马精子冷冻保存方法及其冻融损伤机制的研究

With the rapid rise of China's horse industry, lacking of superior genetic breed has become the bottle-neck of industrial development. the introduction of the outstanding stallions from foreign and insemination by frozen semen is an effective way to genetic improvement. But sperm frozen technology is still immature in equine. Preliminary studies, we found that crude separation of the ingredients of the milk and egg yolks is play a important role in Sperm freezing process. Based on this, this study intends to fully explore some factors, including PH value, osmotic pressure, the proportion of sugar and salt, different animal milk, effect on sperm freezing. In order to study the impact of the different components of milk and egg yolks on sperm freeze, isolation and purification the different components of milk and egg yolks will be performance by using the isoelectric point precipitation, ultra-filtration, dialysis and other methods, aimed to develop a chemically defined extender .Further ultrastructure, integrity of plasma membrane and acrosome, enzyme activity, DNA damage of the Freezing and thawing sperm be detected to explore the mechanism of its protective role in sperm freezing process, and provide a theoretical basis for the protection of mammalian sperm frozen.

随着我国马业的迅速兴起，良种缺乏已成为产业发展的瓶颈，引进国外优秀种公马，采用冷冻精液配种实施品种改良是行之有效的方法。但从世界范围来看马精子冷冻技术还很不成熟。精子冻融后活力差、个体间冷冻效果不稳定，无法满足生产要求。前期研究的过程中我们发现，在冷冻液中加入奶和蛋黄粗分离的成分后，精子冷冻效果明显提高。基于此，本研究拟在全面探索PH值、渗透压、糖盐比例、抗氧化剂、咖啡因及不同动物奶对马精子冷冻效果影响的基础上，采用等电点沉淀、超过滤、透析等方法对奶和蛋黄中物质进行分离，分析不同成分对精子冷冻保存的影响，旨研制一种成分明确、冷冻效果稳定可靠的马精子冷冻保存液。进一步借助透射电镜、质膜顶体完整性观测、酶活性分析、DNA损伤检测等手探讨其在精子冷冻过程中发挥保护作用的机制，为哺乳动物精子冷冻保护提供理论依据。

中国是养马业大国，马匹数量居世界第二位。但由于受国家产业政策、经济条件的限制，我国马业发展相对滞后。近几年，随着马旅游业、马产品的深度开发、国家对马业支持力度的加大，我国马业已悄然兴起。现代马业的发展迫切需要采用人工授精的方式进行马匹改良，精液的体外保存是目前我国马匹人工授精推广应用过程中的技术瓶颈，本研究针对国内尚无稳定可靠的马精液低温保存稀释液和国际上马精液冷冻效果差的现状。以研制出一种效果稳定、成本相对低廉便于储存和运输马精液专用低温保存稀释液和冷冻液，在此基础上建立与之相适应的低温和冷冻保存方法为主要目标。分别研究了pH、糖、缓冲体系、奶成分、抗氧化剂、抗冻剂、抗生素、抗冻蛋白、不同卵黄成分、大豆卵磷脂及β环糊精胆固醇对精子低温和冷冻保存效果的影响，确定了适宜精液低温保存和冷冻稀释液组成成分；通过研究精清浓度、平衡时间、冷冻方法、解冻程序对精液低温和冷冻保存效果的影响，最终确定了优化的保存方法。通过受胎试验证明了稀释液和保存方法的可靠性。在这些研究的基础上运用iTRAQ技术检测精子冻融前后蛋白表达差异，探索精子冻融损伤的机制。