Determination of Parentage by the Use of Polyvalent Immune Sera in Chickens1

Determination of Parentage by the Use of Polyvalent Immune Sera in Chickens1

Determination of Parentage by the Use of Polyvalent Immune Sera in Chickens 1 TAKATSUGU MITSUMOTO2 AND YUKIO YAMADA The National Institute of Genetics...

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Determination of Parentage by the Use of Polyvalent Immune Sera in Chickens 1 TAKATSUGU MITSUMOTO2 AND YUKIO YAMADA The National Institute of Genetics, Misima, Japan (Received for publication February 22, 1961)

Simplified techniques of blood grouping would be desirable in order to test a large number of offspring of questionable parentage. An example is the case where pure and strain cross progeny without marker genes are the result of single male or multiple male breeding pens. In a laboratory where large numbers of reagents are not available, it is possible to determine parentage by the use of polyvalent immune sera. These would be adsorbed by the red cells of the dam and sire and should react with the red cells of all other individuals except the red cells of Contribution No. 376. 2 Present address: Department of Poultry Husbandry, Iowa State University, Ames.

their offspring (Todd, 1930; Rapacz and Dubiski, 1958). The present paper illustrates the method of determining parentage in chickens by the use of polyvalent immune sera and gives an experimental check on the parentage determinations using marker genes. The utilization of this method is briefly discussed. MATERIALS AND METHODS All parent birds were selected randomly and housed in individual cages for this experiment. Each bird was inseminated every three days with about 0.1 cc. of a mixture of approximately equal amounts of semen from a White Leghorn (WL-1) and a Barred Plymouth Rock (BR-1). The eggs layed by each bird were recorded daily. All offspring were wingbanded at hatching time and were examined when eighty days old. Marker genes were used for the determination of the mated male in order to check with the results found in the serological test. Sex-linked genes for slow (K) and rapid (k) feathering were used as markers for a White Leghorn female inseminated with the mixed semen. Dominant white (I) and Barred (B) plumage were used as the marker genes for a Rhode Island Red female inseminated with the mixed semen. Hetero-immune sera were prepared by injecting washed red cells (approximately 50 percent red cell suspension) of 18 different chickens (6 White Leghorns, 4 Barred Plymouth Rocks, 4 Rhode Island Reds and 4 Nagoyas) into guinea fowl. Iso-immune sera were prepared by injecting washed red 701

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T T IS well known that blood groups, -*- characterized as polymorphism, can be applied to various problems of identity and parentage, because of the antigenic individuality of red cells and sera of man and other animals, and because of the rigid manner of their inheritance (Race and Sanger, 1958). In chickens, also the chance that even two closely related individuals will inherit the same blood groups is small because the number of genes determining blood groups is very large (Briles, McGibbon and Irwin, 1950; Briles, Allen and Millen, 1957; Gilmour, 1959). Although blood groups are effective as a means of solving disputed parentage cases, a large number of reagents must be available for carrying out blood group tests. Furthermore, a considerable volume of each reagent is necessary.

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T . MlTSUMOTO AND Y . YAMADA

were repeated until the supernatant ceased to produce agglutination with the red cells used in the adsorption. The agglutination tests were performed in small glass tubes by adding one drop of a two percent red cell suspension in saline to two drops of the absorbed test fluid. Each mixture of red cells and test fluid was observed for presence of agglutination after standing at room temperature for one hour. A second agglutination score was recorded after three hours of incubation. A third confirmatory observation was made after storage overnight in the refrigerator. Doubtful and negative reactions, and controls were checked microscopically. RESULTS AND DISCUSSION The distribution of paternity between breeds with regard to marker genes is shown in Table 1. The legitimate offspring

TABLE 1.—Determination of paternity between breeds using marker genes as a check Test Cells From: Offspring Test Mating

Sire

Dam

Agglutination reaction with polyvalent sera adsorbed with red cells of sire and dam of each test mating*

