Study On Vitrification -Induced DNA Damage Of Porcine Oocytes And Blastocysts

In this study, the porcine oocytes were collected from the abandoned porcine ovaries in local slaughters. In order to investigate the effect of DNA damage induced by vitrification and cryoprotectant cytotoxicity on porcine oocytes and blastocysts, DNA damage were detected by single cell gel electrophoresis (SCGE) and LMPCR (Ligation mediated PCR). To establish a porcine oocytes and blastocysts vitrification systems and to select the effective method to evaluate oocytes'DNA damage, which provide the further study some theoretical foundation and experiment materials.The results are as follows:1. DNA damage effects of cryoprotectant toxicity on porcine oocytes In vitro maturation (IVM) porcine oocytes DNA damage which caused by cryoprotectant (EDFS40) contains ethylene glycol (EG) and dimethylsulfoxide (DMSO) were detected by single cell gel electrophoresis (SCGE). The EDFS40 treated oocytes and the control oocytes were subjected to morphology evaluation and underwent comet assay (SCGE) to indentify cryoinjury at DNA level. The results showed that the morphology intact rates and DNA damage rates of oocytes which exposured to EDFS40 were 63.36% and 42.06%, and the control shows 84.51% and 12.28%. There is significant morphological damage as well as DNA damage compared to the control (P<0.05). It appears that oocytes that had been treated with cryoprotectant exhibited more morphology abnomality and DNA damage than the control. This experiment demonstrated that the cryoprotectant result in cytotoxicity at DNA level for porcine oocytes to some extent. And a better cryoprotectant should be invented to treat oocytes of porcine. 2. Effects of vitrification-induced DNA damage of IVM porcine oocytesDNA damage of IVM porcine oocytes which induced by cryopreservation in 0.25ml straws were studied on the thesis. The frozen-thawed oocytes and the unfrozen oocytes (control) were immediately subjected to morphology evaluation and underwent comet assay to detect cryoinjury at DNA level. And gene p53 frozen fracture was detected with LMPCR. The results showed that: morphologicaly intact rates and DNA damage rates of vitrified oocytes were 63.366% and 42.06%, which exhibited more DNA damage than the control --morphology intact rates and DNA damage rates were 84.51% and 12.28%. Frozen fracture among exon 7 of p53 gene were investigated in IVM porcine oocytes and purified DNA by LMPCR. The result confirms that LMPCR maybe an efficient method to map DNA strands breaks produced by cryopreservation. This experiment shows that oocyte DNA is a target of cryoinjury and the LMPCR may detect the frozen fracture of porcine oocytes.3. Study on cryoprotectants cytotoxicity and vitrification-induced DNA damage of porcine blastocystsPorcine oocytes were matured in NCSU-37, after in vitro fertilization, the blastocysts were exposure to EDFS40 and vitrificated in 0.25ml straws. The intact rates were recorded and DNA damage were detected by comet assay. Results showed that the vitrification blastocysts morphology intact rates and DNA damage rates were 35.48% and 79.14% which was significantly higher than the cryoprotectant treated group (56.46%, 53.45%, P<0.05). CASP analysis demostrated that there was a big DNA damage difference between the vitrification group/ cryoprotectant treated group and the control group. It appears that the blastocysts vitrificaton and cryoprotectant treated could cause DNA damage to a certain extent and the living rates would decline.