RU2429598C1 - Method to increase immune competence of animals in cattle herds - Google Patents

Abstract

FIELD: veterinary science. ^ SUBSTANCE: method of polymerase chain reaction with further analysis of restriction fragments (PCR/RFLP) is used to establish genotypes of breeding stock cows and herd bulls by gene BoLA-DRB3. Then the animals are divided into groups with genotypes that differ relative to infection diseases: P/P, S/S, P/S,U/U, P/U, S/U. For reproduction of posterity with maximum frequency of heterozygous genotypes occurrence, including a stable (S) allele, parent couples are selected in the following manner: one parent with genotype S/S, and the other one - P/P, one parent with genotype S/S, and the other one - U/U, one parent with genotype P/S, and the other one - P/P (P-alleles must not coincide in the mother and father), one parent with genotype P/S, and the other - U/U (S-alleles must not coincide in the mother and father), one parent with genotype S/U, and the other one - S/S (S-alleles must not coincide in the mother and the father), one parent with genotype P/U, and the other one - S/S. ^ EFFECT: method makes it possible to increase immune competence in cattle herds. ^ 2 tbl

Description

The invention relates to livestock and veterinary medicine and is intended for use in the formation of a herd of cattle with a high level of immune competence.

According to the information and analytical center of the Rosselkhoznadzor [information resource: http: // www.fsvps.ru ], the unfavorable epidemiological state of the cattle population in Russia in 2009 is noted for anthrax, tuberculosis, brucellosis, rabies, leukemia, leptospirosis, etc. diseases. Therefore, increasing the immune competence of animals is an important national economic task.

It is known that the BoLA-DRB3 marker is currently used in the following areas:

- as a highly informative marker (sequenced more than 100 alleles) for studying the genetic diversity of breeds, lines and herds of cattle (5, 9, 10),

- as a marker of the level of the body's immune response to viral and bacterial infections (5, 7, 11). It is known that different alleles of the BoLA-DRB3 gene play a different role in the formation of resistance of cattle to various infectious diseases. For example, it was shown that animals bearing the BoLA-DRB3 * 11, * 23, * 28 alleles are stable (Y-group), are not prone to the transition of leukemia to the stage of persistent lymphocytosis, and animals that carry BoLA- alleles in their genotype DRB3 * 8, * 16, * 22, * 24 - sensitive (H-group), on the contrary, are more often than others in the sample of hematological patients. Neutral - other varieties - or N-alleles are not associated with either resistance or susceptibility to persistent lymphocytosis,

- as a marker of the severity of economically useful signs of cattle (2, 4, 8) (probably due to the close localization of BoLA-DRB3 to some productive loci).

The level of heterozygosity at some loci, in particular, the BoLA-DRB3 gene, should be considered as a non-specific factor of resistance to infectious diseases (1).

The closest to the essence of the proposed is the genetic selection method of creating a highly productive and resistant to persistent lymphocytosis of a herd of cattle (RU 2316207 C2, 02.10.2008) (3), including DNA testing of breeding stock and bulls according to the BoLA-DRB3 gene , then the distribution of animals into groups with different sensitivity to persistent lymphocytosis: with a genotype that includes two alleles that determine sensitivity (H / H); with a genotype that includes two alleles that cause resistance (U / Y); with a genotype that includes one allele that causes sensitivity, and the other - resistance (B / W); with a genotype comprising two neutral alleles (N / H); with a genotype that includes one allele that causes sensitivity, and the other is neutral (H / N); with a genotype including one allele that causes resistance and the other neutral (Y / H) and selection for reproduction of parental pairs so as to ensure maximum saturation of the population with U / H genotypes in the offspring.

The disadvantages of this method are:

- restriction of the use of pedigree animals (applicable only in the herds of Holstein, red steppe breeds and their crosses);

- does not take into account the allelic diversity of the crossed (mated) groups;

- restriction of the use of the BoLA-DRB3 marker only in relation to persistent lymphocytosis.

The technical result of the invention is to increase the competence of the immune system of animals in herds of cattle.

The technical result is achieved by the fact that in a method of increasing the immune competence of animals in herds of cattle using a genetic marker, DNA testing of brood stocks and bulls by the BoLA-DRB3 gene is performed, followed by the distribution of animals into groups with genotypes: H / H, H / U, B / N, U / N, N / N, U / U, selection of parental pairs for the reproduction of offspring with directed selection genotypes and characterized in that for the reproduction of offspring with a maximum frequency of occurrence of heterozygous genotypes including a stable (Y) allele, parental pairs are selected as follows:

one parent with the Y / Y genotype, and the other with H / H, one parent with the Y / Y genotype, and the other with N / N, one parent with the H / Y genotype, and the other with H / H (H alleles should not match for mother and father), one parent with the B / U genotype, and the other for U / U (the U-alleles should not match the mother and father), one parent for the U / N genotype, and the other for U / U (U alleles must not coincide in the mother and father), one parent with the genotype B / N, and the other - U / U.

