抗氧化剂对马精子低温及冷冻保存效果的影响

Effects of Antioxidants on the Quality of Hypothermic Preservation and Frozen-thawed Equine Semen

试验旨在研究不同抗氧化剂对马精子低温保存及冷冻效果的影响。选取6匹英纯血种公马作为试验动物,以INRA82液作为基础稀释液(对照组),分别添加谷氨酰胺(0.015g/mL)、甘氨酸(0.019g/mL)、半胱氨酸(0.024g/mL)、甲硫氨酸(0.015g/mL)、牛磺酸(0.063g/mL)、维生素C(0.4mg/mL)、维生素E(0.5mg/mL)、褪黑素(0.001mg/mL)制备成含有不同抗氧化剂的低温保存和冷冻保存稀释液,将浓缩处理后的马精子分别置于上述稀释液中保存或冷冻,检测低温保存48h后精子运动参数,评价抗氧化剂对精子低温保存的影响;检测精子冻融后运动参数、精子质膜完整性、线粒体膜电势及顶体完整性,评价抗氧化剂对精子冷冻效果的影响。结果表明,精子低温保存48h后,添加牛磺酸组活精子比例和前向运动精子比例显著高于对照组(P<0.05),添加维生素C组前向运动精子比例显著高于对照组(P<0.05)。冷冻解冻后结果表明,添加不同抗氧化剂没有改善精子冻融后活精子比例和前向运动精子比例,但添加甲硫氨酸显著延长了精子体外存活能力(P<0.05);添加不同抗氧化剂精子质膜完整性与对照组间无显著差异(P>0.05),但甲硫氨酸和甘氨酸组有高于对照组的趋势;添加维生素E和甲硫氨酸组精子线粒体膜电势显著高于对照组(P<0.05),不同抗氧化剂组精子顶体完整性与对照组间均无显著差异(P>0.05)。结果提示,低温保存时添加牛磺酸和维生素C可以提高精液低温保存效果;冷冻时添加甲硫氨酸能提高精子质膜完整性和线粒体膜电势,且能够延长精子冻融后的存活时间。

This study was aimed to evaluate the effects of various antioxidants,namely glutamine（0.015g/mL）,glycine（0.019g/mL）,cysteine（0.024g/mL）,methionine（0.015g/mL）,taurine（0.063 g/mL）,vitamin C（0.4 mg/mL）,vitamin E（0.5 mg/mL）and melatonin（0.001mg/mL）on equine sperm quality after chill or freeze-thaw.Semen were collected from 6adult thoroughbred stallions,INRA82 was used as the base extender（control group）,adding INRA82 with different antioxidants was used in experimental group.Assess the effect of antioxidants on semen by detecting motion parameters after storage at 5 ℃for 48 h.Motion parameters,plasma membrane integrity（PMI）and mitochondrial membrane potential were used to evaluate semen quality after thawing.The extender supplemented with 25 mmol/L taurine led to higher TM and PM,and supplemented with 0.4mg/mL vitamin C obtained significant higher PM compared with control group（P〈0.05）after storage at 5 ℃ for 48 h.The freeze extender supplemented with 0.5mg/mL vitamin E or 0.015g/mL methionine significantly increased the mitochondrial membrane potential compare with control group（P〈0.05）.No significant differences were observed for PMI and acrosomes integrity rate after frozen-thawed（P〉0.05）,but there was a trend that PMI of adding methionine and glycine group was higher than control group.The results suggested that extender supplemented with taurine and vitamin C could improve the semen preservation effect,and the extender supplemented with methionine could improve plasma membrane integrity,mitochondrial membrane potential of thawing sperm,and also could prolong the survival time of frozen thawed sperm.