谷胱甘肽对犬精液冷冻保存效果的影响

Effect of Glutathione on Cryopreservation of Canine Semen

为探讨在冷冻稀释液中添加谷胱甘肽(GSH)对犬精液冷冻保存效果的影响,采用按摩法采集5只杂种土犬的精液,离心去精清后,在冷冻液中分别加入0.5、1.0、1.5、2.0、2.5 mmol/L的GSH,制成0.25 mL的冻精进行冷冻保存,以不添加GSH的处理组作为对照组。解冻后在含有5%CO2的空气、37℃、相对饱和湿度条件下孵育10 h,分别在孵育0、2、4、6、8、10 h时检查精子活力。结果显示:冻融后0 h,0.5、1.0 mmol/L GSH处理组的精子活力较高,分别为0.36和0.38,均显著(P<0.05)高于对照组和2.0、2.5 mmol/L处理组,且两者之间差异不显著(P>0.05);1.0 mmol/L处理组的精子顶体完整率最高,为85.10%,显著(P<0.05)高于对照组,同时,其精子畸形率最低,为23.00%,显著(P<0.05)低于对照组。冻融后体外孵育2、4 h时,0.5、1.0 mmol/L处理组的精子活力均较高,其中,0.5 mmol/L处理组在体外孵育4 h时,其精子活力仍可达到0.30;孵育6 h时,1.0 mmol/L处理组精子活力最高,显著(P<0.05)高于对照组和2.0、2.5 mmol/L处理组;孵育8 h时,各GSH处理组的精子活力均显著(P<0.05)高于对照组;在孵育至10 h时,各GSH处理组的精子活力较其他孵育时间均有较大幅度的下降,未检测到对照组中有呈直线运动的精子。综上提示,在犬精液冷冻液中添加0.5~1.0 mmol/L的GSH能够显著提高冻融后的精子质量和体外存活时间。

In order to investigate the influence of adding glutathione(GSH)in frozen diluent on cryopreservation effect of canine semen,the seminal samples were collected from 5 hybrid dogs by massage method.After centrifugation to remove the seminal plasma,0.5,1.0,1.5,2.0 and 2.5 mmol/L of GSH was supplemented in the frozen diluent,respectively.All of the above samples with volume of 0.25 mL containing different concentrations of GSH were prepared for cryopreservation.The seminal samples without addition of GSH was used as a control group.After thawing,the samples were incubated for 10 hours in air containing 5%CO2 at 37℃ with relative saturated humidity.The sperm motility of the samples was determined at 0,2,4,6,8,10 h after incubation,respectively.The results showed that at 0 h after freezing and thawing,the sperm motility of the 0.5 and 1.0 mmol/L GSH treatment groups was 0.36 and 0.38,respectively,which were both significantly(P<0.05)higher than those of the control group and 2.0 and 2.5 mmol/L GSH treatment groups,and no significant difference(P>0.05)were observed between them;the highest(85.10%)sperm acrosome integrity rate and the lowest(23.00%)sperm deformity rate were simultaneously observed in the 1.0 mmol/L GSH treatment group,and they were significantly(P<0.05)higher and significantly(P<0.05)lower than those of the control group,respectively.After incubation for 2 and 4 hours after freezing and thawing,the sperm motility of the 0.5 and 1.0 mmol/L GSH treatment groups was relatively higher,and that of the 0.5 mmol/L GSH treatment group after in vitro incubation for 4 hours still reached up to 0.30;after in vitro incubation for 6 hours,the highest sperm motility was observed in the 1.0 mmol/L GSH treatment group,which was significantly(P<0.05)higher than those of the control group and 2.0 and 2.5 mmol/L GSH treatment groups;after in vitro incubation for 8 hours,the sperm motility of all GSH treatment groups was significantly(P<0.05)higher than that of the control group;after in vitro incubation for 10 hours,the sperm motility of each GSH treatment group was all decreased compared with other incubation time points,and there was no sperm with linear motion in the control group.In summary,the addition of 0.5-1.0 mmol/L GSH in freezing solution of canine semen can significantly improve the sperm quality and in vitro survival time after freezing and thawing.