If necessary, individual DNA testing of the entire maternal population is replaced by testing a representative sample from the herd; or, the genetic profile of the maternal population is determined by analyzing the genetic profile of the ancestors.

Moreover, the U-alleles provide the initial level of protection of the body against bacterial and viral infections, and the combination of U-alleys with H- or H-alleles provides additional protection due to the high degree of combinatorial variation of the genotype. These two factors generally contribute to enhancing the competence of the immune system.

The method is as follows.

Sperm samples are taken from breeding bulls for DNA analysis. Exon 2 of the BoLA-DRB 3 gene is amplified (the size of the PCR product is 284 bp), and then restriction polymorphism of this region is analyzed using endonucleases Rsa I, Nae III, BstYI, Bst2UI. The animal genotype is determined by the BoLA-DRB 3 gene. A bullfile is created, in which, in addition to the main zootechnical characteristics, the BoLA DRB3 gene is taken into account.

Blood samples are taken from breeding cows for DNA analysis. Exon 2 of the BoLA DRB3 gene is amplified (the size of the PNR product is 284 bp), and then restriction polymorphism of this region is analyzed using endonucleases Rsa I, Nae III, BstYI, Bst2UI. The animal genotype is determined by the BoLA DRB3 gene.

Then the animals are divided into groups: with a genotype that includes two alleles that determine sensitivity (H / H); with a genotype that includes two alleles that cause resistance (U / Y); with a genotype that includes one allele that causes sensitivity, and the other - resistance (B / W); with a genotype comprising two neutral alleles (N / H); with a genotype that includes one allele that causes sensitivity, and the other is neutral (H / N); with a genotype including one allele that causes resistance and the other neutral (Y / H), and for reproduction, the parental pairs are selected so as to ensure maximum saturation of the population in the offspring with heterozygous genotypes including the Y-allele (Y / H, H). Moreover, the profile of paternal alleys should differ as much as possible from the profile of maternal ones, to exclude the appearance of homozygous descendants. In other words, even if alleles belonging to the same group (for example, the H-group) are found in crosses in the genotypes of fathers and mothers, it is necessary that they be different alleles (for example, if the BoLA-DRB3 * alleles are mainly found in the mother genotype 16 and BoLA-DRB3 * 8, fathers with Ch-alleles BoLA-DRB3 * 22 and BoLA-DRB3 * 24 should be selected).

When genotyping samples of bulls - producers of Holstein (n = 293) and Ayrshire (n = 113) breeds belonging to various Russian breeding enterprises, we found that the N / N genotypes (65%) prevail in the profile of the Ayrshire breed, and Ch / N and H / H (33 and 35%, respectively) (table 1). It should be noted that BoLA-DRB3 * 24 belongs to the absolute leader in distribution among Holstein (the allele was found with a frequency of 22.02%); among ayrshirs, the monomorphism is even more pronounced - the frequency of occurrence of the BoLA-DRB3 * 7 allele is 26, 99%. If we talk about absolute values, then 115 out of 293 (or 39.25%) bulls of Holstein breed in various combinations carry the BoLA-DRB3 * 24 allele and 49 out of 113 (43.36%) bulls of Ayrshire breed in their genotype - BoLA -DRB3 * 7. Of the 293 Holstein bulls, 30 (or 10.23%) were found to be homozygous (with 46.67% of them carrying the 24 * 24 genotype and 20.00% of the 22 * 22 genotype); and in ayrshirs, 17 (or 15.04%) of 113 bulls are homozygous (of 17 homozygous bulls, 12 or 70, 59% carry the 7 * 7 genotype). Thus, the level of homozygosity is very high in Holstein and Ayrshire rocks.

Similar results were obtained in various farms of the Krasnodar Territory (CJSC SKVO, OJSC Plemzavod Kuban, CJSCSS Plemzavod Beisug, APF Niva), where a variety of cattle herds of Holstein, Ayrshire and red steppe breeds were studied in BoLA-DRB3 gene. We found that the various BoLA-DRB3 genotypes are not equivalent in terms of the length of the period of economic use. Thus, the combination of the Ch- and Y-alleles of the BoLA-DRB3 locus in the genotype of Holstein cattle increases the period of their productive use. A 7.9–9.2% reduction in the frequency of occurrence of cows with the B / W genotype in the sample of retired animals was noted.

Table 1 Frequency of BoLA-DRB3 genotypes in various groups of cattle Group of animals The frequency of occurrence of genotypes,% H / h B / w H / n U / U U / N N / A red steppe cows (n = 60) fifteen eighteen fifteen 8 21 23 Holstein cows (n = 417) 42 twenty 25 2 6 5 production bulls of Holstein breed (n = 293) 35 fifteen 33 2 8 7 Ayrshire cows (n = 55) - - fifteen - 12 73 Ayrshire bulls (n = 113) 3 four 17 eleven 65

When analyzing the distribution of BoLA-DRB3 genotypes in three groups of Ayrshire cows in the farm of OJSC “Plemzavod im. Chapaeva V.I. ”: in a group of cows with manifestations of persistent lymphocytosis; in a group of cows with high productivity; in a random sample of cows, a significant predominance of the frequency of occurrence of one of the BoLA-DRB3 genotypes — U / N in the group of healthy highly productive Ayrshire cows (milk yield for lactation over 7500 kg of milk) compared with the group of cows with persistent lymphocytosis was found to be 4.6 times.

