体外操作对小鼠精子参数及内源性ROS的影响

The Effect of In Vitro Manipulation on Sperm Parameters and ROS

目的:研究体外物理操作对小鼠精子回收率、活动精子百分比、膜完整率以及细胞内活性氧簇(DCFMF)I的影响,探索最佳的小鼠精子体外处理方法。方法:采用不同离心力及离心时间(200,400,600,800×g;5,10,15,20 min),1 m L微量加样器不同抽吸次数(2,4,6,8,10次)以及不同Percoll密度梯度离心条件(600,800,1 000,1 200×g;15,30min)处理小鼠精子,探讨导致各指标变化的原因以及最优的处理条件。结果:析因分析结果提示,离心力及离心时间双重因素影响离心操作时精子回收率以及Percoll密度梯度离心活动精子百分比(P<0.05);离心力单一因素影响离心操作时活动精子百分比以及Percoll密度梯度离心精子膜完整率(P<0.05);加样抽吸操作可影响精子膜完整率以及活动精子百分比(P<0.05)。离心操作时,当离心力大于600×g时活动精子百分比下降(P<0.05);加样器抽吸小鼠精子4次以上,小鼠精子膜完整率、活动精子百分比均下降(P<0.05);Percoll密度梯度离心时间30 min,离心力达到800×g以上活动精子百分比与对照组比较升高(P<0.05),离心力达到1 000×g以上时精子膜完整性与对照组比较降低(P<0.05)。各组存活精子DCFMFI无变化(P>0.05)。结论:在小鼠精子离心时,离心力应控制在600×g以下;抽吸时次数应控制在4次以下;Percoll密度梯度离心最优条件为800×g 30 min。

Objective：To study the effects of various physical interventions [such as centrifugation, repeated pipetting, Percoll gradient centrifugation （PGC）] on the recovery rate, motility, membrane integrity （MI） and intercellular ROS （DCFMFI） of mice spermatozoa. Methods： The mice sperms were treated by different centrifugation force and time （200×g, 400×g, 600×g, 800×g; 5 min, 10 min, 15 min, 20 min）, different pipetting times （2, 4, 6, 8, 10 times） and PGC condition （600×g, 800×g, 1 000×g, 1 200×g, 15 min, 30 min）, so as to explore the impact factors and optimal procedure of sperm treatment. Results： The factorial analysis showed that the centrifugation force and centrifugation duration were two factors of the sperm recovery rate in centrifugation procedure and the percentage of motile sperm in PGC procedure （P〈0.05）. The eentrifugation force was a single factor of the percentage of motile sperm in centrifugation procedure and the MI in PGC procedure （P〈0.05）. The pipetting procedure was related to the MI and the percentage of motile sperm （P〈0.05）. The percentage of motile sperm was significantly decreased when the eentrifugation force reached to 600×g （P〈0.05）. The MI and percentage of motility sperm were significantly decreased when the pipetting times were more than 4 （P〈0.05）. The percentage of motile sperm was significantly increased when the centrifugation force was more than 800×g and the centrifugation duration was 30 min in PGC procedure （P〈0.05）. However, the MI was significantly decreased when the centrifugation force was over 1 000×g （P〈0.05）. There was not significant change in the DCFMFI after different sperm treatments （P〉0.05）. Conclusions： During mouse sperm treatment, the eentrifugation force should be controlled under 600×g, the pipetting times less than 4, and the optimal PGC procedure is 800×g 30 min.