精液生产中温度管理对猪精子质量的影响

Effect of temperature management during boar semen production on sperm quality

为研究精液生产中温度管理对猪精子质量的影响及机制,将采集后的猪精液样品等分成3份并分别于17℃(对照组)、22℃(公猪站实验室温度)和37℃(用于稀释液预温的水浴锅温度)下保存24 h。在0,6,18和24 h分别取样检测猪精子活率、活力、精子质膜和顶体膜完整性、细胞内ROS水平及精子凋亡率。在保存24 h后取样检测精子p-AMPKα、LKB-1、CaMKKβ、Bax、Bcl-2、Hsp70和Hsp90蛋白表达量。结果表明,与17℃相比,22℃下保存精子活率和活力在24 h后显著下降(P<0.05)。与17℃和22℃相比,37℃下保存精子各质量参数均显著下降(P<0.05)且出现质量损伤的时间更早,细胞内ROS水平显著升高(P<0.01),保存18 h后精子早期凋亡水平显著降低(P<0.05)而晚期凋亡水平显著升高(P<0.05)。与17℃和37℃相比,22℃保存24 h后精子p-AMPKα水平显著升高(P<0.05)。与17℃和22℃相比,37℃保存24 h后AMPK上游激酶LKB-1和CaMKKβ的表达量显著降低(P<0.05),凋亡蛋白Bcl-2、Bax和Caspase-3及热休克蛋白Hsp70表达量显著下降(P<0.05)。综合以上结果,精液生产过程中17~22℃下保存不会显著影响猪精子质量,精子通过LKB-1途径激活AMPK调节凋亡和热休克蛋白的表达来应答温度的升高。37℃下保存促进了精子早期凋亡向晚期凋亡的转化,破坏了精子的调节机制,不利于精子的存活。

This study aims to investigate the effect of temperature management during boar semen production on sperm quality and its underlying mechanism.Ejaculates was collected and aliquoted into 3 parts and stored respectively at 17℃as control,22℃(temperature of AI laboratory)and 37℃(temperature of water bath used to prewarm semen extender)for 24 h.Total and progressive motility,plasmatic and acrosomal membrane integrity,intracellular ROS level and sperm apoptosis of boar semen were evaluated after storage for 0,6,18,24 h.Sperm p-AMPKα,LKB-1,CaMKKβ,Bax,Bcl-2,Caspase-3,Hsp70 and Hsp90 expression levels were determined after 24 h of storage.The results showed that storage at 22℃induced a significant decrease in sperm total and progressive motility after 24hin comparison with 17℃(P<0.05).Compared with 17℃and 22℃,storage at 37℃caused a significant decline in all sperm quality parameters(P<0.05),manifesting damage at earlier time point,a higher intracellular ROS level(P<0.05),and lower early apoptotic level(P<0.05)and higher late apoptotic level(P<0.05)after 18hof storage.Compared with 17℃and37℃,storage at 22℃for 24hinduced higher p-AMPKαlevel(P<0.05).In comparison with 17℃and 22℃,storage at 37℃for 24hcaused markable decrease in LKB-1and CaMKKβ,upstream kinases of AMPK(P<0.05),apoptosis-related proteins Bax,Bcl-2and Caspase-3(P<0.05),and Hsp70(P<0.05).Taken together,storage of boar semen at 17-22℃has no significant influence on sperm quality,within which sperm response to temperature increase through LKB-1-induced AMPK activation regulating expression levels of apoptosis-related and heat shock proteins.While storage at 37℃deteriorates sperm quality by promoting transformation of sperm early apoptosis to late apoptosis,breaking the regulatory mechanism of sperm to thermal stress.