杜洛克猪精子体外4 ℃保存过程中活力参数及蛋白修饰变化
Research on Motility and Protein Modifications of Duroc Sperm Preserved at 4 ℃ in Vitro

为探究体外4 ℃保存对猪精子活力参数及蛋白修饰的影响,使用猪精液保存稀释液体外4 ℃保存猪精子7 d,利用计算机辅助精子活力分析仪(CASA)分析测定新鲜精液和保存7 d后精液的精子活力指标;使用相应试剂盒检测精子中ATP水平及甘油醛-3-磷酸脱氢酶(GAPDH)活性;应用蛋白免疫印迹(WB)技术,比较新鲜精液在4 ℃保存7 d后蛋白翻译后修饰水平的变化。结果表明,猪精液体外4 ℃保存7 d后,精子活力参数呈现下降趋势。其中,运动力(MOT)、快速前进性运动速率(PRO)、平均路径速度(VAP)、平均直线运动速率(VSL)与新鲜精液相比分别降低59.99%、37.03%、12.34%、18.97%,且差异显著(P<0.05);发现精子中ATP水平和GAPDH酶活性在4 ℃保存7 d后显著降低(P<0.05),即随着体外保存时间的延长,精子细胞内的能量供应不足,代谢水平降低;猪精液4 ℃保存7 d后精子蛋白的磷酸化和酰化修饰明显减弱,这一结果与精子活力、代谢酶活性结果的变化趋势一致。研究结果证实,在体外4 ℃保存条件下,可能因精子细胞内能量代谢受阻而影响了精子活力及蛋白修饰水平。因此,本研究论对于丰富精子的能量代谢基础理论,还是对于延长猪精液低温保存时间,提高人工授精效率,均具有重要的理论和实践意义。
To explore the effects of preservation at 4 ℃ on motility and protein modifications of Duroc boar sperm during liquid storage,spermatozoa were incubated in boar semen extender at 4 ℃ in vitro for 7 days.Sperm motility,metabolic enzymes activity and protein modifications were analyzed using computer-assisted sperm analysis(CASA),commercial kits and western blot,respectively.The results showed that during preservation in extender at 4 ℃ in vitro,the sperm motility parameters decreased.In particular,motility(MOT),progressive(PRO),average path velocity(VAP)and average straight line velocity(VSL)declined by 59.99%,37.03%,12.34%,18.97%,respectively(P<0.05);Moreover,sperm cellular ATP level and GAPDH activity were both significantly decreased(P<0.05).Similarly,protein phosphorylation,acetylation and succinylation were significantly reduced when sperm were incubated in media at 4 ℃(P<0.05).These molecular changes were in line with sperm motility and metabolic enzymes activity.The results suggested that preservation at 4 ℃ may decrease sperm motility by inhibiting the energy metabolism through protein modifications.Therefore,the present study is of theoretical and practical significance for understanding the basic energy metabolism theory,extending the preservation of boar semen in vitro and improving the efficiency of artificial insemination.