Research On The Porcine Oocytes Vitrification At MⅡ Stage

The development of mammalian oocytes and embryos vitrification was considered as the important achievement in the field of developmental biology and assisted reproductive technology. The of technology of embryo and oocytes cryopreservation was not only used for genetic resource conservation,but also the possibility of gamete transportation in the domestic and universe. Technology of oocytes and embryos cryopreservation can help people with reproductive disorder have babies. The technology of oocytes and embryos vitrification was development since 1985. With the research deeply, vitrification has been used for rat,bovine and ovine oocytes and embryos preservation successfully. The effect of porcine oocytes and embryos vitrification were poorer than the other mammalian species.To establish the cryopreservation system of porcine oocyes at MII stage, different cryoprotectants, different vitrified carriers and different sugars add to the cryopretectants have been used for porcine oocytes vitrification. To investigate the mechanisem that subsequent development of porcine oocytes after vitrification was significantly lower than the fresh oocytes, oocytes were cultured in vitro and delipated through micromanipulation method, the normal rate of mitochondria distribution, ROS activity and viability were assessed after vitrification. The maternal factors m RNA expression in porcine oocytes at MII stage were assessed by Realtime PCR to investigate the effect of porcine oocytes vitrification on the maternal factors m RNA expression.All the results as fellows below:(1)The effect of EG and DMSO were used alone or combained as cryoprotectants on porcine oocytes vitrification. The results showed that there was no difference between use EG and DMSO alone and used together during porcine oocytes vitrification(2)To investigate the best sugar can be used for porcine oocytes vitrification,the sucrose, ficoll and trehalose were added to the cryoprotectants.The result showed that different sugars add to the cryoprotectant did not affect the result of oocyte vitrification(3)In this experiment,the treatment groups were separated into three groups in accordance with the layers of culumus cells. The results indicated that culumus cells did not affect the results of porcine oocytes vitrification(4)In order to choose the best vitrified carriers for vitrification, Cryotop,Cryotip,GMP and OPS were used for porcine oocytes cryopreservation. The results indicated that different vitrified carriers affected the results of vitrified oocytes.Cryotop was much better than GMP and OPS. But there was no different between Cryotop group and Cryotip group.(5)In this experiment, the macromoleculers were added to cryoprotectants to investigate the the effect of macromolecules on the porcine oocytes vitrification. The result showed that the macromolecules add to the cryoprotectants did not affect the development of porcine oocyte. The normal rate of mitochondrial distribution in SPS group was significantly higher than the other treatment groups, but it was significantly lower than the control. The morphology normal rate and survival rate in SPS group were significantly higher than the other group after vitrification. The cryoprotectant used for vitrification which added to SPS induced the vitrified oocytes developed to the cleavage stage after parthenogenetic activation(6)To investigate the effect of mechanical lipid droplets on porcine oocytes vitrification, the lipid droplets were removed through micromanipulation. The result indicated that normal rate of mitochondrial distribution in vitrified group was significantly lower than that in control.Porcine oocytes vitrification increased the level of ROS activity, but there was no difference in delipated group and undelipated group. The delipated oocytes without vitrification may developed to the blastocyst stage, but the blastocyst formation rate was significantly lower than control.Vitrification induced the poor subsequent development capacity of porcine oocytes,,but the capacity of development in delipated group was significantly than the intact group(21.43% versus 10.38%).(7) In this experiment, to investigate the maternal factors expression variation, maternal factors mRNA expression were assessed by Real-time PCR..The m RNA expression of maternal factors(DNMT1、DNMT3a、IGF2、IGF2R、BAX、BCL2 �'� ZAR1)in porcine oocytes has the tendency of rising after vitrification and the m RNA expression of four maternal factors(DNMT1、IGF2R、IGF2 and BCL2)in porcine oocytes was significantly higher than the unvitrified group.