhibitors, u EQUINE VETERINARY SCIENCE
A SPECIAL, NON-REVIEWED SECTION
TABLE 3
TABLE 4
Equine Albumin Phenotypes Expected and Not Expected In the Six Possible Kinds of Matlngs
Probability of Excluding an Incorrectly Assigned Parent*
Kinds of Matings
AxA AxAB AxB AB x AB AB x B B xB
Expected
A only 1A: lAB AB only 1A : lAB : 1A lAB : 1B B only
Not expected*
AB and B B A and B none A A and AB
*The occurrenceof any one of these typeswould excludethe particular mating.
erably between species, ]3 with over 80 recognizable in cattle contrasted with some 35 in horses. Isoimmune or alloimmune antisera are the main source of equine blood typing reagents (antibodies). Some of these antibodies react best or exclusively as agglutinins whereas others react best or exclusively as hemolysins. Indeed, it was the introduction of the hemolytic testing procedures Is that brought to light many hitherto unrecognized equine blood factors. Genetic analysis of family data reveals that the segregating blood factors fall into one or another of a limited number of blood group systems. In the horse, eight such systems have been described. 15These are named A, C, D, K, P, Q, T, and U. All of the systems, except C, K, and U, involve multiple blood factors. As new or previously undetected blood factors are brought to light, the systems continue to expand. For example, the D system originally involved only two blood factors, namely, D (now called Da in the international nomenclature) and J (or De), but presently it involves at least 14 factors. In the multi-factor blood group systems, the blood factors are inherited in a variety of combinations that we refer to as phenogroups. ",19 Examples are phenogroups A-(the absence of all factors), A', A ~f, etc., of the A system of horses. Each phenogroup of any multi-factor system is encoded by one of a series of allelic genes. Each of the single factor systems involves apair of alleles~ one encoding the factor, and one encoding its absence, which is signified by a dash (-). In Table 1 are shown the symbols of the allelic genes for each of the eight blood group systems of horses. Every horse posses ses a pair of alleles at each of the eight blood group loci, one transmitted by its sire and the other transmitted by its darn. If, for example, a foal possesses one or more alleles not present in its putative parents, the mating is excluded.
The Electrophoretically Defined Genetic Markers In the horse, variants in some 20 systems of blood Volume 8, Number 2, 1988
Systems A
TB 0.01
AR 0.04
ST 0.08
All) Tf Es Pa PGD Pi
0.02 0.59 0.02 0.16 0.01 0.06 0.13 0.53 0.09 0.02 0.18 0.51
0.00 0.46 0.00 0.09 0.08 0.08 0.18 0.42 0.06 0.05 0.18 0.61
0.02 0.61 0.07 0.12 0.06 0.05 0.18 0.38 0.27 0.00 0.11 0A3
Gc
0.05
Hb
0.13
Cumulative Totals 0.96
C D K P Q U
MH 0.06
QH
PF
PP
0.14 0.01 0.00 0.62 0.67 0.01 0.03 0.15 0.08 0.08 0.08 0.08 0.06 1 . 1 9 0.18 0.36 0.58 0.28 0.17 0.08 0.06 0.12 0.18 0.58 0.54
0.13 0.06 0.69 0.00 0.14 0.06 0.06 0.18 0.67 0.26 0.09 0.10 0.50
0.09 0.00 0.61 0.02 0.11 0.20 0.07 0.16 0.59 0.43 0.14 0.16 0.46
0.03 0.15
0.I0
0.I0
0.02
0.02
0.18
0.21
0.18
0.21
--
--
0.96
0.97
0.98
0.99 >0.98 >0.98
*using data from~*when consideringall the systems listed in Tables2 and 3, exceptT. TBffiThoroughbreds,ARfArabians, MH=Morgan Horses,QHfQuarterHorses,PFfPaso Finos, PPffiPeruvianPasos.
