Genetic structure and genome-wide association study of the traditional Kazakh horses.

Horses are traditionally used in Kazakhstan as a source of food and as working and saddle animals as well. Here, for the first time, microarray-based medium-density single nucleotide polymorphism (SNP) genotyping of six traditionally defined types and breeds of indigenous Kazakh horses was conducted to reveal their genetic structure and find markers associated with animal size and weight. The results showed that the predefined separation between breeds and sampled populations was not supported by the molecular data. The lack of genetic variation between breeds and populations was revealed by the principal component analysis, ADMIXTURE, and distance-based analyses, as well as the general population parameters expected and observed heterozygosity (He and Ho) and between-group fixation index (Fst). The analysis revealed that the studied types and breeds should be considered as a single breed, namely the 'Kazakh horse'. The comparison with previously published data on global horse breed diversity revealed the relatively high level of individual diversity of Kazakh horses in comparison with the well-known foreign breeds. The Mongolian and Tuva breeds were identified as the closest horse landraces, demonstrating similar patterns of internal variability. The genome-wide association analysis was performed for animal size and weight as the traits directly related with the meat productivity of horses. The analysis identified a set of 60 SNPs linked with horse genes involved in the regulation of processes of development of connective tissues and the bone system, neural system, immune system regulation, and other processes. The present study is novel and introduces Kazakh horses as a promising genetic source for horse breeding and selection both on the domestic and international levels.

Genetic polymorphism of prolactin and nitric oxide synthase in Holstein cattle.

Background and Aim: Bacterial and viral infections affect the welfare of animals and lead to large economic losses in dairy cattle breeding due to decreased productive indicators and increased culling rates. In modern dairy farming, farmers are looking for effective solutions to prevent and minimize infectious disease risks. To this end, the most relevant study field is the search for gene sites that impact production and health. This study aimed to determine the nature of the distribution of the relative frequencies of alleles and genotypes of polymorphic prolactin (PRL) and nitric oxide synthase (NOS2) in Holstein cows and identify the relationship of these genes with resistance to mastitis and bovine leukemia. Materials and Methods: For this study, we chose cows because infectious diseases affect the amount of lactation and milk quality. Holstein cattle with mastitis and bovine leukemia were selected. Animal genotypes were determined by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. The results were analyzed using a nonparametric statistical method using Microsoft Excel 2010 and Statistica 6.0. Results: In healthy animals, 94 genotypes were identified for both genes under study. For bPRL, bPRL-RsaIAA (72) was the most common genotype and bPRL-RsaIBB (4) the least; for NOS2, bNOS2 -HinfIAB (47) was the most common genotype and bNOS2 -HinfIAA the least (21). In animals with leukemia, 34 genotypes were identified. For PRL, bPRL-RsaIAA (25) was the most common genotype and bPRL-RsaIBB (2) the least; for NOS2, bNOS2 -HinfIBB (17) was the most common genotype and bNOS2 -HinfIAA (3) the least. In animals with mastitis, 67 genotypes were identified. For PRL, bPRL-RsaIAA (43) was the most common genotype and bPRL-RsaIBB (6) the least; for NOS2, bNOS2 -HinfIBB (31) was the most common genotype and bNOS2-HinfIAA (7) the least. The distribution of genotypes of polymorphic bPRL and bNOS2 generally coincides, and bPRL-RsaIBB is the most common genotype. In groups of sick animals, the number of bNOS2 -HinfIAA homozygotes was lower than that of the control group. In particular, the proportion of animals with the bNOS2 -HinfIAA genotype with bovine leukemia was 8.7% and with mastitis was 10.3% compared with 22.4% in healthy animals. These data support the possible association of the bNOS2 -HinfIAA genotype with resistance to infection. The frequency of the bPRL-RsaIB allele was higher in groups of sick animals. This allele is associated with increased milk productivity, suggesting that highly productive animals are less resistant to the incidence of viral bovine leukemia and mastitis of bacterial etiology. Conclusion: DNA amplification of Holstein cattle for the polymorphic regions of PRL and NOS2 using the PCR-RFLP method revealed a possible connection between the distribution of relative allele frequencies of bPRL and bNOS2 and resistance to viral and bacterial infections. Thus, in groups of sick animals, the frequency of bPRL-RsaIBB, associated with increased milk production compared with the theoretically calculated equilibrium value was higher and the number of homozygotes bNOS2 -HinfIAA was lower than in the control group. In conclusion, animals with increased milk production were more prone to diseases, such as mastitis and bovine leukemia.

α4-Integrin deficiency in human CD34+ cells engenders precocious erythroid differentiation but inhibits enucleation.

