【摘要】 精液冷冻保存对畜禽遗传资源保护和人工授精技术推广具有十分重大的意义。但是，冷冻解冻过程严重损害精子结构功能的完整性。为了降低冷冻解冻过程对山羊精子的冷冻损伤，以新培育的云上黑山羊为实验对象，观察在冷冻稀释液中添加不同浓度[0 mM（对照组）、0.5 m M、1.0 m M、2.0 mM]的丁基羟基甲苯（BHT）对山羊精液的冷冻保存效果，评估BHT对冷冻精子运动能力、质膜和顶体完整性、ROS生成等参数的影响。采用假阴道法采集6只公羊精液，将每次采集的精液混合均匀以降低个体差异对实验结果的影响，按照BHT浓度分别加入稀释液进行精液稀释，稀释后的密度为2.0×10~8个/mL。将稀释后的精液于5℃平衡3 h后，在液氮蒸汽中预冻7 min，最后投入液氮中保存。解冻时，将冷冻细管投入37℃水浴锅中30 s。结果表明，与对照组相比，向冷冻稀释液中添加0.5 mM BHT可显著改善山羊冻精的总活力（TM）、前项运动活力（PM）和快速前项运动活力（FPM），而且，0.5 m M BHT对冻精质膜和顶体完整性的冷冻保护效果极显著高于其他处理组（P<0.01）。另外，BHT也显著抑制了精子冷冻解冻过程中ROS的产生。但是，当BHT浓度高于0.5 mM时，各项指标呈下降的趋势。综上，该研究首次证实了BHT对山羊精液的冷冻保护效果，且确定其最佳作用浓度为0.5 m M，这为改善山羊冻精品质和加快人工授精技术推广提供了必要的技术支撑。更多还原

【Abstract】 Semen cryopreservation has significant meaning for the protection of livestock genetic resources and the popularization of artificial insemination in goats.However,the integrity of spermatozoa structure and function will be seriously damaged during the process of freezing and thawing.The purpose of this experiment is to reduce the cryodamage of goat sperm during the freezing and thawing process.The newly bred Yunshang Black goat was used to observe the cryopreservative effects of butylated hydroxytoluene(BHT)with different concentrations[0 m M(control group),0.5 mM,1.0 m M,2.0 mM]on goat semen.The effects of BHT on the motility,plasma membrane and acrosome integrity and ROS production of frozen sperm were evaluated.The semen of 6 rams was collected by artificial vagina,and the semen collected each time was pooled evenly to reduce the impact of individual differences on the final experimental results.The pooled semen was diluted using the freezing extenders containing BHT to a final spermatozoa concentration of 2.0×10~8/mL.The diluted semen was equilibrated at 5℃for 3 hours.Then,the diluted semen was pre-frozen in liquid nitrogen vapor for 7 min,and finally stored in liquid nitrogen.When thawing,the frozen tube loaded with the diluted semen was thawed a 37℃water bath for 30 seconds.The result showed that in comparison with the control group,the post-thaw total motility(TM),progressive motility(PM),and rapid progressive motility(FPM)of goat spermatozoawere significantly improved by adding 0.5 mM BHT into the freezing extender.The cryopreservative effects of 0.5 mM BHT were significantly higher than the other treatments on the integrity of plasma membrane and acrosome (P<0.01).In addition,BHT could also significantly inhibit the production of ROS during the freezing and thawing process.However,when the concentration of BHT was greater than 0.5 mM,there was a reducing trend for all indexes mentioned above.In summary,this study first confirmed the cryopreservative effects of BHT on goat semen,and determined that opitimal concentration was 0.5 m M,which provided necessary technical supports for improving the quality of goat frozen semen and enhancing the extension of artificial insemination.更多还原