Number

Phenotypic marker gene

Mating A

Mating B

BR-1 WL-1

WL-4

4 2

K k

all 0 all + +

all + + all 0

BR-1 WL-1

WL-5

5 none

K

all

0

all + +

B

3A ^B

BR-1 WL-1

WL-6

4 2

K k

all 0 all + +

all + + all 0

4A *B

BR-1 WL-1

WL-7

5 none

K

all

0

all + +

A

BR-1 WL-1

WL-8

2 1

K k

all

0

B

all + + 0

BR-1 WL-1

RIR-1

B

2 3

I B

all + + all 0

7A ' B

BR-1 WL-1

RIR-2

1 1

I B

++

0

++

BR-1 WL-1

RIR-3

1 none

I

++

0

11

2 Z

55

6

88

A

B A

A

B

++

all 0 all + +

* The symbols ( + + ) , ( + ) and (0) indicate strong, weak and no agglutination, respectively.

0

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cells from a White Leghorn male into a Barred Plymouth Rock male and a Rhode Island Red male and from a Barred Plymouth Rock male into a White Leghorn male. The polyvalent sera used in this study were a mixture of the iso-immune sera and hetero-immune sera. The inactivated polyvalent sera strongly agglutinated the red cells of all chickens at dilutions of 1:64 to 1:128. To prepare test fluids, polyvalent sera were adsorbed with a suitable quantity of washed red cells from those chickens assumed to be disputed parents. Usually 3 cc. of red cells to 1 cc. of polyvalent sera were used in the adsorption process. Sera were adsorbed at dilutions of 1:64 and 1:128. The mixtures were allowed to set at 25° to 30°C. with frequent agitation for two hours and stored overnight in a refrigerator at 0° to 5°C. The adsorptions

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DETERMINATION OF PARENTAGE

The exclusion of paternity and maternity within breed without marker genes are shown in Tables 2 and 3. These tables show TABLE 2.—Determination of paternity within a breed without marker genes

BR-1 cTXWL-5 9

1 2 3 4 S

+++++

Offspring

++ + +++++

Mating

Agglutination wit h polyvalent sera adsorbed wi:th red cells from WL-! ) ana. I-l BR-2 BR-3 ooooo

Test Cells F r o m :

WL-1 WL-1 t f X W L - 4 9

6 7 8 9 10

0 0 0 0 0

WL-2

WL-3

+++++ +++++

Agglutination with polyvalent sera adsorbed with red cells from WL-4 and:

+ + + + ++

TABLE 3.—Determination of maternity within a breed without marker genes Agglutination reaction with polyvalent sera adsorbed with red cells from BR-1 and:

Test Cells From: Offspring 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

WL-4

WL-5

WL-6

WL-7

0 0 0 0

++ ++ ++ +0

++ ++ ++ ++ + + ++ ++ ++0

++ ++ ++ + + ++ ++ ++ + ++ ++ ++ + +0

++ ++ ++ ++ + + ++ ++ ++ ++ ++ ++ ++ ++

0 0 0 0

+ ++ ++ ++ ++ ++ ++ ++ ++

0 0 0

++ ++ ++ ++ ++

0 0 0 0

that the relationship of parent to offspring can be resolved within a breed as well as between breeds by the serological method. This method may be particularly applicable in determining paternity in a breeding program using reciprocal recurrent selection where pure and strain cross progeny result from the same set of single male breeding pens or in a breeding scheme using shifts of males in the same pen during short breeding periods. Also the method could be used to determine maternity in non-trapnested breeding pens or in the case of chicks from unidentified floor eggs. The accuracy of solving parentage cases depends on the variability of blood group genes within the breed, and also on the lack of dominance between blood group alleles. In chickens it appears that all of the multiple alleles of each series of blood groups show a complete lack of dominance in any combination (Briles, McGibbon and Irwin, 1950). Exception to Todd's (1930) rule, that no individual reacts with a serum to which neither parent will react, have been reported (Thomsen, 1936; Boyd and Alley, 1940). However, at least one of these exceptions may have been due to improper serological procedures (Irwin, 1952). Allen (1960) reported an antiserum produced

