不同发情阶段的绵羊血清对绵羊精子获能和体外受精的影响

Comparison of the Ovine Sperm Capacitation and Fertilization in vitro among the Different Concentrations of Serum Collected from Their Different Estrous Stages

为比较不同发情阶段和不同浓度的绵羊血清在绵羊体外受精过程中对绵羊精子体外获能及存活时间、受精效果的影响,试验以绵羊输卵管合成液(SOF)为基础获能液,在其中分别添加发情当天、发情结束和发情间期的绵羊血清,血清浓度分别为0%、2%、5%、10%和20%。利用金霉素荧光染色(CTC,chlortetracycline)法及体外受精2种方法检测不同血清及其浓度对绵羊精子获能的影响。结果表明:1)当在获能液中添加发情当天绵羊血清时,在10%和20%血清浓度下作用45 min的获能效果最好,其获能而且未完成顶体反应的精子率(45.28%和44.52%)显著高于0%、2%和5%血清浓度组(30.14%、28.71%和35.49%,P<0.05);2)当在获能液中添加发情结束时或发情间期的绵羊血清时,在含不同浓度的这2种血清的获能液中作用45 min后,各浓度组引起的获能精子百分率变化均不显著(P>0.05);3)在含20%发情当天绵羊血清的获能液中,精子的存活时间(18 h)显著高于含20%发情结束和发情间期的绵羊血清的获能液(11 h;11 h);4)在不同血清诱导获能后进行的体外受精的试验中,添加发情当天绵羊血清组的卵裂率和囊胚率(62.5%和26.25%)均显著高于发情结束时血清组(42.22%和11.11%,P<0.05)和发情间期血清组(37.36%和15.38%,P<005)。以上结果表明:在进行绵羊体外受精试验中,采用20%发情当天绵羊血清作用45 min比采用发情结束和发情间期的绵羊血清能更好地诱导精子获能。

This study was aimed to provide some experimental references for improving the in vitro fertilization system of sheep. The effect of different ovine serum concentrations during their different estrous stages on their sperm capacitation, enduring durations and fertilization were analyzed. After the basic sperm capacitation medium（Sheep Oviduct Fluid, SOF medium） was supplied respectively with 0%, 2%, 5%, 10% and 20% ovine serum collected during their different estrous stages （the first day of estrus, the last day of estrus, and diestrus ）, the effects of their sperm capacitation, enduring durations were compared and analyzed reasonably by CTC（chlortetracycline）staining and appropriate fertilization in vitro. The results showed as following： （1）While the SOF medium was supplied with the ovine serum collected on the first day of their estrus, its best effect was initiated by treating for 45 min with 10% and 20% serum. Their sperm rates （45.28% and 44. 52%） by capacitation hut without acrosome reaction（AR） were significantly higher than those among the other groups treated with 0%, 2% and 5% serum, respectively （30.14%, 28.71% and 35.49%,P〈0.05） ; （2）While the SOF medium was supplied with the ovine serum collected on the last day of estrus and diestrus, there were no significant differences of the ovine sperm capacitation percentage among all the groups treated for 45 minutes（P〉0. 05）;（2）While the SOF medium was supplied with 20% ovine serum collected on the first day of their estrus, enduring duration of sperm （18 h）was much longer than that of the other two groups with 20% serum collected on their last day of estrus or diestrus respectively（11 h, 11 h） ; （4）Following their appropriate fertilization in vitro, the cleavage and blastocyst rates（62.5% and 26. 25%）in the group treated with the serum collected on the first day of their estrus, were significantly higher than those in the other two groups treated with the serum collected on the last day of estrus （42.22% and 11.11%,P〈 0.05）or diestrus（37.36% and 15. 38%,P〈 0. 05 ） , respectively. It can be recommended that the serum be applicable for improving ovine IVF system, while the sheep sperm is treated for 45 minutes by SOF medium with 20% ovine estrous serum collected on the first day of their estrus.