| The collection method of ovine oocytes, and its in vitro maturation (IVM), in vitro fertilization (IVF) . in vitro development and vitrification of ovine oocytes were investigated in the present study, focusing on some factors which are important in the process of in vitro production of ovine embryos, for the purpose of establishing an excellent manipulative system on bovine embryo production in vitro.In oocyte collection, two different collection methods (sucking method and slicing method) was tested and the effect of different seasons on the oocytes collection efficiency was investigated by recorded the number of oocytes collected per ovary, results indicate: the slicing method is better than the sucking method (2.02 vs 1.05, P<0.05) on the collection efficiency of oocytes;In Sep and Oct, the number of collected oocytes per ovary is 1.50 and 1.68, respectively, significantly higher than other monthes(Jan,Feb,Mar,Apr,May,Jun,Nov,Dec)(P<0.01).In the in vitro maturation of oocyte, effects of adding FBS to the maturation medium on the maturation efficiency of oocytes were investigated. The result indicate: adding ESS to he maturation medium can reached high maturation rate of the oocytes than FBS as an addition (72.7% vs 60.2%, P<0.05).In the in vitro fertilization, effects of different presermvative methods of sperm and different co-cultrue time of oocyte and sperm on the fertilization cleavage rate of oocytes was investegate. Results indicate: oocytes co-cultured with sperm for 24h reached significant higher cleavage rate of oocytes than which co-cultured for 6h (60.4% vs 22.7%, P<0.05), but showing no significant difference to that co-culture for 18h (60.4% vs 58.6%, P>0.05);the sperm stored at low temperature and up-swim is the best treatment of sperm and reached a high cleavage rate (78.6%), significantly higher than the groups, which used cryopreserved (60.2%, P<0.05) and low temperature stored (62.3%, P<0.05) and Percoll centrifuged sperm, or cryopreserved and up-swim sperm(21.1%, P<0.01).In the in vitro cultrue of embryo, the effects of the cultrue medium with or without EDTA and glucose and different medium-chang modes on the develoment of embryos was investigate. The results indicate: adding 10 μM EDTA to the cluture medium could significantly improved the cleavage rate (55.73% vs 43.66%, P<0.05) and morulae rate(24.6% vs 16.1%, P<0.05) of embryo;adding different concentration of glucose to the culture medium at diferent develomental stage of embryos could significantly improve the cleavage rate (66.12% vs 44.76%, P<0.01) and morulae rate (33.75 vs 17.19%, P<0.05);half-renew of the culture medium during the culture procedure can reach higher morulae-blastocyst rate of embryo than that renew the culture medium completely or without renew (42.0% vs 33.3% and 16.2%, P>0.05).In the present study, VEGF is added to the maturation medium, fertilization medium and culture medium in 4 levels (1, 2, 5 and lOng/ml) to test the effect of different addition levels of VEGF on the efficiency of in vitro production of ovine embryos. The results indicate:among the four levels of VEGF addition, the third group (5ng/ml VEGF) and the second group (2ng/ml) reached higher cleavage rate (86%,69%) and marulae rate(31%,25.0%), significantly higher than other groups (P<0.05).The cryopreservation of bovine oocyte was also studyed in the present study. The result indicate: the cleavage rate of the control after fertilization or parthenogenetic activation is higher than the vitrification group (65.3% and 68.2% vs 42.3% and 50.0%, P<0.05). The rates of embryo that develop to morulae were 33.3% and 27.0% for the groups of vitrification-activation and contro-fertilization, respectively, significantly higher than other groups (P<0.05).
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