For maximum saturation of the population with heterozygous genotypes, it is necessary to select parental pairs as follows (Table 2):

table 2 Possible selection of parental pairs and distribution of genotypes in the offspring Parents Possible distribution of genotypes in the offspring H / h U / U 100% U / H H (* 24, * 16) / U H (* 8) / H (* 22) 50% B / W + 50% B / H or y / y (50% Black and White + 50% Black) H / n U / U 50% B / W + 50% Y / N U / U H / H (N / N) 100% U / H (100% U / N) Y (* 11, * 28) / N Y (* 23) / Y (* 23) 50% U / N + 50% U / U N / A U / U 100% Y / N

Then in the second generation you can get the maximum possible number of animals with heterozygous genotypes.

Information sources

1. Genzhieva O., Ruzina M., Sulimova G. Analysis of the genetic resistance of Kalmyk cattle to leukemia // Dairy and beef cattle breeding. - 2008. - No. 8. - S.9-10.

2. Gorkovenko L.G., Kovalyuk N.V., Satsuk V.F. The relationship of the genotype at the BoLA DRB3 locus of the main histocompatibility complex of cattle with milk productivity // Vestnik RAAS. - 2007. - No. 3. - S. 73-74.

3. Gorkovenko L.G., Kovalyuk N.V., Satsuk V.F. RF patent for invention No. 2316207 "Breeding-genetic method of creating a highly productive and stable to herd of lymphocytosis herd of cattle", application No. 2005112494. Applicant and patent holder North Caucasian Research Institute of Livestock. Declared April 25, 2005, registered in the state register of inventions of the Russian Federation February 10, 2008

4. Kovalyuk N.V., Satsuk V.F., Machulskaya E.V. The use of genetic markers to increase the milk production of cows // Zootechny. - 2007. - No. 8. - C.2-4.

5. Sulimova G.E., Udina I.G., Shaykhaev G.O., Zakharov I.A. DNA polymorphism of the Bola-DRB3 gene in cattle due to resistance and susceptibility to leukemia // Genetics. - 1995. - No. 9. - S. 1294-1299.

6. Groenen M.A.M. et al. The nucleotide sequence of bovine MHC class II DQB and DRB genes // Immunogenetics. - 1990. - No. 31. - P.37.

7. Ripoli M.V., Villegas-Castagnasso E.E., Peral-Garcia P., Giovambattista G. New polymorphisms for the BoLA-DRB3 upstream regulatory region // Tissue Antigens. - 2005. - No. 66 (2). - P.136-137.

8. Rupp R. et al. Association of bovine leukocyte antigen (BoLA) DRB3.2 with immune response, mastitis, and production and type traits in Canadian Holsteins // J Dairy Sci. - 2007. - Feb. - 90 (2). - P.1029-1038.

9. Van Eijk M.J.T., Stewart-Haynes J.A., Lewin H.A. Extensive polymorphism of the BoLA DRB3 gene distinguished PCR-RFLP // Anim. Genet. - 1992. - 23. - 483.

10. Xu A., Van Eijk V.J.T., Park Ch. Polymorphism in BoLA-DRB3 Exon 2 correlates with resistance to persistent lymphocytosis caused by bovine leukemia virus // J. of Immunology. - 1993. - V.151. - No. 12. - P.6977-6985.

11. Zanotti M. et al. Association of BoLA class II haplotypes with subclinical progression of bovine leukaemia virus infection in Holstein - Friesian cattle // Anim. Genet. - 1996. - 27. - P.337-341.

Claims (1)

A method of increasing the immune competence of animals in cattle herds, including the analysis of the genotypes of brood stocks and bulls according to the BoLA-DRB3 gene, the subsequent distribution of animals into groups with genotypes: H / H, H / U, H / N, U / N, N / N, Y / Y, selection of parental pairs for reproduction of offspring with directed selection genotypes, characterized in that for reproduction of offspring with the maximum frequency of heterozygous genotypes including a stable (Y) allele, parental pairs select the following image ohm:
one parent with the Y / Y genotype, and the other with H / H, one parent with the Y / Y genotype, and the other with N / N, one parent with the H / Y genotype and the other with H / H (H alleles should not match for mother and father), one parent with the B / U genotype, and the other for U / U (the U-alleles should not match the mother and father), one parent for the U / N genotype, and the other for U / U (U alleles must not coincide in the mother and father), one parent with the genotype B / N, and the other - U / U.