The Effect Of Two-step Maturation In Vitro On Developmental Capacity Of Oocytes From Sheep

At present, the subsequent embryo development ability of ovine oocytes maturated in vitro is far lower than the embryos derived from in vivo matured oocytes, the main reason may be the nuclear maturation earlier than cytoplasmic maturation. It is to say oocyte nuclear and cytoplasmic maturation is not synchronous. In this paper, ovine oocytes were matured by two step mature method which using a variety of nuclear maturation inhibitors, delayed oocyte nuclear maturation, in order to make the oocyte nuclear and cytoplasmic matured as synchronous as possible, so as to improve the quality of oocytes matured in vitro and subsequent embryo development competence.At first, researched the different concentrations of Cilostamide delayed oocyte nuclear maturation time and inhibit effect, the influence of Follicle stimulating hormone (FSH) on Cilostamide maturation inhibition and the effect of Cilostamide inhibits oocyte nuclear maturation on subsequent embryonic development ability. The study shows that Cilostamide on sheep oocyte nuclear maturation inhibition in a dose-dependent manner, and the 5μmol/L Cilostamide is the suitable concentration for sheep oocyte maturation in vitro, the oocyte nuclear maturation can be delayed about 4 hours. While the high concentration (10μmol/L and 20μmol/L) Cilostamide will lead to some oocytes lost the ability to resume meiosis. FSH shows synergistic effect on Cilostamide inhibited oocyte nuclear maturation. When Cilostamide exists, with FSH concentration increase, the oocyte maturation rate of each group showed a downward trend. Ovine oocytes treaded with 5μmol/L Cilostamide 28 h after chemical activation, the blastocyst rate was 40.4% higher than that in the control group 29.0% (P<0.05).In addition, oocyte were treaded during per-IVM 2 h (the first maturation step) with the culture medium contained IBMX and FSK for, then transfered to the culture medium contained 5μmol/L Cilostamide matured in vitro (the second maturation step). The study shows that 100μmol/L IBMX+100μmol/L FSK or 100μmol/L+100μmol/L FSK was the better inhibitor combination choosed for the first step, the blastocyst rate were (51.6% and 49.8%) higher than that in the control group (33.1%) (P<0.05). Compared with the control group,the blastocyst cell number of two step group (151.6vs138) has no significant difference (P>0.05), so that two step maturation in vitro does not reduce the blastocyst cell number. After the first step mature treatment, cAMP levels were significantly increased in COCs and higher than that of FSK alone and IBMX treatment group. With the extension of maturity time, both the control group and experimental group, oocyte cAMP levels are decreased. At the IVM 9 h、20 h, the cAMP levels of control group oocyte have been reduced to lower,but the cAMP level of experimental group oocytes remained high level. At IVM 30 h,the cAMP level of two step group and the control group was no longer significant.