海藻糖和果糖联合应用提高了乌骨羊冷冻精子质量

Quality Improvement of Frozen Sperm by Combined Usage of Trehalose and Fructose in Black-Bone Sheep

该文探讨了海藻糖和果糖联合应用对乌骨羊精子冷冻保存的影响。在含有82.65 mmol/L果糖的Tris-柠檬酸–卵黄稀释液中分别添加0、5、10、15、25、50和100 mmol/L海藻糖,并将其分别命名为T0、T5、T10、T15、T25、T50和T100,采用两步稀释法对来自12只乌骨羊的27份精液进行冷冻保存。在精子冷冻前和解冻后,利用精子质量分析仪评估精子运动情况,采用伊红–苯胺黑染色法评估精子存活百分率,采用羧基荧光素二乙酸酯–碘化丙碇双重染色评估精子质膜状况,利用络合异硫氰酸荧光素的碗豆凝集素染色对精子顶体状况进行评估。采用过氧化氢含量测定试剂盒和丙二醛含量测定试剂盒检测各实验组解冻后精子的过氧化氢含量和丙二醛含量,以评估精子脂质过氧化状况。结果显示,T5、T10和T15组解冻后的精子运动和存活率有显著提高;T5组精子质膜状况有显著改善;和T0组相比,添加海藻糖并不能改善精子顶体状况;添加海藻糖不能降低解冻后精子的过氧化氢含量,但是可以显著降低精子的丙二醛含量。该研究的结果说明:在Tris-柠檬酸–卵黄稀释液中添加5 mmol/L海藻糖和82.65 mmol/L果糖能提高乌骨羊冷冻精子质量,能够改善解冻后精子的运动、存活和质膜完整性,减少解冻后精子膜的脂质过氧化。

The impact of trehalose and fructose on cryopreservation of Black-bone sheep sperm was researched.Tris-citric acid-egg yolk diluent containing 82.65 mmol/L fructose was supplemented with 0,5,10,15,25,50 and 100 mmol/L trehalose,respectively.The assay was divided into T0,T5,T10,T15,T25,T50 and T100 groups.Twenty-seven semen samples from 12 Black-bone sheeps were cryopreserved by two-step dilution method.Before and after thawing,sperm motility was evaluated by sperm quality analyzer.The percentage of survival sperm was evaluated by eosin aniline black staining.Sperm plasma membrane status was evaluated by carboxyl fluorescein diacetate/propidium iodide double staining.Sperm acrosome status was evaluated by pea agglutinin staining complexed with fluorescein isothiocyanate.The hydrogen peroxide content and malondialdehyde content of thawed sperm in each experimental group were detected by hydrogen peroxide content determination kit and malondialdehyde content determination kit to evaluate the lipid peroxidation of sperm.The results showed that the sperm movement and survival rate of T5,T10 and T15 groups were significantly improved after freezing.The status of sperm plasma membrane of T5 group was significantly improved.Compared with the T0 group,adding trehalose could not improve sperm acrosome status.The addition of trehalose could not reduce the hydrogen peroxide content of thawed sperm,but could significantly reduce the malondialdehyde content of sperm.The results suggest that adding 5 mmol/L trehalose and 82.65 mmol/L fructose to Tris-citric acid-yolk dilution can improve the quality of frozen sperm of Black-bone sheep,increase the sperm movement,survival and plasma membrane integrity after thawing,and reduce the lipid peroxidation of sperm membrane after thawing.