proteins have been brought to light using the methods of gel electrophoresis. In the author's laboratory we routinely test for eight of those systems (Table 2). Albumin constitutes the majorprotein in blood plasma. In the initial report on genetic variation in albumin phenotypes of horses, we described two molecular forms that we named A and B, with the A zone migrating just ahead of the B zone. Family studies revealed that the two forms are encoded by a pair of codominant alleles named Alb A and Alb B. The two alleles combine to form three genotypes that produce the phenotypes A, AB andB. There are six possible mating types and these are shown in Table 4 along with the kinds of offspring expected and not expected in each of the possible matings. For example, A x A can produce only A offspring. Hence, with an offspring of type AB or B, the mating would be excluded. Only one of the matings, namely, AB x AB, can produce offspring of all three types. The Alb system is a particularly effective system to work with because the two original alleles are well balanced in frequency in most breeds of horses. The maximum chance of excluding a mating when considering the two original alleles is 18.75 percent, and it occurs when each allele has a frequency of 0.5. And this brings us to'the subject of the combined efficacy of the blood group systems and the eight systems of electrophoretically defined markers in detecting errors in parentage assignments.
177
A SPECIAL. NON-REVIEWED SECTION
DISCUSSION Efficacy of the Blood Typing Tests in Detecting Errors in Parentage Assignments The efficacy of the blood typing tests in detecting errors in parentage assignment is based on the assumption that only one of the parents is misassigned and this is almost invariably the paternal parent. It is exactly the same as calculating the likelihood of solving a paternity case involving two males. In our initial reports, ~7,16which involved only two breeds, namely, Shetland Ponies and Thoroughbreds, we showed that the combined efficacy of the alleles then being tested for in the eight blood group systems and the Alb and Tf systems was 77.80 percent for Shetlands and 62.01 for Thoroughbreds, with the Tf system being the most effective of all. However, these were underestimates. In actual practice we were able to solve 82 percent of 55 Shetland paternity cases and 70 percent of 73 paternity cases in Thoroughbreds. Since then, with the expansion of the blood group systems to include numerous additional blood factors and the inclusion of additional systems of electrophoretically defined markers, the utility of the equine blood typing tests in detecting errors in parentage assignments and solving cases of questionable parentage has improved considerably. Trommershausen-Bowling and Clark have recently published2°data from my former laboratory on blood groups and protein polymorphism gene frequencies for seven breeds of horses in the United States. The data were collected over the years 1973 to 1983 and covered all the blood group systems except T. Their data also included 20 systems of electrophoretic markers. They calculated the effectiveness of the tests for detecting incorrect parentage or paternity at each of 20 loci (seven blood group loci, or systems, and 13 gel systems that included a variety of gel systems not routinely tested for). As expected, the D system of blood groups, and Tf and Pi systems of electrophoretic markers, each with numerous alleles, turned out to be the most effective in detecting errors in parentage assignments. The D system, when averaged over the seven breeds, would theoretically detect 61 percent of the errors in parentage assignments. The figures for the Tfand P i systems are, respectively, 50 percent and 52 percent. When calculating the cumulative effectiveness for just those three systems, the percent ranged from 86 percent in Standardbreds to 95 percent in Paso Finos. Using the data of Trommershausen-Bowling and Clark, the author calculated the cumulative effectiveness of all the systems in Table 2 (excepting T for which no data were available) and Table 3, a total of 15 systems. The resulting figures for the seven breeds are shown in Table 4. These figures were either equal to or within a percentage point of those reported by Trommershausen-Bowling and Clark using 178
data on 20 systems. Thus, there was virtually nothing to be gained in efficacy by running gels to reveal genetic variants in such systems as CA (carbonic anhydrase), AP (acid phosphotase) and PHI (phosphohexose isomerase). Moreover, the costs to the industry of adding such tests to those routinely performed would be prohibitive in many instances. As can easil~ be shown mathematically, the increments of increasing efficacy become smaller and smaller as additional systems are added to the tests. In most if not all breeds, the 95 percent level of efficacy can be closely approached or exceeded by using the five most effective systems. For Thoroughbreds these would presently be D, P, Alb, Tf and Pi, and the cumulative or combined efficacy, based on the data of Trommershausen-Bowling and Clark, would be 94.34 percent.
