胆固醇对内蒙古绒山羊精子冷冻保存的影响

Effect of cholesterol supplementation on cryosurvival of Inner Mongolia cashmere goats

为了探究胆固醇对内蒙古绒山羊精子冷冻保存过程中酪氨酸磷酸化水平的影响,试验在精液冷冻前用不同浓度(1,2,3,4 mg/mL)胆固醇-环糊精(CLC)进行处理,精液冷冻解冻后用计算机辅助精子分析系统(CASAS)检测精子活力,用流式细胞仪检测精子的DNA完整性、顶体和质膜完整性及游离Ca^(2+)水平,采用Western blot检测精子蛋白酪氨酸磷酸化水平。结果表明:与对照组相比,CLC组精液冷冻解冻后精子活力、精子顶体和质膜的完整性显著提高(P<0.05);CLC组的精子DNA完整性极显著高于对照组(P<0.01);2 mg/mL CLC组的精子细胞中游离Ca^(2+)水平显著低于对照组(P<0.05);Western blot结果显示,CLC处理明显降低了解冻后精子中30,37,40 kD的蛋白酪氨酸磷酸化水平。说明精子冷冻前用CLC处理能够降低内蒙古绒山羊精子蛋白酪氨酸磷酸化水平,提高精液冷冻保存效果。

The aim of this study was to investigate the effect of cholesterol on the level of tyrosine phosphorylation of Inner Mongolian cashmere goat semen during cryopreservation.In the experiment,the spermatozoa were treated with different concentrations(1 mg/mL,2 mg/mL,3 mg/mL and 4 mg/mL)of cholesterol-cyclodextrin(CLC)before cryopreservation,the viability of frozen-thawed spermatozoa was detected using the computer-assisted semen analysis system(CASAS),the DNA integrity,acrosome and plasma membrane integrity,and free Ca^(2+)level and the sperm protein tyrosine phosphorylation level of frozen-thawed spermatozoa were detected by flow cytometry and Western blot,respectively.The results showed that the CLC supplementation significantly improved the viability,acrosome and plasma membrane integrity(P<0.05)and the DNA integrity of frozen-thawed sperms in CLC groups(P<0.01),compared with the control group;The free Ca^(2+)levels in the sperms of the 2 mg/mL CLC group were significantly lower than that of the control group(P<0.05);the CLC treatment significantly reduced the protein tyrosine phosphorylation level of 30 kD、37 kD、40 kD in frozen-thawed sperms.It could be deduced that the CLC supplementation in Inner Mongolian cashmere goat semen before cryopreservation could reduce the level of sperm protein tyrosine phosphorylation,and improve the cryosurvival of sperms.