白藜芦醇对山羊冷冻精子质膜、DNA完整性和温度耐受性的影响

Effects of Resveratrol on Plasma Membrane,DNA Integrity, and Thermal Tolerance of Frozen Goat Sperm

试验旨在研究白藜芦醇对山羊冷冻精子质膜、DNA完整性和温度耐受性的影响。采用假阴道法采集8只云上黑山羊精液,用含不同浓度(0、0.1、1、10和20μmol//L)白藜芦醇的Optidyl稀释液稀释后进行细管分装,对照组不添加白藜芦醇。在5℃平衡4 h后,将细管于液氮蒸气中预冻10 min,最后在液氮中保存30 d。37℃水浴解冻后,采用低渗耐受性试验检测质膜完整性和温度耐受性,精子染色质扩散法检测DNA碎片率等指标。结果显示,10μmol/L白藜芦醇冷冻组精子弯尾率显著高于其他各处理组(P<0.05),而其他各冷冻组之间无显著性差异(P>0.05);10μmol/L白藜芦醇冷冻组精子DNA碎片率极显著高于鲜精组(P<0.01),极显著低于未添加白藜芦醇冷冻组(P<0.01)。温度耐受性试验结果表明,不同浓度白藜芦醇冷冻组精子37℃水浴1 h后的精子弯尾率以10μmol/L冷冻组最高,与其他各冷冻组间差异极显著(P<0.01);精子弯尾率随着孵育时间的延长而呈逐步下降的趋势,当孵育4 h时,10μmol/L白藜芦醇冷冻组精子弯尾率与对照组无显著差异(P>0.05)。透射电镜结果表明,10μmol/L白藜芦醇组精子质膜完整率显著高于不添加白藜芦醇对照组(P<0.05)。上述结果表明,在冷冻稀释液中添加白藜芦醇可显著改善山羊冻精质膜状态、DNA完整率和温度耐受性,其最佳作用浓度为10μmol/L,但白藜芦醇是否能改善山羊冻精的人工授精效果还有待进一步研究。

The aim of this study was to investigate the effects of resveratrol on plasma membrane,DNA integrity and thermal tolerance of frozen goat sperm.Semen was collected from 8 Yunshang Black goats using artificial vagina.Semen was diluted with the Optidyl extender supplemented with resveratrol at 0,0.1,1,10 and 20μmol/L.Then the diluted semen was floaded into IMV plastic straw.The semen frozen without resveratrol was used as control group.After equilibration at 5℃for 4 h,the straws were pre-frozen in liquid nitrogen vapor for 10 min,followed by storage in liquid nitrogen for 30 d.After thawing in 37℃water bath,the plasma membrane integrity and temperature tolerance were measured by low osmolality tolerance test and sperm chromatin diffusion method was used to detect DNA fragmentation rate.The results showed that the rate of sperm with curled tail in 10μmol/L resveratrol freezing group was significantly higher than that in other groups(P<0.05),but there was no significant difference in other groups(P>0.05).The DNA fragmentation rate of 10μmol/L resveratrol freezing group was extremely significantly higher than that of fresh sperm(P<0.01),and was extremely significantly lower than that in non-resveratrol control group(P<0.01).The results of thermal tolerance test showed that the sperm tail-bending rate was the highest when sperm frozen with 10μmol/L resveratrol were equilibrated in 37℃water bath for 1 h,and there was extremely significant difference between 10μmol/L resveratrol freezing group and the other freezing groups(P<0.01).The rate of sperm with curled tail decreased gradually with prolonging of the incubation time.After incubation for 4 h,there was no significant difference in the rate of sperm with curled tail between 10μmol/L resveratrol freezing group and control group(P>0.05).The rate of sperm with intact plasma membrane in 10μmol/L resveratrol freezing group was significantly higher than that in control group(P<0.05).In conclusion,resveratrol could improve the plasma membrane status,DNA integrity and thermal tolerance of frozen goat semen.The optimally functional concentration of resveratrol was 10μmol/L.However,whether resveratrol could improve the effect of artificial insemination of frozen goat semen remains to be studied.