Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Reservation and Utilization, Ministry of Education and Sichuan Province, Southwest Minzu University, Chengdu, 610041, China; Key Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu, 610041, China.
Longri Breeding Stock Farm of Sichuan Province, Dujiangyan, 611800, China.
Theriogenology. 2024 Feb;215:249-258. doi: 10.1016/j.theriogenology.2023.12.011. Epub 2023 Dec 12.
Sperm survival and activity depend on the provision of energy and nutrients from seminal plasma (SP). This study aimed to investigate the variations of metabolites within SP before and after freezing and subsequently explore the potential regulatory mechanisms affecting yak sperm cryodamage due to changes in metabolites in the SP. Untargeted metabolomics analysis was performed to screen for differential metabolites, followed by KEGG analysis to identify enriched signaling pathways. The combinatorial analysis of metabolomics and sperm proteomics revealed the influence of key SP metabolites on sperm proteins. Subsequently, the relevant differentially expressed proteins were verified by Western blot analysis. Finally, the mechanism underlying the positive effect of galactose on sperm motility was determined by assessing the change in ATP content in sperm before and after freezing and thawing. The data showed that a total of 425 and 269 metabolites were identified in the positive and negative ion modes, respectively. Freezing and thawing resulted in the up-regulation of 70 metabolites and the down-regulation of 29 metabolites in SP. The primary impact of freezing and thawing was observed in carbohydrate metabolism, including pyruvate metabolism, pentose phosphate pathway, galactose metabolism, the TCA cycle, and butanoate metabolism. In the combined analysis and Western blot results, a significant positive correlation was observed between galactose and Aldo-keto reductase family 1 member B1 (AKR1B1) (P < 0.05), which has the ability to convert galactose into galactol. Furthermore, the addition of galactose to thawed semen improved sperm motility by increasing AKR1B1 protein in sperm and was associated with the content of ATP. These data identify differential metabolites between fresh and frozen-thawed SP and suggest that galactose is a valuable additive for cryopreserved sperm, providing a theoretical basis for further exploration of the refrigerant formula for yak sperm cryopreservation.
精子的存活和活力依赖于精浆（SP）提供的能量和营养物质。本研究旨在探讨冷冻前后 SP 中代谢物的变化，并探索由于 SP 中代谢物的变化而影响牦牛精子冷冻损伤的潜在调节机制。采用非靶向代谢组学分析筛选差异代谢物，然后进行 KEGG 分析以鉴定富集的信号通路。代谢组学和精子蛋白质组学的组合分析揭示了关键 SP 代谢物对精子蛋白质的影响。随后，通过 Western blot 分析验证了相关差异表达蛋白。最后，通过评估冷冻和解冻前后精子中 ATP 含量的变化，确定了半乳糖对精子活力的积极影响的机制。数据显示，在正离子模式和负离子模式下分别鉴定出 425 种和 269 种代谢物。冷冻和解冻导致 SP 中 70 种代谢物上调和 29 种代谢物下调。冷冻和解冻的主要影响发生在碳水化合物代谢中，包括丙酮酸代谢、戊糖磷酸途径、半乳糖代谢、三羧酸循环和丁酸盐代谢。在联合分析和 Western blot 结果中，观察到半乳糖与醛酮还原酶家族 1 成员 B1（AKR1B1）之间存在显著的正相关（P<0.05），AKR1B1 具有将半乳糖转化为半乳糖醇的能力。此外，在解冻精液中添加半乳糖可通过增加精子中的 AKR1B1 蛋白来提高精子活力，并与 ATP 含量相关。这些数据确定了新鲜和冷冻解冻 SP 之间的差异代谢物，并表明半乳糖是冷冻保存精子的有价值的添加剂，为进一步探索牦牛精子冷冻保存的制冷剂配方提供了理论依据。
