平衡时间、冷冻方法及解冻程序对马精液冷冻效果的影响  

The Effect of Equilibrium Time and Freezing-thawing Method on Equine (Equus caballus) Sperm Cryopreservation

作　　者：毋状元[1] 郑新宝[1] 霍飞[1] 董红[1] 罗永明[1] 于维浩 徐文慧 陈静波[1] 

机构地区：[1]新疆畜牧科学院畜牧研究所,乌鲁木齐830011 [2]昭苏县畜牧兽医局,伊犁昭苏835600

出　　处：《动物学杂志》2016年第6期1092-1100,共9页Chinese Journal of Zoology

基　　金：国家自然科学基金项目(No.31360550)

摘　　要：精液平衡、冷冻及解冻是冻精制作过程中三个必不可少的环节,对精液冷冻效果起着决定性作用。在马（Equus caballus）精子冷冻中针对这三个过程的研究较少,为进一步优化马精液冷冻方法,提高精液冻后质量,本研究比较不同平衡时间、冷冻方法及解冻程序对冻融后精子运动参数、质膜完整性及线粒体膜电势的影响。平衡120 min、180 min和240 min组冻融后精液活力及质膜完整性明显高于平衡0 min、45 min、90 min及8 h平衡组;距离液氮面2 cm和4 cm高度熏蒸冷冻获得了与程序冷冻仪冷冻法相似的冷冻效果;采用高温瞬时解冻法（75℃7 s和46℃20 s）比常规方法（37℃30 s）获得了更高的冻后精液活力（P〈0.05）。综合上述结果,在马精液冷冻过程中综合采用120-240 min平衡,距离液氮面2-4 cm熏蒸法和高温瞬时解冻法（75℃7s和46℃20 s）可获得更好的精液冷冻效果。Equilibrium, freezing and thawing are three essential steps which play a critical role in semen freezing process. In order to optimize Horse （Equus caballus） sperm freezing methods, we investigated the impact of the equilibrium, freezing and thawing manipulations on sperm motility, membrane integrity and mitochondrial membrane potential after freeze-thawing. Semen was collected from 4 adult stallions, diluted in INRA82 ＋ 5% clarified egg yolk ＋ 3.5% combined cryoprotectant and then frozen. In excrement 1, we compared the effect of equilibration for 0, 45, 90, 120, 180, 240 min and 8 h on equine sperm cryopreservation; in experiment 2, we aimed to find a suitable height of liquid nitrogen steam for freezing; in exprement 3, we assessed the effect of different thawing procedures on post-thaw semen quality. Statistical analysis was conducted with ANOVA SPSS 13. Differences between means of parameters were subjected to an analysis of variance using the Tukey′s test, with P 〈 0.05 considered significant. The results showed that the equilibrium time of 120, 180 and 240 min resulted in significantly higher sperm motility and membrane integrity than the equilibrium for 0, 45, 90 min and 8 h group after thawing （Fig. 2）; liquid nitrogen steam frozen at 2 cm and 4 cm from liquid nitrogen surface obtained similar results with program freezing method （Fig. 3）; high temperature rapid thawing method （75℃ 7 s and 46℃ 20 s） gained a higher post-thaw motility than the conventional thawing method （37℃ 30 s） （Fig. 4）. In summary, we believe that combination of equilibrium for 120﹣240 min, liquid nitrogen steam frozen at 2 cm and 4 cm from liquid nitrogen surface and high-temperature rapid thawing method （75℃ 7 s and 46℃ 20 s） can get better effect in horse sperm freezing process.

关 键 词：马 精液冷冻 精液平衡 熏蒸法 解冻 

分 类 号：S821[农业科学—畜牧学]