Parentage Verification Tests All parentage tests are based on the principle of genetic exclusion. The burden of proof rests in showing that a given animal (or animals) could not be a parent of the animal in question, a6 The observation that all the known genetic markers in the blood of an animal may be genetically compatible with those of its stated parents does not constitute (absolute) proof that the animal is in fact the offspring of those parents. The reason for this is simple. We know if we look long enough we are likely to find another pair of parents whose genetic markers are fully compatible genetically with those of the animal in question. ~6 Generally, however, mitigating circumstances would rule out the possibility that the second pair of animals could be the true parents of the animal in question. Today, all breed registries and certainly most if not all courts would accept the blood typing evidence as proof of parentage, knowing that the tests have a better than 95 percent chance of detecting incorrectly as signed parentage. Hence, we can rightully speak of these tests as parentage verification tests.
CONCLUSION Positive Horse Identification by Blood Typing We ask the simple question, "What is the probability of drawing two horses from the same breeding population that have identical blood types for all systems under test?" Some years ago, I calculated 12the probability of drawing two horses at random from each of four breeding populations that would have identical blood types. This statistic (Pi or the probability of identity) turned out to be 1/25,000 for Thoroughbreds, 1/200,000 for Arabians, 1/250,000 for Quarter Horses, and 1/500,000 for Standardbreds. If recalculated on the basis of the systems considered in this report, the P~indices would exceed by several fold the fractions previously arrived
EQUINEVETERINARYSCIENCE
A SPECIAL, NON-REVIEWEDSECTION
at. It is hardly necessary to do so at this time. All we need to say is that the probability of drawing at random two horses with identical blood types from the same breeding population is exceedingly remote. Hence, the blood type serves well as a permanent and indelble means of horse identification. REFERENCES 1. Bengtsson S, Sandberg K: A method for simultaneous electrophoresis of four horse red cell enzyme systems. Animal Blood Groups and Biochemical Genetics 4:83-87, 1973. 2. Braend M: Genetics of horse acidic prealbumins. Genetics 65:495503, 1970. 3. Braend M, Johansen KE: Haemoglobin types in Norwegian horses. Animal Blood Groups and Biochemical Genetics 14:305-307, 1983. 4. Braend M, Stormont C: Studies on haemoglobin and transferrin types of horses. Nord Vet Med 16:31-37, 1964. 5. EkN: Identification of the Pr prealbumin proteins in horses. Acta Vet Scan
9. Kitchen HD, Boreson D, Malkin S, Brett I: Horse hemoglobin. Proceedings of the First International Symposium on Equine Hematology pp 42-47, 1975. 10. Sandberg K: A third allele in the horse albumin system. An/mal Blood Groups and Biochemical Genetics 3:20%210, 1972. 11. Stormont C: Linked genes, pseudoalleles and blood groups. Amer Naturalist 89:105-116, 1955. 12. Stormont C: Positive horse identification, Part 2: blood typing. Equine Practice 1:48-54, 1979. 13. Stormont C: Blood groups in animals. J Amer Vet Med Assoc 181:1120-1124, 1982. 14. Stormont C and Suzuki Y: Genetic control of albumin phenotypes in horses. Proc Soc Exp Biol bled 144:673-675, 1963. 15. Stormont C and Suzuki Y: Genetic systems of blood groups in horses. Genetics 50:915-929, 1964. 16. Stormont C and Suzuki Y: Paternity tests in horses. Cornell Vet 55:365-377, 1965. 17. Stormont C, Suzuki Y, Rendel J: Application of blood typing and protein tests in horses, pp 221-228 in Blood Groups in Animals Ed by J. Matousek. Publishing House Czechoslovak Acad Sci, Prague, 1965. 18. Stormont C, Suzuki Y, Rhode EA: Serology of horse blood groups. Cornell Vet 54~439-452, 1964. 19. Suzuki Y: Studies on blood groups of horses. Memoirs Tokyo University af Agriculture 20:1-50, 1978. 20. Tmmmershausen-Bowling A, Clark RS: Blood group and protein polymorphism gene frequencies for seven breeds of horses in the United States. Animal Blood Groups and Biochemical Genetics 16:93-103, 1985. 21. Trommershausen-SmithA, SuzukiY: Identity ofXk and Pa systems in equine blood. Animal Blood Groups and Biochemical Genetics 9:127128, 1978.