The functional impact of integrin expression in erythropoiesis has been previously emphasized through its decisive influence on erythroid cell-microenvironment (matrix and cellular) interactions, especially under conditions of stress. Beyond that, in several in vitro studies the relationship between the two erythroid integrins, α4 and α5, has been incongruous in terms of a proliferative support, either synergistic or antagonistic, whereas a dominant influence of α4 integrin on terminal erythropoiesis in vitro and in vivo has been consistently emphasized. However, the specific cellular and molecular details of this effect have not been defined, especially for human cells. In the study described here, we cultured human CD34+ progenitor cells with induced deficiency of α4 integrin (shRNAα4) under erythroid differentiation conditions, in which expanded erythroid progenitor cells are directed to terminal erythroid maturation stages in the absence of any microenvironmental influence. Our data indicate that early proliferative expansion in cells lacking α4 expression is significantly limited, but although erythroid cell differentiation can proceed normally to terminal stages, their enucleation is drastically impaired. This novel aspect of α4 integrin participation in the enucleation process in vitro resonates on the lack of in vivo enucleation of primitive erythroid cells lacking any integrin expression but affecting adult cells only under stress conditions.

Reappraising the role of α5 integrin and the microenvironmental support in stress erythropoiesis.

We previously studied the role of ß1 integrin and some of its different α partners relevant to erythropoiesis. Although clear and consistent answers regarding the role of α4ß1 (VLA-4) were evident, the role of its companion integrin α5ß1 (VLA-5) was clouded by inconsistent outcomes in all prior publications. Furthermore, the functional consequences of integrin deficiencies only in microenvironmental (ME) cells supporting erythroid cell expansion and maturation post stress have never been explored. In the study described here, we created several additional mouse models in the aim of addressing unanswered questions regarding functional consequences of single or combined integrin deficiencies in erythroid cells or only in ME supporting cells. Our novel and expansive data solidified the intrinsic requirement of both α4 and α5 integrins in erythroid cells for their proliferative expansion and maturation in response to stress; α5 integrin alone, deleted either early in all hematopoietic cells or only in erythroid cell, has only a redundant role in proliferative expansion and is dispensable for erythroid maturation. By contrast, α4 integrin, on its own, exerts a dominant effect on timely and optimal erythroid maturation. Deficiency of both α4 and α5 integrins in ME cells, including macrophages, does not negatively influence stress response by normal erythroid cells, in great contrast to the effect of ME cells deficient in all ß1 integrins. Collectively the present data offer deeper insight into the coordination of different ß1 integrin functional activities in erythroid cells or in ME cells for optimal erythroid stress response.

Stage-specific functional roles of integrins in murine erythropoiesis.

When the erythroid integrins α5ß1 and α4ß1 were each deleted previously at the stem cell level, they yielded distinct physiologic responses to stress by affecting erythoid expansion and terminal differentiation or only the latter, respectively. To test at what stage of differentiation the integrin effects were exerted, we created mice with α4- or α5-integrin deletions only in erythroid cells and characterized them at homeostasis and after phenylhydrazine-induced hemolytic stress. Unlike our prior data, the phenotype of mice with α5-erythroid deletions was similar to controls, especially after stress. These outcomes seem to reconcile divergent prior views on the role of α5-integrin in erythropoiesis. By contrast, α4 integrins whether deleted early or late have a dominant effect on bone marrow retention of erythroblasts and on terminal erythroid maturation at homeostasis and after stress.

Erythroid cells generated in the absence of specific ß1-integrin heterodimers accumulate reactive oxygen species at homeostasis and are unable to mount effective antioxidant defenses.

We have previously reported that ß1(Δ/Δ) mice have a markedly impaired response to hemolytic stress, but the mechanisms of this were unclear. In the present study we explored in detail quantitative, phenotypic and functional aspects of erythropoiesis at homeostasis in a large number of animals for each of 3 murine models with specific ß1 heterodimer integrin deficiencies. We found that, at homeostasis, ß1-deficient mice have a modest uncompensated anemia with ineffective erythropoiesis and decreased red blood cell survival. Mice lacking only α4 integrins (α4ß1/α4ß7) do not share this phenotype. There is an increased tendency for reactive oxygen species accumulation in ß1(Δ/Δ) erythroid cells with decreased anti-oxidant defenses at homeostasis which are exaggerated after stress. Furthermore, expansion of erythroid cells in spleen post-stress is dependent on α5ß1, likely through mechanisms activating focal adhesion kinase complexes that are distinct from α4ß1-mediated responses. In vivo inhibition of focal adhesion kinase activation partially recapitulates the ß1(Δ/Δ) stress response. Mice lacking all α4 and ß1 integrins (double knockouts) had, at homeostasis, the most severe phenotype with selective impairment of erythroid responses. The fact that integrins participate in mitigating stress in erythroid cells through redox activation of distinct signaling pathways by specific integrin heterodimers is a link that has not been appreciated until now.