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The effect of polymorphic variants of the LCORL gene in many species of farm animals was revealed. It is believed that the LCORL gene is associated with skeleton sizes in chickens, but has not yet been adequately studied. Therefore, we are faced with the task of finding new polymorphic variants in the LCORL gene in gene pool breeds. The aim of the work is to search for and analyze polymorphic variants in the LCORL gene using Senger sequencing in breeds of various types of productivity. Methodology and methods. The studies were carried out on the basis of the laboratory of molecular genetics of All-Russian Scientific Research Institute of Genetics and Breeding of Agricultural Animals. The object of the experiment was populations of 4 breeds of chickens of different directions in productivity of the All-Russian Scientific Research Institute of Genetics and Breeding of Agricultural Animals Biological Resource Collection “Genetic Collection of Rare and Endangered Breeds of Chickens” (Pushkin, St. Petersburg): Kornish, Kitayskaya shelkovaya, Ital’yanskaya kuropatchataya, Pushkinskaya. 61 DNA samples served as the material for the study. amplification was performed on a Thermal Cycler T100 instrument (Bio-Rad, USA). Nucleotide sequences were determined on an Applied Biosystems 3500 automated sequencer (Thermo Fisher Scientific Inc., USA) at the All-Russian Scientific Research Institute of Genetics and Breeding of Agricultural Animals Laboratory of Molecular Genetics. The Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) was used for sequencing. Biometric data processing was performed using Microsoft Excel. Results. The study revealed genetic variation in polymorphic variants A30G, G82C, G236T, A342G, A450C, A503G, A508G in the intron of the LCORL gene of chicken populations of different directions of productivity. The results suggest that the polymorphic A30G variant of the LCORL gene can be considered as a DNA marker for the sign of “skeleton size” in chickens.

Genetic resistance to sperm cryopreservation is an important factor in the sires selection. In our study, we studied the cryoresistance of the sperm in Gallus gallus, Bos taurus, Equus ferus caballus. Sperm quality was assessed using computer-assisted semen analysis (CASA). Genetic studies were performed using Affymetrix Axiom®600k Array chips for cocks, Affymetrix Axiom®600k Array for stallions and BovineSNP50 for bulls. For the analysis of genotypes, the GWAS (Genome-Wide Association Studies) was used. The calculations were carried out using the PLINK and EMMAX programs. The candidate genes found as a result of our work were compared with the functions of orthologous genes, according to the publications of other researchers. As a result of comparing different animal species, a common candidate gene was found, localized on chromosome 3 (Gallus gallus) and on BTA9 (Bos taurus) BCKDHB gene encodes the beta subunit E1 of branched chain keto acid dehydrogenase, which is a multienzyme complex associated with the inner membrane of mitochondria. In roosters, it was associated with the preservation of sperm motility after cryopreservation, and it was found in the analysis of the integrity of the sperm head in bulls. The functioning of genes associated with meiosis and sperm maturation is an important condition for maintaining its quality after freezing. The work of genes responsible for the formation of membranes and various cellular structures associated with the functions of spermatozoa is also an important factor in cryoresistance. Another important condition for the functioning of the sperm is the expression of genes that prepare the sperm for fertilization. These are calcium metabolism genes involved in preparation for capacitation. Changes in the structure of proteins can lead to premature capacitation and disruption of sperm integrity. The found molecular genetic markers will be used to select sires whose sperm are suitable for freezing. Project of RSF № 18-16-00071.