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O 179
IDENTIFICATION A N D PARENTAGE VERIFICATION OF INDIVIDUAL H O R S E S BY BLOOD TYPING TESTS C l y d e J. S t o r m o n t
INTRODUCTION Horse blood typing as a means of identifying individual animals, verifying parentage, and solving problems of questionable parentage did not come into its own until the 1960s. T M Since then the amount of equine blood typing, performed primarily for the registries, has increased considerably. Following the recent lead of The Jockey Club (USA), more and more equine registries will be requiring a parentage check by blood typing on all horses to enter their Stud Books. From a forensic point of view this is as it should be, because when a well-established method is available to authenticate parentage assignments, and to serve as a bulwark of breed integrity, that methodology should be used to assure the accuracy of all pedigrees. In this article I shall briefly describe the kinds of blood typing tests performed in this laboratory. Then I shall turn to a discussion of the efficacy of the tests in detecting errors in parentage assignments, followed by a section on the burden of truth as it relates to parentage verification cases, concluding with a section on positive horse identification by blood typing.
MATERIALS AND METHODS Two blood samples are required from each horse, one collected in a yellow-stoppered Vacutainer tube, which contains ACD anticoagulant, and the other in a dry, red-stoppered Vacutainer tube. We recommend the use of Vacutainers that draw approximately 7 ml of blood. The blood sample drawn in ACD solution serves as a source of red blood cells (RBCs) that are tested not only for a variety ofserologically deffmed antigenic markers, but also are used in electrophoretic tests that define molecular differences in hemoglobin and 6-phosphogluconate dehydrogenase. The blood sample drawn in the dry tube serves as a source of serum for use in the electrophoretic tests. W e are often asked by veterinarians if they should spin the tubes down and decant the serum into separate tubes. The answer is no, because in order for the samples to remain uncontaminated the stoppers should not be removed. Author's address: Stormont Laboratories, Inc., 1237 E. Beamer SL, Suite D, Woodland, California 95695 USA. Acknowledgement: I am indebted to Dr. Yoshiko Suzuki for suggestions in the preparation of the manuscript and to Jonathan Dodd for his work in putting the manuscript and tables in final form. 176
The Serologically Defined Genetic Markers The membrane of the RBC is literally peppered with a variety of antigenic determinants, referred to as blood factors, which occur at multiple sites on the membrane. Some of the blood factors are genetically fixed, i.e., they occur on the RBCs of all members of the species under study. Others segregate genetically; they appear on the RBCs of some but not all members of the species under study, a classic example being blood factors A and B of the ABO system of human blood groups (after Stormont13). The number of segregating blood factors varies considTABLE 1 Symbols of Allelic Genes Which Encode Phenogroups in the Blood Group Systems of Horses Sy~ems
Alleles*
Ae¢, A~:e' Ac, A~,A co, AO...etc.
A
g , A a , A a ~ , A a d g , A b,
C
C', Ce
D
D- D adt D adln D bcm D ~ D cgm D c°rr° D eefgm Dehoimn,'DdelO,'Ddl., bOkl,'Ddfla, bdol...etc.
K
K',K k
P
'Daghm,
p-,p~,pc, pal,p, paca,p~, p~...etc.
Q
Q-, Qae, QabC, Qb, Qe ...etc.
T U
TW, Trw, TV, "I~ U-, Uu
* The number of alleles and their specific symbols are brought up-to-date at the workshops held at the biennial meetings of the International Society of Animal Blood Group Research. The T system s' is not as yet officially recognized by the ISABR.
