解冻稀释液中添加精浆对冻融猪精子品质的影响

Effect of Adding Seminal Plasma to the Thawing Diluent onthe Quality of Frozen-thawed Boar Sperm

【目的】探究冷冻前添加热休克蛋白A8(heat shock protein A8,HSPA8)和解冻后添加不同浓度精浆(seminal plasma,SP)对冻融猪精子的影响。【方法】采用手握法采集长白猪精液,添加0.5μg/mL HSPA8到猪精液冷冻保护剂中进行细管分装,投入液氮中保存3周后进行解冻,解冻后添加不同浓度精浆(0、10%、30%和50%),对冻融后长白猪精子的运动能力、质膜完整性、顶体完整性、细胞凋亡、线粒体膜电位、鱼精蛋白缺乏及体外获能水平等进行评估。【结果】与对照组相比(无HSPA8和精浆),添加0.5μg/mL HSPA8处理组(无精浆)的精子直线速度(VSL)、曲线速度(VCL)、平均路径速度(VAP)和前向性运动(STR)均显著提升(P<0.05),精子直线性运动(LIN)和运动的摆动性(WOB)均无显著差异(P>0.05);精子质量参数中活力、质膜完整性和顶体完整性均显著升高(P<0.05),细胞凋亡水平与线粒体膜电位均显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。之后在解冻液中添加不同浓度的精浆,与添加0.5μg/mL HSPA8处理组(无精浆)相比,精浆添加量达到50%时,精子VSL、VCL、VAP、LIN、STR和WOB均显著提升(P<0.05);精子活力、质膜完整性、顶体完整性和线粒体膜电位均显著提高(P<0.05),细胞凋亡水平显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。【结论】在冷冻基础液中添加0.5μg/mL HSPA8和解冻稀释液中添加50%精浆联合使用可以有效改善冻融精子质量,将会对猪精液的冷冻保存及商业化生产提供一定的参考。

【Objective】The purpose of this experiment was to explore the effects of adding heat shock protein A8(HSPA8)before freezing and adding different concentrations of seminal plasma(SP)after thawing on frozen-thawed boar sperm.【Method】The semen of Landrace boars was collected by hand holding method,0.5μg/mL HSPA8 was added to the boar semen cryopreserved agent for French straws,and then stored in liquid nitrogen for 3 weeks before thawing.After thawing,different concentrations of seminal plasma(0,10%,30%and 50%)were added again.The athletic ability,plasma membrane integrity,acrosome integrity,apoptosis,mitochondrial membrane potential,protamine deficiency and in vitro capacitation level of Landrace boar sperm after freezing and thawing were evaluated.【Result】Compared with control group(no HSPA8 and sperm plasma),in 0.5μg/mL HSPA8 treatment group(no sperm plasma),the sperm kinetic parameters VSL,VCL,VAP and STR were significantly increased(P<0.05),and LIN and WOB were not significantly different(P>0.05),the sperm quality parameters such as sperm motility,plasma membrane integrity and acrosome integrity were significantly increased(P<0.05),the sperm apoptosis level and mitochondrial membrane potential were significantly reduced(P<0.05),the sperm protamine deletion rate was significantly decreased(P<0.05),the tyrosine phosphorylation level of sperm protein was significantly increased(P<0.05).After that,adding different concentrations of seminal plasma treatment group to the thawing solution,compared with 0.5μg/mL HSPA8 treatment group(no seminal plasma),when the amount of seminal plasma reached 50%,VSL,VCL,VAP,LIN,STR and WOB were significantly increased(P<0.05),the sperm motility,plasma membrane integrity,acrosome integrity and mitochondrial membrane potential were significantly increased(P<0.05),the sperm apoptosis level was significantly reduced(P<0.05),the sperm protamine deletion rate was significantly decreased(P<0.05),the tyrosine phosphorylation level of sperm protein was significantly increased(P<0.05).【Conclusion】In conclusion,the combination of adding 0.5μg/mL HSPA8 to the freezing base solution and adding 50%seminal plasma to the thawing diluent could effectively improve the quality of frozen-thawed sperm,which would provide certain reference value for the cryopreservation and commercial production of boar semen.