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of any male in each group were previously determined by the marker genes K and k or B and I as shown in Table 1. The results obtained show clearly that test fluids adsorbed by both parents never reacted with the red cells of legitimate offspring. On the other hand, the test fluids adsorbed by the dam and sire very clearly reacted with the red cells of non-legitimate offspring. As an example, K and k progeny in test mating 1 are the offspring of BR-1 ( S ) X WL-4 ( ? ) and WL-1 ( $ ) X WL-4 ( 9 ), respectively. The test fluid adsorbed by BR-1 and WL-4 did not react with the red cells of K progeny, but reacted with the red cells of k progeny. The test fluid adsorbed by WL-1 and WL-4 contrast completely with that by BR-1 and WL-4. Accordingly, the serological test indicates that K and k progeny are the legitimate offspring of BR-1 and WL-4 and WL-1 and WL-4, respectively. Because the results of the exclusion of paternity by the serological test agreed with that determined by marker genes, it seems that this method may be useful to determine causes of questionable parentage in chickens.

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T . MlTSUMOTO AND Y . YAMADA

SUMMARY A method of determining parentage in chickens by the use of polyvalent immune sera appropriately adsorbed is outlined. Marker genes were used to identify the sires of two breeds. This made possible a genetic check on the accuracy of the serological test. The legitimate parent can be determined from disputed dams and sires within breeds as well as between breeds by the use of polyvalent sera. ACKNOWLEDGMENT

The authors are indebted to Dr. Arne W. Nordskog, Iowa State University, for suggestions made in the preparation of the

manuscript and the senior author wishes to express his gratitude to Dr. Shusaku Nishida, Tohoku University, for his interest and encouragement during the course of the present work. REFERENCES Allen, C. P., 1960. A specific iso-immune chicken antiserum which identifies a locus alleles and B locus heterozygotes within Leghorn lines. Genetics, 45: 971-972. Boyd, W. L., and O. E. Alley, 1940. Individual blood differences in chickens. J. Hered. 3 1 : 135136. Briles, W. E., W. H. McGibbon and M. R. Irwin, 1950. On multiple alleles effecting cellular antigens in the chickens. Genetics, 35: 633-652. Briles, W. E., C. P. Allen and T. W. Millen, 1957. The B blood group system of chickens. 1. Heterozygosity in closed populations. Genetics, 42: 631-648. Gilmour, D. G., 1959. Segregation of genes determining red cell antigens at high levels of inbreeding in chickens. Genetics, 44: 14-33. Irwin, M. R., 1932. Dissimilarities between antigenic properties of red blood cells of dove hybrids and parents. Proc. Soc. Exp. Biol. Med. 29: 850-851. Irwin, M. R., 1952. Heterosis, edit, by J. W. Gowen. Iowa State College Press, Ames. Race, R. R., and S. Sanger, 1958. Blood Groups in Man. Blackwell Scientific Publications. Oxford. Rapacz, J., and S. Dubiski, 1958. Serological test for determination of parentage in cattle. Nature, 182: 1176. Thomsen, O., 1936. Untersuchungen iiber erbliche Blutgruppenantigene bei Huhnern. II. Hereditas, 22: 129-144. Todd, C. H., 1930. Cellular individuality in the higher animals with special reference to the individuality of the red blood corpuscle. Proc. Roy. Soc. B. 107: 197-205.

NEWS AND NOTES (Continued from page 1597) Assam, Bihar, Orissa and West Bengal. L. C. Bora and U. Rahman of Assam, India, and J. B. Lai of Rajasthan, India, have arrived for Professor Maynard Yoes, on leave from Sam Houston State College, Huntsville, Texas, will take graduate study in poultry husbandry at the University of Missouri. Two other students from India, over Professor Kinder's teaching duties this year. He is working on his Ph.D. degree in poultry breed- N. C. Panda and Gopal Pandey, are completing their master's degrees during the fall semester. ing. (Continued on page 1750)

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within a Leghorn that does not agglutinate the parents but can agglutinate the progeny. A hybrid specificity (Irwin, 1932) in the antigen-antibody reaction would reduce the probability of solving parentage by the method presented in this study. However, the frequency of such phenomena is thought to be sufficiently small so that in the majority of cases the method could be considered determinative. This method could be applicable to other animals including cattle, swine and sheep (Rapacz and Dubiske, 1958). The probability of solving cases by this method could be increased if several polyvalent sera were used. However, this method is somewhat dependent upon the survival of both disputed parents. Less conclusive evidence would be obtained after the death of one of the parents.