Study On The Factors Affecting The Efficiency Of Vitrified Oocytes In Sheep

Oocyte vitrification is of great significance for rare and endangered animal germplasm conservation , "egg bank" established, and the development of embryonic biotechnology. In this study, we take ovine oocytes as the research model to study the effects of different developmental stages of oocytes, with or without cumulus cells and pre-treatment with Taxol and Cytochalasin B on the cryopreservation efficiency of ovine oocytes, and the frozen-thawed oocytes are used for in vitro fertilization and parthenogenetic activation,status the cleavage rate and blastocyst rate for evaluation.The results show that: (1) in vitro maturation 24 h ovine oocytes(MⅡstage) and immature oocytes(GV stage) vitrification are compared, and the thawed viability for FAD staining viability rate (70.63%vs.58.51%), after IVF, cleavage rate(38.42%vs.22.68%), blastocyst rate (5.89%vs.0)are significant differences (P<0.05). It indicated that: mature ovine oocytes is more suitable for cryopreservation. (2) with or without culumus cells has no significant difference for MII oocytes vitrification in survival rate and cleavage rate or blastocyst rate (P>0.05).(3)Pre-treatment the ovine oocytes with the concentration(0.5μmol/L) of taxol cryopreservation best effect, after thawed, the efficience was verified by parthenogenetic activation and in vitro fertilization.The results showed that: the survival rate(82.38%)in 0.5μmol/L taxol group was significantly higher (P < 0.05) than those in three groups; there was significant diferences between 0.5μmol/L taxol group and those three treatment groups in cleavage rate ( 51.37% ) and morula-blastocyst rate(20.30%) of in vitro fertilization of frozen-thaw oocytes(P < 0.05), but was significantly lower (P < 0.05) than the control(69.42%,34.77%)(P<0.05). And pre-treatment with 0.5μmol/L taxol mature in vitro ovine oocytes, the cleavage rate(53.61%)and morula-blastocyst rate(22.64%) of parthenogenetic activation of frozen-thaw oocytes was higher than those three treatment groups(P < 0.05), but was significantly lower than the control(73.40%,33.79)(P<0.05). (4)The survive rate of frozen-thawed oocytes between in vitrification group without cytochalansin B treatment and with cytochalansin B treatment (64.50% vs.68.87%) was no significant difference(P>0.05). After in vitro fertilization (IVF) and parthenogenetic activation, the cleavage and morula-blastocyst rates of the frozen-thawed oocytes in both the vitrification (without cytochalansin B treatment) and cytochalansin B treatment group were also no significant difference(P>0.05),but also lower than those in the control(P<0.05).In conclusion,compare with GV stage ovine oocytes, MⅡstage oocytes are more suitable for cryopreservation; 0.5μmol/L taxol is benificial to improve the cryopreservation effect of in vitro maturation sheep oocytes .