TABLE 2 The Eight Genetic Systems of Electrophoretic Markers that are Routinely Tested for in the Author's Laboratory Systems
Symbol
Albumin Transferrin Esterase Postalbumin or Xk 6--phosphogluconate dehydrogenase Protease inhibitor* Vitamin D binding protein Hemoglobin
No. of Alleles References
Alb Tf Es Pa PGD
3 12 6 3 3
8,9 10,11 11 12,13 14
Pi Gc Hb
8 2 4
11,12,15,16 17 18,19
*This system was originally called the prealbumin (Pr) system prior to the discovery that the zones of prealbumin proteins are actually a l protease inhibitors, u EQUINE VETERINARY SCIENCE
A SPECIAL, NON-REVIEWED SECTION
TABLE 3
TABLE 4
Equine Albumin Phenotypes Expected and Not Expected In the Six Possible Kinds of Matlngs
Probability of Excluding an Incorrectly Assigned Parent*
Kinds of Matings
AxA AxAB AxB AB x AB AB x B B xB
Expected
A only 1A: lAB AB only 1A : lAB : 1A lAB : 1B B only
Not expected*
AB and B B A and B none A A and AB
*The occurrenceof any one of these typeswould excludethe particular mating.
erably between species, ]3 with over 80 recognizable in cattle contrasted with some 35 in horses. Isoimmune or alloimmune antisera are the main source of equine blood typing reagents (antibodies). Some of these antibodies react best or exclusively as agglutinins whereas others react best or exclusively as hemolysins. Indeed, it was the introduction of the hemolytic testing procedures Is that brought to light many hitherto unrecognized equine blood factors. Genetic analysis of family data reveals that the segregating blood factors fall into one or another of a limited number of blood group systems. In the horse, eight such systems have been described. 15These are named A, C, D, K, P, Q, T, and U. All of the systems, except C, K, and U, involve multiple blood factors. As new or previously undetected blood factors are brought to light, the systems continue to expand. For example, the D system originally involved only two blood factors, namely, D (now called Da in the international nomenclature) and J (or De), but presently it involves at least 14 factors. In the multi-factor blood group systems, the blood factors are inherited in a variety of combinations that we refer to as phenogroups. ",19 Examples are phenogroups A-(the absence of all factors), A', A ~f, etc., of the A system of horses. Each phenogroup of any multi-factor system is encoded by one of a series of allelic genes. Each of the single factor systems involves apair of alleles~ one encoding the factor, and one encoding its absence, which is signified by a dash (-). In Table 1 are shown the symbols of the allelic genes for each of the eight blood group systems of horses. Every horse posses ses a pair of alleles at each of the eight blood group loci, one transmitted by its sire and the other transmitted by its darn. If, for example, a foal possesses one or more alleles not present in its putative parents, the mating is excluded.
The Electrophoretically Defined Genetic Markers In the horse, variants in some 20 systems of blood Volume 8, Number 2, 1988
Systems A
TB 0.01
AR 0.04
ST 0.08
All) Tf Es Pa PGD Pi
0.02 0.59 0.02 0.16 0.01 0.06 0.13 0.53 0.09 0.02 0.18 0.51
0.00 0.46 0.00 0.09 0.08 0.08 0.18 0.42 0.06 0.05 0.18 0.61
0.02 0.61 0.07 0.12 0.06 0.05 0.18 0.38 0.27 0.00 0.11 0A3
Gc
0.05
Hb
0.13
Cumulative Totals 0.96
C D K P Q U
MH 0.06
QH
PF
PP
0.14 0.01 0.00 0.62 0.67 0.01 0.03 0.15 0.08 0.08 0.08 0.08 0.06 1 . 1 9 0.18 0.36 0.58 0.28 0.17 0.08 0.06 0.12 0.18 0.58 0.54
0.13 0.06 0.69 0.00 0.14 0.06 0.06 0.18 0.67 0.26 0.09 0.10 0.50
0.09 0.00 0.61 0.02 0.11 0.20 0.07 0.16 0.59 0.43 0.14 0.16 0.46
0.03 0.15
0.I0
0.I0
0.02
0.02
0.18
0.21
0.18
0.21
--
--
0.96
0.97
0.98
0.99 >0.98 >0.98
*using data from~*when consideringall the systems listed in Tables2 and 3, exceptT. TBffiThoroughbreds,ARfArabians, MH=Morgan Horses,QHfQuarterHorses,PFfPaso Finos, PPffiPeruvianPasos.
