The second exon of the BoLA-DRB3 gene has the highest level of polymorphism among all studied loci of the major histocompatibility complex (MHC) in cattle, which allows it to be used for studying population-genetic structure and assessing the level of biodiversity of populations or comparing the biodiversity of particular herds. According to the results of typing the blood samples of 293 cows using the method of PCR-RLFP, we determined allele frequencies of the BoLA-DRB3 gene for the Ukrainian black-pied dairy breed. The study was conducted on three herds in Khmelnytskyi Oblast: LLC “Kozatska Dolyna 2006” (herd A, n = 122), agrofirm “Perlyna Podillya” (herd B, n = 82) and branch “Ridnyy kray” (herd C, n = 89). In total, 37 alleles were found: herd A 31, herd B 25 and herd C 28. In total, in the three subpopulations seven alleles were found with frequency of over 5%, the total share of which equaled 55.8%. The most widely distributed allele was BoLA-DRB3.2*24, which composed 22.2% of the allele pool of the breed. We determined a high level of observed (0.89 to 0.95) and expected (0.93 to 0.94) heterozygosity. In herds A and B, there was determined domination of homozygotes. Deviation from HWE, calculated using the value of Wright`s individual fixation index, equaled FIS(A) = 0.016 (χ2 = 0.03; P > 0.05) and FIS(B) = 0.044 (χ2 = 0.076; P > 0.05). In herd C, we found excess of heterozygotes FIS(C) = -0.017 (χ2 = 0.026; P > 0.05). Rather low values were determined for the subpopulation fixation index: FST(A) = 0.009 (χ2 = 65.9; P < 0.01), FST(B) = 0.012 (χ2 = 47.2; P < 0.05) and FST(C) = 0.003 (χ2 = 14.4; P > 0.05), which were significantly different from the mean value for cattle (FST = 0.078), indicating insignificant reduction of heterozygosity and divergence between the subpopulations by the BoLA-DRB3 gene. To assess genetic diversity, we calculated parameters of effective allele number (Ae) and Shannon’s information index (I). In spite of the different numbers of alleles found in the selections, it was suggested that for assessing their diversity, an efficiency index will be used which shows the share of effective alleles among all alleles found in a subpopulation (Ae/Nа). The calculated values of the parameters equaled: herd A Ae = 14.9, Ae/Nа = 0.48, I = 3.05; herd B Ae = 14.5, Ae/Nа = 0.58, I = 2.87; herd C Ae = 16.4, Ae/Nа = 0.59, I = 3.01. Frequencies of BoLA-DRB3 alleles were used for calculating genetic similarity and standard genetic distances according to Nei. Cows of herds B and C were found to be more genetically affinitive by the BoLA-DRB3 gene. Standard genetic distance between them was the lowest D = 0.13, which coincides with the geographic locations and historical development of these populations. The results of the study prove that the studied herds have a high level of polymorphism. Frequency characteristics, values of expected heterozygosity, effective allele number, efficiency index and Shannon’s information index compared to the similar parameters for Holstein, black-pied and some other local breeds of cattle indicate the high genetic diversity of the studied subpopulations of the Ukrainian black-pied dairy breed.

The major histocompatibility complex (MHC) determines the immune response, and the MHC genes are promising candidate genes for identifying associations with diseases. The decisive role in the resistance of cattle to diseases belongs to the major histocompatibility complex of (BoLA). The BoLA system consists of several jointly operating genes that provide antigen presentation by MHC system molecules followed by an immune response to pathogenic microorganisms. The most functional is the BoLA-DRB3 gene. Its exon 2 is highly polymorphic and encodes the peptide antigen-binding cleft. Alleles, for which a close connection with disease susceptibility or disease resistance has been detected, are considered as DNA markers. These play a decisive role in the breeding of cattle to create herds resistant to diseases, including mastitis. This paper presents the results of a study of BoLA-DRB3 gene polymorphism in two commercial cattle breeds: the Ukrainian black-pied dairy (UBPD) and the Ukrainian red-pied dairy (URPD) and its association with mastitis. The UBPD and the URPD cows were genotyped at the bovine lymphocyte antigen DRB3.2 locus by a genotyping system that used polymerase chain reaction and restriction fragment length polymorphisms (PCR-RLFP). In 276 UBPD cows, 32 BoLA-DRB alleles have been found. Six alleles (*03, *08, *10, *22, *24 and *28) were identified with a frequency of more than 5% (total amount of 50.4%). The allele BoLA-DRB3.2*24 was the most frequent (19.2%). In the UBPD population (n = 162), four BoLA-DRB3.2 alleles are truly associated with mastitis: *24 and *26 with susceptibility and *13 and *22 with resistance. In 117 URPD cows, 22 alleles were identified, of which the most frequent were *07, *22, *11, *24, *01, *03 and *16 (total frequency 64.5%). Allele BoLA-DRB3.2*07 (present in 25.6% of cows) was the most commonly found. In the URPD population studied, four alleles truly associated with mastitis were identified. Animals susceptible to the disease had alleles *07 and *08, and resistant animals had alleles *22 and *24. Breeding activities for the creation of cattle resistant to mastitis using alleles of the BoLA-DRB3 gene are much more effective than treatment and special care for animals. Similar research should be carried out for other Ukrainian breeds in relation to various diseases (leukemia, necrobacteriosis, etc.).

