Research On MⅡ Stage Porcine Oocytes Vitrification And In Vitro Fertilization

Vitrification freezing of oocytes is the effective measure to protect species resources, at the same time can also provide lots of experiment materials to the research of modern biological technology such as cloning. But as a result of porcine oocytes is sensitive to low temperature, the vitrification progress is very slow. This experiment with MⅡ stage porcine oocytes as the research object, studied several kinds of vitrification cryoprotectants toxic effects on oocyte and influence of different cryoprotectants, carriers on the vitrified effect, and test viability of oocytes after vitrification by in vitro fertilization, to investigate damage mechanism of the porcine oocytes in the process of vitrification and the influence factors of in vitro fertilization. In order to select a suitable combination of the vitrification cryoprotectants and carrier, and lay a foundation for further study.This research mainly includes the following main content:1. Adopt three different kinds of vitrification cryoprotectants to deal with MⅡ stage porcine oocytes according to the corresponding process of vitrification and do parthenogenetic activation without vitrification to research effects of different cryoprotectant combinations toxic on oocytes.2. Adopt three different kinds of vitrification cryoprotectants to vitrify MⅡ stage porcine oocytes according to the corresponding process of vitrification by GMP to research effects of different cryoprotectant combinations on oocyte survival rate.3. Choosing the cryoprotectant combination which makes the best effect of vitrification to vitrify MⅡ stage porcine oocytes with GMP and Cryotop as the carriers to research effects of different carriers on oocyte survival rate.4. To research the effect of vitrification on oocyte cortical granule distribution by FITC-PNA staining.5In vitro fertilization to MⅡ stage porcine oocytes after making different treatment to the cumulus cells to research the effects of cumulus cells on in vitro fertilization.6. To research the influence of vitrification on oocytes developmental ability by doing parthenogenetic and in vitro fertilization to MⅡ stage porcine oocytes after vitrification.The results of the research show that:(1) All of three kinds of cryoprotectant combinations have acentain degree of toxicity.the toxicity of ES3+VS3(TCM-199(Hepes)+20%SPS+15%EG+15%DMSO+0.5mol/L sucrose as cryoprotectant combination)is minimal of all.(2) The rate of embryos normoplasia (82.31%), the rate of embryos surviving by FDA stain (59.40%) and the rate of embryos surviving by TB stain (62.18%) of oocytes vitrified with ES3+VS3cryoprotectants combination are significantly higher than the other two groups (p<0.05),(3) The rate of embryos normoplasia (96.67%), the rate of embryos surviving by FDA stain (83.33%) and the rate of embryos surviving by TB stain (86.67%) of oocytes vitrified with Cryotop as the carrier are significantly higher than the oocytes vitrified with GMP as the cairier.(4) The rate of cortical granule release in advance of porcine oocytes vitrified(20.35%)is higher significantry than oocytes without vitrification.(5) The developmental ability after in vitro fertilization of the oocytes removed all of the cumulus cells is poor, the rate of embryos cleaved is30.26%and the rate of blastocysts is6.56%. The developmental ability of the oocytes removed all of the cumulus cells are significantry lower than the oocytes removed part of the cumulus cells and the oocytes keep all of the cumulus cells(p<0.05). It proved that cumulus cells play an important role in the in vitro fertilization.