Difference Analysis On Expression Lever Of Maternal Genes In Oocytes In Sheep Before And After BCB Screening And Vitrified Freezing

Vitrified freezing had effect on the development potency of oocyte.The expression of maternal genes not only had influenced on growing of oocyte,but also played a decisive role to the early development of embryo.The expression content of mRNA in oocyte was detected by Real-time PCR after vitrified freezing and BCB.The expression mode of maternal gene in oocyte befor and after vitrified freezing,the reliability of oobyte's qualititative judged by BCB and molecule mechanism were researched.The depedablity between four maternal genes (Gdf9,Zarl,Mater and Dnmtl) and oobyte's qualititative were studied.The results were as follows:After screening by BCB dying,the maturing rate,fertility rate and blastula rate of BCB+ oocytes which cytoplasm was blue (86.16%,85.29%,34.4%) were higher than BCB-(50.94%,36.19%,6.73%),which cytoplasm was not blue (P<0.05).The results showed that BCB method was feasible and dependable.The expression content of four maternal, genes in BCB+ oocytes were lower than BCB-(P<0.05),which showed that it was negative correlation between the expression content of mRNA in four maternal genes and oobyte's qualititative.Vitrified-warmed different ripeness period oocytes and then vitro fertifilized,we found that the survival rate,the maturing rate and the cleavage rate of oocytes in GV(45.93%,14.07%,10.37%) was lower than M24h control group (—,91.11%,82.96%), M18h+V experimental group(68.29%,36.59%,23.17%), M24h+V experimental group (72.44%,48.82%,33.07%). The differences of vitrified oocytes in M18h and M24h were not apperent (P<0.05). The expression content of maternal genes in oocyte was highest in GV+V(P<0.01).The content of maternal genes in oocytes in GV was lower than in GV+V,but higher than the other experimental groups (P<0.01). The content of maternal genes in oocytes in M18h+V was more than in M24h+V. The content of maternal genes in oocytes in V+M24h was more than in M24h+V (P<0.01). The content of maternal genes in oocytes in the control group was the lowest.This showed that the maturity time get closer to 24h,the better the results of preservation by vitrified freezing were. The expression content of mRNA in Gdf9, Zarl, Mater and Dnmtl genes had some relationships with vitrified freezing. It was negative correlation with oobyte's qualititative and efficiency of vitro cultivation.Screened BCB+ and not screened oocytes were matured for 24 hours in vitro and defrosted respectively, and then cultured in vitro.The results showed that the survival rate, the maturing rate and the cleavage rate of oocytes in experimental group (BCB(+)M24h+ V) (87.83%,64.22%,48.78%) were much more than in (M24h+V) group(P<0.05). The content of maternal genes in oocytes in (BCB(+)M24h+V) was less than in (M24h+V) group (P<0.05). We discovered that BCB dying could be used in the technology of vitrified freezing. Before oocytes were vitrified freezed, the better ones were screened by BCB dying and immature cytoplasm of oocytes were replaced directly.Thus the workload of vitrified freezing were greatly reduced and improved availability rate of unfreezed oocytes and developmental rate of embryos.This method could be considered in futuer application in production.