Abstract: Abstract: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:①While 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 but 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); ②While 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）; ③While 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); ④Following 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.