Mastitis of cows causes significant harm to dairy farming, a susceptibility to which is partly determined by genetic factors. Therefore, identifying the susceptibility or resistance of cows to mastitis at an early stage of postnatal ontogenesis has both practical and scientific importance. In this work, we examined a model grounded on the collegial use of lymphocyte antigens class I MHC cattle and DNA markers based on alleles BoLA-DRB3 gene to identify the sensitivity of heifers to mastitis to their use in a milking herd. On the data of testing of 649 cows of the Ukrainian black-and-white breed, the antigens of histocompatibility were revealed, and the statusmetric model facilitated determining the integral estimation of sensitiveness (Z) to mastitis was constructed. The greater the positive Z value, the higher the predicted resistance to mastitis and vice versa. The model yields 69.2% correct mastitis susceptibility decisions based on 17 class I antigens (antigens W2, W6, W31, W14, W19, W15, A9, A12, A13 and A24 indicate susceptibility and W10, A1, A3, A6, A16, A17 and A22 – indicate resistance to the disease). Some exon 2 alleles of the BoLA-DRB3 gene were found to be associated with mastitis. For 162 cows from the preliminary sample DRB3.2*18, *24, *26 and *48 alleles characterize susceptibility to mastitis, and BoLA-DRB3.2*08, *13 and *22 – characterize resistance to the disease. A comparative analysis of the association of lymphocyte and DNA-markers was performed by comparing the diagnosis, status score, and the presence of associated alleles in the genotype. Two of the four possible variants unambiguously indicate the immune status of the cow: – diagnosis and integral score (by sign) coincide, and there is an allele in the genotype that coincides with the established diagnosis (65,7%); – diagnosis and integral score sign do not coincide, and there is a DNA marker in the genotype that coincides with the immune status of the animal by Z (13,3%). For 83 animals out of 105 in which DNA markers were detected, the immune status established by the statusmetric model was confirmed, for a total of 79%. The accuracy in predicting the susceptibility of cows to mastitis increased by 9.8%. Following the obtained results, the model for predicting sensitivity of heifers of Ukrainian black-and-white breed to mastitis at the stage of early postembryonic ontogenesis has been proposed. The model is universal and can be applied to different cattle breeds after appropriate research.

Introduction. The main direction of increasing the productivity of milk is to increase the proportion of heredity of the Holstein breed in the genotype of cows. Industrial breeds in Ukraine are improving due to the increase in the Holstein inheritance in the genotype of cows. The "holsteinization" of the most widespread domestic Black-and-White diary breed is intensively conducted. Currently, the percentage of heredity from Holstein is 90% or more. The negative effect of "holsteinization" appeared in reducing the resistance of animals to diseases that led to the spread of necrobacterial pathology. The control of the spread of necrobacteriosis can be based on genetic markers. Important markers can be the allele of the BoLA-DRB3.2 gene responsible for the formation of adaptive immunity. Due to the ambiguity of the results of "holsteinization", the following tasks were solved: To study the genetic structure of the herd for the BoLA-DRB3.2 gene at the beginning of the "holsteinization" and now. To compare the detected genetic structures with the alleles spectrum of North American Holstein and identify quantitative and qualitative changes in the structure of the herd genotype. To determine the effect of "holsteinization" on the dynamics of milk production and the state of morbidity by necrobacteriosis. Materials and methods of research. Comparison of alleles of population of the Ukrainian Black-Pied Dairy (UBPD) breed and Holstein breed was conducted to detect the consequences of "holsteinization" on milk yield and incidence of necrobacteriosis. The data of the allelic polymorphism of the BoLA-DRB3.2 gene of the UBPD10 (2010, n = 162), UBPD15 (2015, n = 114) and two Holstein populations of the USA and Canada were collected. The allelic spectrum was determined by the PCR-RFLP method. The amplification of the BoLA-DRB3.2 gene was performed using 2-step PCR with the use of primers HLO-30, HLO-31 and HLO-32 and allele-specific PCR. Restriction analysis was performed with endonuclease RsaI, HaeIII, BstYI (XhoII). Restriction fragments were separated by electrophoresis in 4% agarose gel. Counting of allele frequencies was