α-酮戊二酸对奶山羊精液冷冻保存及精子受精能力的影响

Effects of α-ketoglutaric acid on the cryopreservation of dairy goat semen and sperm fertilization capacity

摘要:

本研究旨在探究α-酮戊二酸(alpha-ketoglutaric acid, AKG)对奶山羊精液冷冻保存效果的影响，并深入揭示其潜在的作用机制。选取10只健康萨能奶山羊种公羊，采集并混合活率大于80%的精液。将精液与含有不同浓度AKG (0、10、100、1 000 μg/mL)的冷冻液混合，采用液氮熏蒸法进行冷冻保存。解冻后分别检测精子运动参数、生存指数、质膜完整率、顶体完整率、抗氧化酶活性、ATP水平及凋亡发生率等关键指标。基于上述结果筛选出最佳浓度，并利用非靶向代谢组学技术分析其引起的代谢物与代谢通路变化。最后，通过体外受精和人工授精试验，对冷冻精液的受精能力进行综合评价。结果表明，与对照组相比，添加100 μg/mL AKG显著提升了冷冻-解冻后精子质量，其精子活力达到58.71%，质膜与顶体完整率分别显著提高至61.56%和62.49%。同时，该浓度的AKG显著提高了精子的总抗氧化能力、过氧化氢酶及超氧化物歧化酶活性，同时显著降低了丙二醛和活性氧水平。此外，精子内ATP水平显著升高，而早期凋亡发生率显著降低。非靶向代谢组学分析显示，100 μg/mL AKG组与对照组之间存在397种显著差异代谢物，这些代谢物主要富集于三羧酸循环、丙酮酸代谢、谷胱甘肽代谢和NF-κB信号通路等154条关键代谢通路。体外受精结果显示，AKG处理显著提高了体外受精卵裂率，但对囊胚率无显著影响，同时人工授精妊娠率显著提高(34.13% vs. 21.97%)，妊娠率提高12.16%。综上所述，在山羊精液冷冻液中添加100 μg/mL AKG可通过调控三羧酸循环、丙酮酸代谢、谷胱甘肽代谢和NF-κB信号通路等代谢过程，有效提升解冻后精子的运动能力、抗氧化能力及能量供应，并抑制凋亡发生，最终显著改善精子的受精能力与人工授精妊娠率。本研究为优化奶山羊精液冷冻保存技术提供了新的有效策略和理论依据。

Abstract:

The role of alpha-ketoglutarate (AKG), a crucial metabolic intermediate, in enhancing the cryotolerance of mammalian sperm remains poorly understood. This study systematically investigated the effects of AKG on semen cryopreservation in dairy goats and elucidated its underlying mechanisms. Semen samples were collected from 10 healthy Saanen dairy goats, pooled after screening for motility (>80%), and cryopreserved in liquid nitrogen vapor with extender supplemented with varying AKG concentrations (0, 10, 100, and 1 000 μg/mL). Post-thaw assessments included sperm motility parameters, survival index, plasma and acrosome membrane integrity, antioxidant enzyme activities, ATP levels, and apoptosis rate. The optimal AKG concentration (100 μg/mL) was selected for subsequent analysis using non-targeted metabolomics to identify altered metabolites and pathways. In vitro fertilization and artificial insemination were conducted to comprehensively assess fertilizing capacity. Compared to the control, 100 μg/mL AKG most effectively enhanced post-thaw sperm quality, improving motility to 58.71%, and plasma and acrosome integrity to 61.56% and 62.49%, respectively. This concentration significantly elevated total antioxidant capacity, catalase, and superoxide dismutase activities, while reducing malondialdehyde and reactive oxygen species levels. Additionally, ATP content increased and the early apoptosis rate decreased. Non-targeted metabolomics revealed 397 significantly differential metabolites, predominantly enriched in 154 key pathways including the tricarboxylic acid cycle, pyruvate metabolism, glutathione metabolism, and the NF-κB signaling pathway. In vitro fertilization results showed that the addition of 100 μg/mL AKG significantly improved the cleavage rate of in vitro fertilized embryos but had no significant effect on the blastocyst rate. Additionally, the pregnancy rate from artificial insemination was significantly improved (34.13% vs. 21.97%), with a 12.16% increase. In conclusion, supplementing cryopreservation extender with 100 μg/mL AKG enhances post-thaw sperm function and fertility in dairy goats by modulating critical metabolic pathways related to energy metabolism and oxidative defense. This study provides a novel strategy and theoretical foundation for optimizing semen cryopreservation.