proteins have been brought to light using the methods of gel electrophoresis. In the author's laboratory we routinely test for eight of those systems (Table 2). Albumin constitutes the majorprotein in blood plasma. In the initial report on genetic variation in albumin phenotypes of horses, we described two molecular forms that we named A and B, with the A zone migrating just ahead of the B zone. Family studies revealed that the two forms are encoded by a pair of codominant alleles named Alb A and Alb B. The two alleles combine to form three genotypes that produce the phenotypes A, AB andB. There are six possible mating types and these are shown in Table 4 along with the kinds of offspring expected and not expected in each of the possible matings. For example, A x A can produce only A offspring. Hence, with an offspring of type AB or B, the mating would be excluded. Only one of the matings, namely, AB x AB, can produce offspring of all three types. The Alb system is a particularly effective system to work with because the two original alleles are well balanced in frequency in most breeds of horses. The maximum chance of excluding a mating when considering the two original alleles is 18.75 percent, and it occurs when each allele has a frequency of 0.5. And this brings us to'the subject of the combined efficacy of the blood group systems and the eight systems of electrophoretically defined markers in detecting errors in parentage assignments.
177
A SPECIAL. NON-REVIEWED SECTION
DISCUSSION Efficacy of the Blood Typing Tests in Detecting Errors in Parentage Assignments The efficacy of the blood typing tests in detecting errors in parentage assignment is based on the assumption that only one of the parents is misassigned and this is almost invariably the paternal parent. It is exactly the same as calculating the likelihood of solving a paternity case involving two males. In our initial reports, ~7,16which involved only two breeds, namely, Shetland Ponies and Thoroughbreds, we showed that the combined efficacy of the alleles then being tested for in the eight blood group systems and the Alb and Tf systems was 77.80 percent for Shetlands and 62.01 for Thoroughbreds, with the Tf system being the most effective of all. However, these were underestimates. In actual practice we were able to solve 82 percent of 55 Shetland paternity cases and 70 percent of 73 paternity cases in Thoroughbreds. Since then, with the expansion of the blood group systems to include numerous additional blood factors and the inclusion of additional systems of electrophoretically defined markers, the utility of the equine blood typing tests in detecting errors in parentage assignments and solving cases of questionable parentage has improved considerably. Trommershausen-Bowling and Clark have recently published2°data from my former laboratory on blood groups and protein polymorphism gene frequencies for seven breeds of horses in the United States. The data were collected over the years 1973 to 1983 and covered all the blood group systems except T. Their data also included 20 systems of electrophoretic markers. They calculated the effectiveness of the tests for detecting incorrect parentage or paternity at each of 20 loci (seven blood group loci, or systems, and 13 gel systems that included a variety of gel systems not routinely tested for). As expected, the D system of blood groups, and Tf and Pi systems of electrophoretic markers, each with numerous alleles, turned out to be the most effective in detecting errors in parentage assignments. The D system, when averaged over the seven breeds, would theoretically detect 61 percent of the errors in parentage assignments. The figures for the Tfand P i systems are, respectively, 50 percent and 52 percent. When calculating the cumulative effectiveness for just those three systems, the percent ranged from 86 percent in Standardbreds to 95 percent in Paso Finos. Using the data of Trommershausen-Bowling and Clark, the author calculated the cumulative effectiveness of all the systems in Table 2 (excepting T for which no data were available) and Table 3, a total of 15 systems. The resulting figures for the seven breeds are shown in Table 4. These figures were either equal to or within a percentage point of those reported by Trommershausen-Bowling and Clark using 178
data on 20 systems. Thus, there was virtually nothing to be gained in efficacy by running gels to reveal genetic variants in such systems as CA (carbonic anhydrase), AP (acid phosphotase) and PHI (phosphohexose isomerase). Moreover, the costs to the industry of adding such tests to those routinely performed would be prohibitive in many instances. As can easil~ be shown mathematically, the increments of increasing efficacy become smaller and smaller as additional systems are added to the tests. In most if not all breeds, the 95 percent level of efficacy can be closely approached or exceeded by using the five most effective systems. For Thoroughbreds these would presently be D, P, Alb, Tf and Pi, and the cumulative or combined efficacy, based on the data of Trommershausen-Bowling and Clark, would be 94.34 percent.
