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® (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 ± 1.72%, and with Steridyl, 72.4 ± 2.10% (p < 0.01). Semen frozen in Steridyl showed good stimulation of respiration by 2.4-DNP, which indicates that oxidative phosphorylation was retained after freezing–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.
Objective:The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds.Methods: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), progressive motility after freezing (FPM), and the difference between PM and FPM. The respective DNA samples from these stallions were genotyped using Axiom™ Equine Genotyping Array. Results:We performed a GWAS search for single nucleotide polymorphism (SNP) markers and potential genes related to motility properties of frozen-thawed semen in the stallions of various breeds.As a result of the GWAS analysis, two SNP markers, rs1141327473 and rs1149048772, were identified that were associated with preservation of the frozen-thawed stallion sperm motility, the relevant putative candidate genes being NME8, OR2AP1 and OR6C4. Potential implications of effects of these genes on sperm motility are herein discussed. Conclusion:The GWAS results enabled us to localize novel SNPs and candidate genes for sperm motility in stallions. Implications of the study for horse breeding and genetics are a better understanding of genomic regions and candidate genes underlying stallion sperm quality, and improvement in horse reproduction and breeding techniques. The identified markers and genes for sperm cryotolerance and the respective genomic regions are promising candidates for further studying the biological processes in the formation and function of the stallion reproductive system.
Поиск геномных ассоциаций с качеством спермы быков голштинской и черно-пестрой породы Аннотация. Одним из важных факторов, влияющих на репродуктивный процесс, является генотип животного. В последнее время активно ведутся исследования с целью выявления полиморфных участков генома, ассоциированных с теми или иными признаками, с перспективой использования в селекции. Целью работы был поиск геномных ассоциаций с качеством спермы быков голштинской и черно-пестрой породы. В наших исследованиях проведена оценка качества спермы 129 быков голштинской и черно-пестрой породы. Были выбраны наивысшие показатели спермопродукции каждого быка по зоотехническому учету ЗАО «Невское». Были проанализированы данные за период не менее трехлетнего использования каждого быка. Максимальный дуплетный объем эякулята варьировал от 3 до 27 мл, максимальная концентрация сперматозоидов в дуплетном эякуляте от 0,7 до 2 млрд/мл, общее количество сперматозоидов в эякуляте-от 2,7 до 26,4 млрд. Дополнительно исследовали морфологию, дыхательную активность и окислительное фосфорилирование, состояние мембран сперматозоидов. Наблюдалась индивидуальная изменчивость по количеству клеток с повреждениями в области хвоста и шейки (от 0,25 до 6%). Стимуляция дыхания сукцинатом калия в сперме исследуемых быков варьировала от 1,0 до 1,3 раз, что свидетельствует о разной степени нарушений проницаемости мембран. Не характерное для свежей спермы невысокое увеличение скорости дыхания клеток после стимуляции 2,4 ДНФ в 1,5 раза у некоторых быков возможно произошло из-за нарушений сперматогенеза. Все исследуемые показатели имели индивидуальную изменчивость, что дает возможность провести поиск генетических ассоциаций с качеством спермы быков. Для проведения поиска геномных ассоциаций с качеством спермопродукции быков сформирована база данных объединяющая генотипы, определенные на чипах Illumina Bovine IBDv3 и фенотипические признаки качества спермы. По таким показателям как объем дуплетного эякулята, подвижность и общее количество сперматозоидов в дуплетном эякуляте найдены потенциальные SNP. По концентрации сперматозоидов потенциальные SNP не найдены. Отбор по генетическому полиморфизму генов, используемых в роли маркеров, позволит выделять быков с хорошим сперматогенезом и качеством спермы в раннем возрасте, до начала физиологической зрелости и получения спермы.
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To assess sperm quality, it is important to evaluate energy metabolism. The test substance 2.4-dinitrophenol (2.4-DNP) is an agent for destroying oxidative phosphorylation. 2.4-DNP shuts off the production of adenosine triphosphate (ATP) from oxidation and then, the respiration rate increases. If the respiratory chain is damaged, there is little or no response to adding 2.4-DNP. The aim of this study was to analyze the respiratory activity and oxidative phosphorylation in semen before and after freezing and compare the obtained data with the fertilizing ability of sperm. There was a reduction in sperm respiration rates in all species after thawing. The respiration of spermatozoa of boars, bulls, stallions, reindeers and chicken showed responses to 2.4-dinitrophenol. The only difference is in the strength of the response to the test substance. After freezing and thawing, respiratory stimulation by 2.4-DNP decreased. The results of our study show that respiration rate is not correlated with pregnancy rates and egg fertility. However, there was a high correlation between the stimulation of respiration by 2.4-dinitrophenol and pregnancy rates. The test for an increase in respiration rate after adding 2.4-dinitrophenol could be a suitable test of the fertilizing ability of sperm.
Damaging effects of low temperatures on the functionality of sperm is well known. Stallion sperm cryoresistance has a high individual variability. The genetic basis of cryoresistance and its heritability are little studied. The aim of the study was to search for genome-wide associations with sperm motility after freezing. 96 sperm samples were collected. The collected sperm was diluted to a final concentration 100 mln/ml and frozen in 0.5 ml straws or 18–20 ml tubes. The evaluation of semen was carried out no earlier than 24 hours after freezing. Sperm motility was assessed by computer-assisted semen analysis (CASA). Genomic DNA was purified form semen samples and genotyped using Affymetrix Equine HD array. Genotypes quality control and association studies were performed in Plink 1.9 and EMMAX software respectively. Evaluation of sperm motility showed high individual variability in both total and progressive motility after freezing. Total motility varied from 0.5 to 92.5%, and progressive motility from 0.5 to 70.8%. Sufficient associations of PTF with SNP’s were detected on chromosome (Chr) 1 (P < 1.33e-09) and 4 (P < 2.00e-09). Found SNP’s were located in genes PAS domain-containing protein 3 (Chr1) and UBAP1-MVB12 (Chr4). Expression of these genes in human body was found in cerebrum and male genitals. Suggestive SNPs were found lying nearby to genes responsible for formation of cell wall and proteins affecting mitochondrial activity. Performed studies are presenting first step of understanding genetic background of cryoresistancy of semen in horses. Found markers could be used for selection of stallions based on cryopreservation ability. Authors acknowledge financial support from Russian Science Foundation, Grant No: 18-16-00071.
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 < 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 < 0.05) with sperm concentration. Polymorphism in the INHAB gene was significantly associated (p < 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.
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