Preliminary Study Of Vitrification On Porcine Oocytes

In order to explore the lowest toxicity, optimal freezing effects of cryoprotectant recipes to different periods of porcine oocytes. To find the most suitable method of freezing porcine oocytes at different stages. To analyze reasons which reduced developmental capacity on porcine oocytes after freezing. To provide theoretical support for the success of frozen pig oocytes can be applied to actual production. Therefore, GV and MII oocytes were taken in this experiment as the test materials. Vitrification freezing method used to filtrate seven vitrification solutions, to compare different effects of three carriers on freezing oocytes. Ultrastructural changes were observed of vitrified-thawed oocytes by transmission electron microscopy. DNA damage caused by freezing oocytes were analyzed by comet assay methods. The results showed that:1. Seven groups of cryoprotectants had low toxicity on MII oocytes. The rate of split and blastocyst, the number of blastocyst cell in each group was lower than in the control group. And cleavage rate of group5compared to the control group had significant difference(P<0.05). The blastocyst rate of group1compared to the control group had significant difference(P<0.05). There was no significant difference of split rate and blastocyst rate among other groups. Cryoprotectant with smallest damage to MII oocytes development ability was selected from group3, group6and group7. Results showed that:either Ethylene glycol used singly or Ethyl ene glycol and dimethyl sulfoxide mixed can be used for MII oocytes vitrification. Taken together, HM+7.5%(DMSO+EG), HM+17%(DMSO+EG)+0.4M Su was selected as the cryoprotectant to compare the effect of three carriers. That the normal rate of oocytes vitrification by using OPS was significant higher than using GMP or using Hemi-straw(P<0.05). The split rate of using OPS was significant lower than Hemi-straw(P<0.05). There was no significant difference of blastocyst rate between three methods. Hemi-straw method has obtained five blastocysts. OPS method has obtained1blastocyst. GMP method has no blastocyst obtained. What’s more, the split rate of Hemi-straw was53.67%which was significant higher than GMP(35.00%) and OPS(29.33%). So Hemi-straw was more suitable for MII porcine oocytes vitrification.2. The effect of seven cryoprotectants on GV oocytes was studied. The results showed that there was no significant difference of PBI rate between each group and control. Cryoprotectant of each group has low influence on GV oocytes maturation. For the rate of split and blastocyst and the number of blastocysts, significant difference was found in group1, group7with control(P<0.05). Cryoprotectant with the smallest damage to GV oocytes development ability was selected from group3, group5and group6. Results showed that:group2has six blastocysts, the mean number of blastocyst number was higher than other groups. Taken together, HM+7.5%(DMSO+EG), HM+15%(DMSO+EG)+0.5M Su was selected as the cryoprotectant to compare the effect of three carriers on porcine GV oocytes vitrification. The results showed that Hemi-straw has obvious better survival rate than OPS and GMP(P<0.05). There was no significant difference between tree methods about the rate of PBI cleavage, blastocysts and blastocyst number. So Hemi-straw was more suitable for GV porcine oocytes vitrification.3. Under scanning transmission electron microscopy showed that GV oocyte were compact connected with Zona Pellucida(ZP) Microvilli extended towards the ZP. There was a large number of mitochondria and clusters of ER, some Golgi apparatus and vesicles in the cortex. There were two kinds of Lipid Droplets (gray lipid droplets and dark lipid droplets) in the cytoplasm on porcine oocytes(MII stage, GVstage). The LD were surrounded by ER. MII oocytes with PBI and obvious PVS could be observed. A large number of cortical granules distributed under plasma membrane. Microvilli shortened to low columnar. Mitochondria often gathered lied in the cytoplasm.4. Vitrified porcine GV oocytes showed that the microvilli, ZP and cytomembrane were fractured or disappeared. Mitochondria swelled with vesicle structure. Dark type lipid droplets were mainly distributed in the cortex. The ultrastructure injury of ER. Cytoplasm of GV oocyte was flocculent. For vitrified MII oocytes ZP was damaged and seldom CG was found in the cortex. Dark type lipid droplets were mainly distributed in the cortex. A large supply of mitochondria in the cortex. Mitochondria swelled with misty bridges. Frozen will result in serious injury of ultrastructure in different periods of oocytes.5. DNA damage of porcine GV oocytes and MII oocytes which induced by cryopreservation and vitrification were detected by Comet Assay. The results showed the DNA damage of cryopreservation treated porcine oocytes(GV stage and MII stage) were similar to the control. But vitrification induced obvious DNA damage was observed in both GV oocytes and MII oocytes with the control. Different period of oocytes vitrification can lead to serious damage of DNA(P<0.05).