The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.

中文翻译：

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二维电泳结合质谱分析冷冻保存公羊精子的蛋白质组学特征

本研究的目的是使用二维电泳 (2-DE) 结合质谱分析冷冻保存过程对公羊精子蛋白质谱的影响。从五只公羊中收集精液，并在补充有甘油和蛋黄作为主要冷冻保护剂的基于 Tris 的稀释剂中冷冻保存。新鲜和解冻后的精子总蛋白被提取和纯化，然后是 2-DE。通过质谱法测定染色凝胶中的差异蛋白。结果表明，新鲜精子和冻融精子之间存在39个差异蛋白。在这些蛋白质中，新鲜精子中28种蛋白质的丰度高于解冻后精子中的蛋白质（P < 0.05）。然而，解冻后精子中的 11 种蛋白质反而被上调。基因本体（GO）分析表明，大多数差异蛋白与细胞过程、代谢和生物过程的调节有关。蛋白质-蛋白质相互作用网络表明这些差异蛋白质之间存在很强的相互作用，可能参与精子代谢、顶体功能、精子运动和降低 ROS 水平。总之，冷冻保存过程改变了公羊精子的蛋白质组，这可能与公羊精子冷冻损伤直接相关，从而影响其生育能力。此外，这些差异蛋白可用作评估冷冻公羊精液质量的生物标志物。蛋白质-蛋白质相互作用网络表明这些差异蛋白质之间存在很强的相互作用，可能参与精子代谢、顶体功能、精子运动和降低 ROS 水平。总之，冷冻保存过程改变了公羊精子的蛋白质组，这可能与公羊精子冷冻损伤直接相关，从而影响其生育能力。此外，这些差异蛋白可用作评估冷冻公羊精液质量的生物标志物。蛋白质-蛋白质相互作用网络表明这些差异蛋白质之间存在很强的相互作用，可能参与精子代谢、顶体功能、精子运动和降低 ROS 水平。总之，冷冻保存过程改变了公羊精子的蛋白质组，这可能与公羊精子冷冻损伤直接相关，从而影响其生育能力。此外，这些差异蛋白可用作评估冷冻公羊精液质量的生物标志物。