谷氨酰胺替代部分甘油对冻融猪精子获能和体外受精能力的影响

Effects of Partially Replacing Glycerol with Glutamine on Capacitation and in vitro Fertilization Ability of Frozen-thawed Pig Sperm

本试验旨在探索用谷氨酰胺(Gln)替代部分甘油对冻融猪精子体外获能和受精能力的影响,试验分为6组:3%甘油对照组和5个处理组(Ⅰ~Ⅴ组:2%甘油+谷氨酰胺(0、20、40、80和100 mmol/L))。对冻融松辽黑猪精子的精子活力、质膜完整性、顶体完整性、线粒体膜电位、鱼精蛋白水平、获能及体外受精等指标进行了检测。结果显示,用谷氨酰胺替代部分甘油均对冻融精子质量有一定的改善作用,改善的程度受谷氨酰胺浓度的影响。与对照组相比,Ⅰ组精子的质量参数均显著下降(P<0.05);与Ⅰ组相比,Ⅱ组精子活力、顶体完整性和活率显著提高(P<0.05),Ⅲ组精子线粒体膜电位显著提高(P<0.05),Ⅴ组精子活力、质膜完整性、顶体完整性、活率和线粒体膜电位均显著提高(P<0.05)。说明用谷氨酰胺替代部分甘油对精子质量具有很大的影响,且当谷氨酰胺为100 mmol/L时可得到更高质量的精子,因此,后续试验使用浓度为100 mmol/L的谷氨酰胺进行研究。与对照组相比,2%甘油+100 mmol/L谷氨酰胺处理组精子鱼精蛋白缺失率显著下降(P<0.05),精子获能无显著差异(P>0.05),但胚胎卵裂率显著提高(P<0.05)。综上所述,谷氨酰胺可作为一种新型冷冻保护剂替代部分甘油来提高猪精液的质量,并降低甘油对猪精液的毒性作用,为猪精液的冷冻保存及商业化生产提供技术支撑。

The purpose of this experiment was to explore the effect of glutamine(Gln)instead of partial glycerol on capacitation and in vitro fertilization ability of frozen-thawed pig sperm.The experiment was divided into 6 groups:3%glycerol control group and 5 treatment groups(groupsⅠ-Ⅴ:2%glycerol+glutamine(0,20,40,80 and 100 mmol/L,respectively)).The sperm motility,plasma membrane integrity,acrosome integrity,mitochondrial membrane potential,protamine deficiency,capacitation and in vitro fertilization of frozen-thawed Songliao Black pig sperm were detected.The results showed that replacing part of glycerol with glutamine could improve the quality of frozen thawed sperm,and the degree of improvement was affected by the concentration of glutamine.Compared with control groupⅠ,the sperm quality parameters in groupⅠdecreased significantly(P<0.05).Compared with groupⅠ,the sperm motility,acrosome integrity and viability in groupⅡincreased significantly(P<0.05),the sperm mitochondrial membrane potential in groupⅢincreased significantly(P<0.05),and the sperm motility,plasma membrane integrity,acrosome integrity,viability and mitochondrial membrane potential in groupⅤincreased significantly(P<0.05).It showed that replacing part of glycerol with glutamine had a great impact on sperm quality,and higher quality sperm could be obtained when glutamine was 100 mmol/L.Therefore,100 mmol/L glutamine was used in subsequent experiments.Compared with control group,the sperm protamine deletion rate of 2%glycerol+100 mmol/L glutamine treatment group decreased significantly(P<0.05),there was no significant difference in sperm capacitation(P>0.05),but the cleavage rate of embryo was increased significantly(P<0.05).In conclusion,glutamine could be used as a new cryoprotectant replacing part of glycerol to improve the quality of pig semen,reduce the toxic effect of glycerol on pig semen,and provide technical support for cryopreservation and commercial production.