Reindeer are one of the main representatives of the ecosystem of the Arctic zone and the only possible object of economic activity of the indigenous peoples of the polar regions of Russia. Reindeer meat is a valuable source of complete protein in the diet of the Evenks, Nenets, Yakuts and other peoples of the Far North. Four breeds of reindeer have been bred in Russia - Nenets, Evenk, Chukchi and Even. Evenk and Nenets are the most numerous. They live in different climatic zones and differ significantly in phenotypic characteristics (size and body weight). Populations of wild reindeer are also widespread. The problem of increasing the meat productivity of the main breeds of domesticated reindeer can be solved by searching for casual polymorphisms in candidate genes responsible for the growth and development of muscle tissue. Thus, polymorphism of the LCORL gene (mRNA ligand-dependent nuclear receptor of the corepressor type, which is a transcription factor) is associated with body weight and size in many mammals. We sequenced exon 7 of the LCORL gene and found seven SNPs, six of which are nonsynonymous and lead to a change in the amino acid composition of the protein. There was a significant difference in the occurrence of wild and mutant alleles in the three analyzed populations (wild reindeer, Nenets breed, Evenk breed), which indicates the presence of selection pressure on the LСORL gene region, and also indirectly confirms the significance of the polymorphism of this locus for phenotypic variability in height and size of the reindeer. Studies of associations of LCORL gene polymorphisms on live weight and growth in other animal species suggest that some of the found SNPs may be true causal quantitative trait loci (QTL) in domestic reindeer breeds. Project No. 0045-2021-0010.

Preservation of the genetic material of unique breeds of hens involves the use of semen cryobanks. The aim of the study was to search for genomic associations with rooster sperm quality after freezing. We took into account the decrease in sperm motility after thawing. Genotyping of 96 individuals (roosters) using the Affymetrix Axiom®600k Array chip was performed. Genomic data were obtained using the Plink 1.9 software. DNA samples were taken with genotyping quality at SNP loci more than 95%, SNP with MAF > 0.01. GWAS was performed using EMMAX. SNP information in the corresponding genes was determined using the genomic browsers NCBI and Ensembl. Assessment of rooster sperm quality showed high individual variability in total motility before freezing (20–95%), and after freezing (5–70%). Significant SNPs were detected on the chromosomes GGA1 (rs315598192, rs316943858), GGA2 (rs312981435), GGA3 (rs13694743), GGA11 (rs314024471), GGA19 (rs316346648), GGA22 (rs31797024248, rga31780824824, gga22 (rs3178082424). Candidate genes ZC3HC1, CACNA2D1, BCKDHB, AMFR, and GOSR1 were identified. The annotated genes were associated with cell cycle regulation, the formation of calcium cytoskeleton, and the formation of a multienzyme complex associated with the inner membrane of mitochondria, glycosylated transmembrane receptor. The experimental data and results can be used to study the localization of regions and genes associated with phenotypic traits of frozen sperm quality. Authors acknowledge financial support from Russian Science Foundation № 18-16-00071.

The aim of the work was to study the relationship of testosterone and estradiol with the biochemical parameters of blood. The blood of 39 stallions aged 3 to 20 years was collected during the breeding season (February–May). Determination of testosterone and estradiol in blood plasma was carried out on a chemiluminescent analyzer Immulite 2000 (Siemens Healthcare Diagnostics Inc., USA). The concentration of HDL and LDL cholesterol and the activity of alkaline phosphatase (ALP) were determined using an automatic biochemical analyzer Vegasys (Analyzer Medical System, Italy), the concentration of total protein, albumins, globulins, glucose, urea, cholesterol, triglycerides, total bilirubin, creatinine, the activity of the enzymes alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltranspeptidase were determined using a biochemical analyzer AU 680 (Beckman Coulter, USA). Spearman’s coefficient was used to estimate the rank correlation. Differences were considered statistically significant at P < 0.05. A significant positive correlation between the concentration of testosterone and the activity of ALP was found (r=0.34; P < 0.05). We found a positive correlation between the concentration of estradiol with the activity of ALP (r=0.44; P < 0.01) and the concentration of HDL cholesterol (r=0.31; P < 0.05), as well as a negative correlation between the concentration of estradiol and glucose (r=-0.32; P < 0.05). We assume that estradiol and testosterone induce the synthesis of alkaline phosphatase through the activation of osteoblastic activity. Estradiol is also able to lower blood glucose level. Among the mechanisms of this phenomenon described in the literature, there is an increase in the expression of GLUT4 and their translocation to the cell membrane, the induction of glycolysis enzymes, the pentose-phosphate cycle and glycogen synthesis. We also assume that estradiol increases HDL cholesterol in the blood by increasing the rate of HDL formation. Authors acknowledge financial support from Russian Science Foundation, Grant No: 20-16-00101

