玻璃化冷冻对猪MⅡ期卵母细胞及其DNA的影响

doi:10.16431/j.cnki.1671-7236.2021.07.023

Abstract

Abstract: The aim of this study was to screen the most suitable cryoprotectant for porcine MⅡ oocytes and explore the effect of vitrification on DNA of porcine MⅡ stage oocytes. The oocytes of MⅡ stage were randomly divided into 8 groups. The oocytes of the control group were parthenogenetically activated directly, while the oocytes of the other 7 groups were treated with the most used 7 kinds of cryoprotectant, respectively, and then thawed in the cryoprotectant directly without liquid nitrogen freezing, and parthenogenetically activated after thawing. The suitable cryoprotectant was screened through the statistical results of cleavage rate, blastocyst rate and blastocyst cell number, and porcine MⅡ oocytes were vitrificated using the three selected cryoprotectants. After thawing, porcine MⅡ oocytes were recovered for 2 h, and the normal rate of oocytes morphology and the cleavage rate were acalculated. The ultrastructural changes of porcine MⅡ oocytes after vitrification were observed by transmission electron microscopy. Porcine MⅡ oocytes were randomly divided into control group, cryoprotectant treat group and vitrification group, and the DNA damage of oocytes by vitrification was detected by comet assay. The results showed that compared with control group, the cleavage rate of group 5 and blastocyst rate of group 1 were significantly decreased (P<0.05), but there were no significant differences in the cleavage rate and blastocyst rate of the other groups (P>0.05). The number of blastocyst cells in control group was higher than that of the other groups, but the differences were not significant (P>0.05). The cleavage rate and blastocyst rate in groups 3, 6 and 7 were higher. After vitrification and thawing, the morphological normal rate and cleavage rate of oocytes in group 7 were significantly lower than those in groups 3 and 6 (P<0.05), and the cleavage rate in group 6 was higher than that in group 3. The MⅡ oocytes were obviously shrinkage after being transferred into the pretreatment solution, and quickly dehydrated after being transferred into the freezing solution. After thawing, the zona pellucida of oocytes was broken, and the cytoplasm was shrunk and unevenly distributed. Under transmission electron microscope, the zona pellucida and cell membrane of porcine MⅡ oocytes were damaged after vitrification, microvilli were seriously damaged or even disappeared, cortical granules were arranged under the plasma membrane and decreased in number, morphology of lipid droplets were destroyed and vacuoles were formed, the connection between endoplasmic reticulum and lipid droplets were damaged, mitochondria swelled and cristae were not obvious. Comet assay showed that there were no significant differences in head DNA, tail DNA and Olive tail moment between control group and cryoprotectant-treated group (P>0.05),and there was comet tailing. The head DNA damage, tail DNA damage and Olive tail moment values of vitrification group were significantly higher than those of the cryoprotectant treated group and control group (P<0.05), and there was obvious comet tailing. The results showed that the cryoprotectant with DMSO and EG as main components were suitable for vitrification of porcine MⅡ oocytes, vitrification could damage the ultrastructure and DNA of porcine MⅡ oocytes to some extent, and the damage mechanism needed further study.

Key words: DNA damage; cryoprotectants; vitrification; oocyte

CLC Number:

S852.16

XUE Mengqi, ZHOU Yue, LIU Keke, WANG Xinyu, DONG Yinyu, TANG Xiaochuan, WANG Xiaoli. Effect of Vitrification on MⅡ Oocytes and Their DNA of Porcine[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2475-2483.

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Effect of Vitrification on MⅡ Oocytes and Their DNA of Porcine

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