| Effect of addition of HMG and ITS with different concentration in culture medium on in vitro maturation of cattle oocytes were observed and the relationship between ovaries holding temperature,preservation time and estrous cycle and in vitro maturation of cattle oocytes and in vitro culture of parthenogenetic embryos were analyzed; Effect of droplet size,renew media volume,FBS inactivation and addition time and concentration on the developmental potentiality of cattle parthenogenetic embryos were reviewed, meanwhile four category culture media were compared; Effect of whether co-culture with granulosa cell monolayers or not,different type,different species and culture time of cell monolayers and renew granulosa cell monolayers on the developmental potentiality of cattle parthenogenetic embryos were observed, in order to optimize in vitro culture system of cattle parthenogenetic embryos; Effect of oocytes maturation time and nucleus/cytoplasm action time on in vitro development of reconstructed embryos between granulosa cell and enucleated cattle oocytes were examined, the results as follows:1. Adding 0.05 IU/mL or 0.1 IU/mL HMG to OM media to reach a higher mature rate respectively(71.26%;74.30%) than 0.025IU/mL group(31.52%) (P<0.01); Addition of 10μL/mL ITS to OM media did not significantly improve the mature rate and cleavage rate(71.52%/69.47%; 84.96%/82.42%), but increased the blastocyst rate of parthenogenesis embryos(41.67% / 32.0%); Cattle ovaries to harvest from different batches were preserved in physiologic saline at different temperature, for the mature and blastocyst rate in all treatment groups, there were no striking difference between 20～29℃group(61.11%/33.33%) and 10～19℃(41.18% / 17.65%) or 30～39℃group(71.34% / 41.84%)(P>0.05), 10～19℃group was lower significantly than 30～39℃group(P<0.05); The mature,cleavage and blastocyst rate of oocytes collected from ovaries preserved 8～10h were significantly lower than preserved 1～3h under 30～39℃(68.57%/32.29% , 86.11%/25.81%, 40.32% / 0 ; P<0.05); The mature,cleavage and blastocyst rate of oocytes collected from ovaries in luteal phase with or without corpus luteum were all lower than from the ovaries in follicular phase, but there were no significant difference(P>0.05).2. There were a higher blastocyst rate when parthenogenetic embryos were cultured in 100μL droplet than cultured in 50μL group or 200μL group, but there were no significant difference (43.20%/31.34%/38.82%) (P>0.05); There were a striking difference in terms of blastocyst rate that one-half of the culture medium was replaced with fresh medium every time comparing with 1/4 group or 3/4 group of renewing media volume(41.84%/26.39%/25.71%) (P<0.05); Heat inactivation or not of FBS have no notable influence for in vitro development of parthenogenetic embryos, non-heat-inactivation group was superior to heat inactivation group in terms of blastocyst and hatching blastocyst rate(40.23%/57.14%;38.95%/51.35%); Culture medium supplemented with FBS at Day 3 after parthenogenetic activation was suitable, the mean ratio of blastocyst and hatching blastocyst reached 46.55% and 53.70% respectively; There were a highest blastocyst and hatching blastocyst rate when culture medium supplemented with 10% FBS at Day 3 after parthenogenetic activation(45.97%/54.39%); For the cleavage rate and blastocyst rate in all treatment groups, SOFaa was the best choice for in vitro culture of cattle parthenogenetic embryos.3. For the cleavage,blastocyst and hatching blastocyst rate, GCM group(85.10%, 41.81%, 52.70%) was higher significantly than control group (no co-cultured feed layer) (66.13%,7.32%,0%)(P<0.05); GCM and CCM were suitable for in vitro development of cattle parthenogenetic embryos, the cleavage,blastocyst and hatching blastocyst rate were 86.11%/ 83.72%,40.32% /41.67%,52. 0% /53.33% respectively; There were no species-specificity for CCM come from cattle,pig and mouse in terms of supporting in vitro developmemt of cattle parthenogenetic embryos; Granulosa cell reached 100% confluence as day 0, then cultured another 2d or 4d and co-cultured with parthenogenetic embryos, for the cleavage and blastocyst rate, there was no significant difference between 2d group and 0d goup or 4d group(P>0.05), but a striking difference between 0d group and 4d group was observed(P<0.05), for hatching blastocyst rate, the three group were no notable difference(P>0.05); Granulosa cell monolayers was renewed zero(control),once(4d group) and twice(3/6d group), for the blastocyst rate, there was no significant difference between 3/6d group and control goup or 4d group(P>0.05), but a striking difference between control group and 4d group was observed (P<0.05), for hatching blastocyst rate, the three group were no notable difference(P>0.05).4. The cleavage and blastocyst rate of reconstructed embryos were no striking difference when oocytes were enucleated after matured different time in vitro (17～19h,19～21h and 21～23h group)(P>0.05). for the enucleate rate, 17～19h group and 19～21h group were higher significantly than 21～23h group(P<0.05). for the blastocyst rate, 19～21h group was superior to 17～19h group and 21～23h group(P>0.05); The reconstructed embryos from oocytes matured in OM media for 19～21h were activated after cultured different time in vitro (1～3h,3～5h and 5～7h group), the results demonstrate that the activation of reconstructed embryos after in vitro culture for 3～5h was suitable, the mean ratio of cleavage and blastocyst reached 75.0% and 11.46% respectively.
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