Currently, there is virtually no information on genetic factors affecting the yield of allanto-amniotic fluid, which is the raw material for the production of human and animal vaccines. Association studies including this trait are beneficial for increasing productivity of a biotechnological line of chickens used for the production of ‘Clean Eggs’. We examined here a population of the Russian White breed for the effects of indels in the prolactin (PRL) and dopamine receptor D2 (DRD2) genes on the yield of extraembryonic fluid (YEF) and embryo weight at 12.5 days of development. A 24-bp insertion in the 5' flanking region of the PRL gene significantly (P

The fertilizing ability of stallion sperm after freezing is lower than in other species. The search for the optimal extender, combination of extenders, and the freezing protocol is relevant. The aim of this study was to compare lactose-chelate-citrate-yolk (LCCY) extender, usually used in Russia, and Steridyl&reg, (Minitube) for freezing sperm of stallions. Steridyl is a concentrated extender medium for freezing ruminant semen. It already contains sterilized egg yolk. Semen was collected from nine stallions, aged from 7 to 12 years old. The total and progressive motility of sperm frozen in Steridyl was significantly higher than in semen frozen in LCCY. The number of spermatozoa with normal morphology in samples frozen in LCCY was 60.4 &plusmn, 1.72%, and with Steridyl, 72.4 &plusmn, 2.10% (p &lt, 0.01). Semen frozen in Steridyl showed good stimulation of respiration by 2.4-DNP, which indicates that oxidative phosphorylation was retained after freezing&ndash, thawing. No differences among the extenders were seen with the DNA integrity of spermatozoa. Six out of ten (60%) mares were pregnant after artificial insemination (AI) by LCCY frozen semen, and 9/12 (75%) by Steridyl frozen semen. No differences among extenders were seen in pregnancy rate. In conclusion, Steridyl was proven to be a good diluent for freezing stallion semen, even though it was developed for ruminants.

Dysferlin is associated with the formation of the muscle membrane, is involved in the repair of the muscle membrane and the regulation of myoblast fusion. In humans, mutations in the dysferlin gene lead to various types of muscle pathologies. The effect of dysferlin on the formation of muscle structures was detected in the early stages of embryonic development. We have suggested that mutations in the dysferlin gene in chickens can have a negative effect on the formation of muscle mass and meat traits of chickens. SNP rs16455118 was studied on chickens of different breeds and hybrids of broilers and layers. Body weight was controlled in 7, 49 and 330 days. Layers were used as a contrast. Standard indicators of egg traits (age of the first egg laying, egg production for 6 months, egg weight) were studied. SNP rs16455118 leads to the replacement of adenine by cytosine in the intron of the dysferlin gene. The GWAS method was used. As a result, the association of SNP in the dysferlin gene with meat traits in chickens was not found. But a statistically significant association of certain genotypes with egg trait in Russian white chickens was determined. Chickens with the AA and AC genotypes had more eggs compared to their peers with the SS genotype during the reference period. The difference was 10 eggs an average of (P ≤ 0.05). Project number: AAAA-A18-118021590138-1

The formation and functioning of the animal reproductive system occurs as a result of the coordinated interaction of a wide range of genes. To search for causal mutations, work was carried out to search for polymorphic variants in the marker regions detected using GWAS. The four single-nucleotide substitutions in the exon of the GRM8 gene identified during the studies and the association of these SNPs with sperm quality was carried out. Semen from 22 stallions was collected. Sperm volume, concentration and progressive motility were assessed. Sequencing of the sections of the candidate GRM8 gene was carried out using an Applied Biosystems 3500 genetic analyzer. For the rs1138419111 genotype, no significant differences were found in the studied parameters. According to the identified single nucleotide substitution rs1147388106, the highest ejaculate volume was in stallions with the GG genotype (55.9±26.5 ml) compared to stallions with the GA genotypes (32.5±13.9 ml) and AA (18.0±33,6) (p < 0.05). When analyzing data on SNP rs395286150, stallions with a heterozygous CT genotype had the best sperm quality. Thus, the cell concentration was 317.0±66.5 million/ml in stallions with the CT genotype, 209.6±58.2 and 189.5±74.9 % with the CC and TT genotypes, respectively (P < 0.05). The progressive sperm motility of stallions with the CT genotype was 65.5±20.5% versus 48.7±22.0% in stallions with the TT genotype and 48.4±18.6% with CC. Analysis of data on SNP rs394524550 revealed a significant effect of the genotype on progressive motility. Stallions with the AG genotype had a progressive motility of 64.6±16.3%, and those with GG and AA 32.7±15.7 and 49.6±18.1%, respectively (P < 0.05). Thus, as a result four single nucleotide polymorphisms were identified in the exon of the GRM gene. Three of them were significantly associated with such indicators of sperm quality as ejaculate volume, concentration and progressive motility. Project No. 0445-2021-0011.