performed taking into account the number of homozygotes and heterozygotes found for the corresponding alleles. To determine the phylogenetic relationships between the populations of the studied herds, genetic distance and genetic similarity were determined by the M. Nei method. Individual dairy productivity of cows was estimated for all lactation (regardless of its duration). Average milk yields were determined as the total volume of milk produced divided by the number of dairy cows. Results and discussion. The breeding measures carried out led to the accumulation of alleles characteristic of the Holstein breed. For Holstein, there are eight alleles with a frequency of more than 4%. It is alleles *03, *07, *08, *11, *16, *22, *23, *24. A high degree of consolidation of weighty alleles can be outlined. In total they occupy 84,6% of allele spectrum of the population. Consolidation of such alleles in the herd of the Ukrainian Black-and-White diary breed is much lower - only 52.2%, although it increased by 6.2% over 5 years. Alleles *10, *13 and *28 are "weighty" for the Ukrainian Black-and-White diary breed, but they are almost non-existent in Holsteins. The genetic similarity of the herd UBPD15 and Holstein increased by ΔI = 0,085, and the genetic distance between the herds of the UBPD increased by ΔD = 0,085 for 5 years. The comparison of the allele spectrum of Holstein and the Ukrainian Black-and-White diary breed shows both the accumulation and the elimination of alleles associated with high productivity. The largest consolidation is typical for alleles *24 (+ 6.75%) and *16 (+ 4.65%). The frequency of "milk" alleles *22 and *08 decreased, respectively, by 4.14 and 1.27%. Alleys, which cause low milk productivity, have the following dynamics: * 23 + 2.53%, *11 – 0.67 and *28 – 0.26. The accumulation of alleles *16 and *23 (7.18%) was found that are associated with predisposition to necrobacteriosis and elimination of *03 and *22 alleles (4.75%) that influence on this disease. Conclusions. It is determined that the role of alleles characteristic for Holstein is increasing in the the Ukrainian Black-and-White diary herd. Breeding measures for holsteinization are conducted in the right direction. There is accumulation of alleles associated with high milk productivity and predisposition to necrobacteriosis. It positively affects the growth of milk production and negatively affects the incidence of necrobacteriosis.

The Major Histocompatability Complex (MHC) determines the immune response to pathogens, and its genes are promising candidates for the search of associations with diseases. A special role is played by BoLA-DRB3 gene, the product of which directly participates in the binding of alien antigens and conditions the specificity of the immune response. The second exon of this gene codes β1-domain of class II antigens, which is necessary for binding a broad spectrum of alien antigens. Exon 2 of BoLA-DRB3 gene is extremely polymorphic, giving the possibility to search the associations of its alleles with various diseases. The article provides the results of the study on polymorphism of alleles of BoLA-DRB3.2 gene for detection of its associations with sensitivity to fusobacteriosis (necrobacteriosis) of cows. The survey was performed using PCR-RFLP method with DNA of blood from 176 cows of two herds of Ukrainian black-and-white dairy breed. As a result of the studies, in the first herd, 25 BoLA-DRB3.2 alleles were found. In the selections of nectobacteriosis susceptible and resistant cows, we found 22 and 21 variants respectively. In the second herd, in the general selection and group of healthy animals, 27 alleles were typed, and 22 in the group of susceptible cows. BoLA-DRB3.2*22 allele was the commonest in both herds in both general selections and groups of nectobacteriosis-resistant cows. In the selection of susceptible animals, the commonest was the variant BoLA-DRB3.2*16. We determined statistically significant associations of BoLA-DRB3.2 alleles with sensitivity to nectobacteriosis of cattle. BoLA-DRB3.2*03 and *22 alleles associate with nectobacteriosis-resistant, while *16 and *23 – with nectobacteriosis-susceptible cows of the both studied groups. Also, in the first herd, another allele was found – *24, indicating close relationship with the disease. The studies of polymorphism of BoLA-DRB3 gene expand the knowledge about genetic peculiarities of the Ukrainian black-and-white dairy breed. The identified molecular-genetic markers could be useful for breeders whose work is oriented towards the formation of herds which are resistant to diseases of the limbs in cattle.