Parentage Verification Tests All parentage tests are based on the principle of genetic exclusion. The burden of proof rests in showing that a given animal (or animals) could not be a parent of the animal in question, a6 The observation that all the known genetic markers in the blood of an animal may be genetically compatible with those of its stated parents does not constitute (absolute) proof that the animal is in fact the offspring of those parents. The reason for this is simple. We know if we look long enough we are likely to find another pair of parents whose genetic markers are fully compatible genetically with those of the animal in question. ~6 Generally, however, mitigating circumstances would rule out the possibility that the second pair of animals could be the true parents of the animal in question. Today, all breed registries and certainly most if not all courts would accept the blood typing evidence as proof of parentage, knowing that the tests have a better than 95 percent chance of detecting incorrectly as signed parentage. Hence, we can rightully speak of these tests as parentage verification tests.
CONCLUSION Positive Horse Identification by Blood Typing We ask the simple question, "What is the probability of drawing two horses from the same breeding population that have identical blood types for all systems under test?" Some years ago, I calculated 12the probability of drawing two horses at random from each of four breeding populations that would have identical blood types. This statistic (Pi or the probability of identity) turned out to be 1/25,000 for Thoroughbreds, 1/200,000 for Arabians, 1/250,000 for Quarter Horses, and 1/500,000 for Standardbreds. If recalculated on the basis of the systems considered in this report, the P~indices would exceed by several fold the fractions previously arrived
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at. It is hardly necessary to do so at this time. All we need to say is that the probability of drawing at random two horses with identical blood types from the same breeding population is exceedingly remote. Hence, the blood type serves well as a permanent and indelble means of horse identification. REFERENCES 1. Bengtsson S, Sandberg K: A method for simultaneous electrophoresis of four horse red cell enzyme systems. Animal Blood Groups and Biochemical Genetics 4:83-87, 1973. 2. Braend M: Genetics of horse acidic prealbumins. Genetics 65:495503, 1970. 3. Braend M, Johansen KE: Haemoglobin types in Norwegian horses. Animal Blood Groups and Biochemical Genetics 14:305-307, 1983. 4. Braend M, Stormont C: Studies on haemoglobin and transferrin types of horses. Nord Vet Med 16:31-37, 1964. 5. EkN: Identification of the Pr prealbumin proteins in horses. Acta Vet Scan
9. Kitchen HD, Boreson D, Malkin S, Brett I: Horse hemoglobin. Proceedings of the First International Symposium on Equine Hematology pp 42-47, 1975. 10. Sandberg K: A third allele in the horse albumin system. An/mal Blood Groups and Biochemical Genetics 3:20%210, 1972. 11. Stormont C: Linked genes, pseudoalleles and blood groups. Amer Naturalist 89:105-116, 1955. 12. Stormont C: Positive horse identification, Part 2: blood typing. Equine Practice 1:48-54, 1979. 13. Stormont C: Blood groups in animals. J Amer Vet Med Assoc 181:1120-1124, 1982. 14. Stormont C and Suzuki Y: Genetic control of albumin phenotypes in horses. Proc Soc Exp Biol bled 144:673-675, 1963. 15. Stormont C and Suzuki Y: Genetic systems of blood groups in horses. Genetics 50:915-929, 1964. 16. Stormont C and Suzuki Y: Paternity tests in horses. Cornell Vet 55:365-377, 1965. 17. Stormont C, Suzuki Y, Rendel J: Application of blood typing and protein tests in horses, pp 221-228 in Blood Groups in Animals Ed by J. Matousek. Publishing House Czechoslovak Acad Sci, Prague, 1965. 18. Stormont C, Suzuki Y, Rhode EA: Serology of horse blood groups. Cornell Vet 54~439-452, 1964. 19. Suzuki Y: Studies on blood groups of horses. Memoirs Tokyo University af Agriculture 20:1-50, 1978. 20. Tmmmershausen-Bowling A, Clark RS: Blood group and protein polymorphism gene frequencies for seven breeds of horses in the United States. Animal Blood Groups and Biochemical Genetics 16:93-103, 1985. 21. Trommershausen-SmithA, SuzukiY: Identity ofXk and Pa systems in equine blood. Animal Blood Groups and Biochemical Genetics 9:127128, 1978.
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