The aim of the study was to search for new mutations in the previously studied gene loci of follicle-stimulating hormone receptor (FSHR), inhibin α (INHA), inhibin β A (INHAB), prolactin (PRL), transition protein 2 (TNP2), and sperm flagella 2 (SPEF2) by sequencing, as well as the search for associations of previously identified mutations at these loci with fresh semen quality in Russian Holstein bulls. Phenotypic data from 189 bulls was collected. Data was analyzed for most bulls for three years of semen collection. The maximum value of each semen quality indicator (doublet ejaculate volume, sperm concentration, progressive motility and total number of spermatozoa) were selected. SNPs were identified in the FSHR, INHA, INHAB, TNP2, SPEF2 genes. The PRL gene did not have polymorphism. Significant (p &lt, 0.05) associations of polymorphisms in the FSHR gene with double ejaculate volume, concentration and total number of spermatozoa were identified. Polymorphism in the INHA gene was significantly associated (p &lt, 0.05) with sperm concentration. Polymorphism in the INHAB gene was significantly associated (p &lt, 0.05) with doublet ejaculate volume and total number of spermatozoa. Polymorphisms in the TNP2 and SPEF2 genes did not have significant associations with semen quality. The SNPs studied in our pilot work may be considered as candidate genetic markers in the selection of bulls.

In the northern part of Russia, there are wild and domestic reindeer. There are four breeds of domestic reindeer in Russia: Nenets, Evenki, Even and Chukchi. The breeds are adapted to various climatic zones of habitat (tundra or taiga). Breeds vary in growth and live weight, as well as in areas of application. The aim was to study the polymorphism of the gene for growth hormone in the reindeer. The DNA was extracted by the phenolic method using the standard method using proteinase K from blood and tissues. Specially designed primers were used to amplify the site of growth hormone gene. As a result of amplification, a 422 bp fragment was obtained covering the region of the somatotropin gene containing 2 and 3 exons. The resulting amplification was sequenced on an Applied Biosystems 3500 Genetic Analyzer using the BigDye® Terminator v3.1 Sequencing Standard Kit according to the protocol. Four single-nucleotide polymorphisms of C12T, C72T, A122G, A235G were detected in the investigated region of the growth hormone gene (somatotropin). The positions of the found SNPs were determined from a similar section of the gene in a red deer (Cervus elaphus), the genome of which was completely sequenced. Two SNPs are in the intron, two are in the exon. The frequencies of genotypes and alleles were calculated in the six studied populations of domestic and wild reindeer. The results obtained in the study make it possible to draw conclusions about the genetic difference between the populations of wild and domestic reindeer of Russia, and between the domestic reindeer of different breeds. Theme АААА-А-18-118021590138-1.

The formation and functioning of the animal reproductive system occurs as a result of the coordinated interaction of a wide range of genes. The hormone inhibin (INHA) is a marker of Sertoli cell function and exocrine testicular function (spermatogenesis state). The hormone prolactin (PRL) is a regulatory factor in the male reproductive system and affects gametogenesis. The aim of the work was to search for associations of inhibin alpha (INHA) and prolactin (PRL) gens polymorphism in the population of Russian Holstein bulls. Sperm traits (doublet ejaculate volume, concentration, progressive motility) were evaluated over three years of bull use and the highest rates for each trait were selected. 32 bulls had contrasting traits. DNA was isolated from semen samples. Samples were sequenced using the Applied Biosystems 3500 Genetic Analyzer using the BigDye® Terminator v3.1 Sequencing Standard Kit according to the protocol. Genotyping was then performed by PCR-RFLP. Data was analyzed using ANOVA. All pairwise multiple comparison between means were conducted by t-test. No polymorphism was detected in two regions of the PRL gene. The frequency of genotype AA in INHA gene was 0.813, the genotype AG -0.187. The GG genotype has not been identified. Bulls with the genotype AG had a higher concentration of cells (1.64 ± 0.093 billion/ml) than bulls with the genotype AA (1.35 ± 0.058) (P˂0.05). No significant differences were found in the volume of ejaculate and motility. But bulls with the AG genotype had greater ejaculate volume and motility than bulls with the AA genotype. The study of polymorphism in the genes that determine the formation and functioning of the reproductive system will help to understand the basics of these processes, as well as use them as molecular genetic markers for culling young bulls according to sperm quality. Project № АААА-А18-118021990006-9.

Reindeer herding is a traditional activity of the indigenous peoples of the Arctic. Assisted reproductive technology in reindeer herding are under development. This is due to the difficulty of collecting reindeer sperm (Rangifer tarandus) in the Arctic. The aim of the work was to study the influence of age and rut period on the quality of reindeer (Rangifer tarandus) sperm. Semen was collected in Taimyr in autumn 2017, 2018. Ejaculate volume, total (TM) and progressive motility (PM) and sperm concentration were evaluated. TM, PM and sperm concentration were measured by CASA (computer-assisted semen analysis). Sperm was not found in caudae epididymidae in males at the age of six months. The sperm was collected only from caudae epididymidae from males aged 1.5 and 2.5 years. Collecting of ejaculated semen is possible in males from 3.5 years of age. The volume of ejaculate increased from 0.3±0.09 ml (mean±SD) at the age of 3.5 years to 0.7±0.09 ml at the age of 5.5 years and older (P < 0.05). Concentration of cells increased by 2 times in older males. It is known that reindeer sperm can be collected only during the rut. At the beginning and end of the rut, the collection of sperm by electroejaculation was not always successful, sperm was collected in 3 of 10 cases, while in the middle of the rut in 9 of 10 cases. There was no significant effect of the rut period on the ejaculate volume and sperm concentration. The lowest TM (51.1±2.16%) and PM (33.5±7.90%) were at the beginning of the rut, the highest at the end (73.0±5.02% and 59.0 ± 6,30%) (P < 0.01). Thus, the effect of age and rutting period on the reindeer sperm was observed. Authors acknowledge financial support from Russian Science Foundation, Grant No:17-16-01023.

The biochemical processes associated with the generation of energy (ATP) - oxidative phosphorylation and glycolysis - occupy a central place in the vital activity of cells. Numerous studies have es...

The aim of the work was compare the concentration the of toxic and essential elements in the sperm plasma and blood serum of stallions. Sperm plasma and serum were collected from 39 Arabian stallions, aged 5 to 15 years. Concentration of toxic elements [aluminum (Al), arsenic (As), cadmium (Cd), lead (Pb), tin (Sn), mercury (Hg)] and essential elements (boron (B), lithium (Li), nickel (Ni), strontium (Sr) and vanadium (V)) in the sperm plasma and serum were determined by atomic emission and mass spectrometry (AES-ICP and MS-ICP) using an mass spectrometer Elan 9000 (Perkin Elmer, USA) and an atomic emission spectrometer Optima 2000V (Perkin Elmer, USA). It is established that the concentration of toxic and essential elements in the sperm plasma is higher than in serum. The greatest difference between the average concentrations of elements in the sperm plasma and serum was set for Sr - 252.9 times, Hg - 85.1 times and Al - 20.7 times. The minimum difference between the average concentrations in the sperm plasma and serum was set for B - 0.3 times, Li - 1.1 times and As - 1.8 times. For the remaining elements, the difference between the averages was in the range 2.7 - 8.5 times. It can be assumed that a greater amount of toxic and essential elements accumulate in the accessory gonads and enter the sperm during ejaculation. Authors acknowledge financial support from Russian Science Foundation, Grant No: 17-16-01109, development program of Bioresource collections “Cryobank of genetic recourses the All-Russian Research Institute for Horse Breeding.”