Effects Of Vitrification On Subcellular Structure And Maternal Gene Expression Of Oocytes In Donkey

Donkey is a domesticated animal with high economic value.Donkey’s meat is a delicious nutritious food,the milk composition of the donkey is similar to human milk and the best substitute for human milk.Donkey skin is the main raw material for making gelatin.However,the long natural breeding period and low breeding rate of the donkey seriously restrict the development of the donkey industry.Artificial breeding techniques can improve the reproduction rate of excellent breeds.Cryopreservation of oocytes is one of the basic techniques for artificial breeding of livestock,and it is also an effective way to preserve high-quality livestock genetic resources.Research on the cryopreservation of donkey oocytes has rarely been reported.In this study,vitrification cryopreservation was used to cryopreserve donkeys oocytes at different stages of development(immature GV and in vitro maturation IVM-MII),and we analyzed the oocyte maturation rate,development rate,and subcellular structure before and after cryopreservation.Single-cell RNA-seq was performed on oocytes before and after freezing,and the expression changes of three parental genes which are closely related to oocyte development were detected,including Nlrp5,Nlrp2 and Padi6.To investigate the effects of vitrification on the maturation and development of oocytes.The results of the study are as follows:1.There was no significant difference in the normal rate of morphological oocyte morphology between frozen GV and control.The maturation rate and cleavage rate were significantly lower than the control group(18.34% VS 52.67%,20.83% VS 41.38%).The cleavage rate of frozen IVM-MII stage was significantly lower than the control group(16.36% VS 41.38%),and cell development was blocked after cleavage,and GV oocytes were more suitable for vitrification cryopreservation.2.The normal rates of microfilaments of oocytes in fresh GV(control),frozen GV and frozen IVM-MII were 73.07%,40.00% and 28.20% respectively.The number of depolymerizations in the microfilament area of frozen oocytes increased,and the number of mitochondria also decreased significantly.After cryopreservation,The abnormal distribution of microfilaments and mitochondria was milder in GV phase than in IVM-MII phase,and the developmental state is also good.3.The normal distribution rate of frozen cortical particles in GV was significantly lower than that in the control group(47.22% VS 76.60%),The normal distribution rate of cortical particle distribution in frozen IVM-MII stage oocytes was significantly different compared to the control group(24.24% VS 53.85%).The number and density of cortical particles in oocytes after freezing were significantly lower than those in fresh eggs,and their migration was blocked.4.During maturation of normal oocytes(GV,MI,MII)maternal genes Nlrp5,Nlrp2,and Padi6 were up-regulated at MI stage and down-regulated at MII stage;after freezing,the maternal genes of the oocyte maturation process have been expression was down-regulated,which in turn affected the normal development of oocytes.All above showed that the GV period is the best period for vitrification of donkey oocytes.Vitrification affects the subcellular structure of donkey oocytes and results in the down-regulation of maternal gene expression,possibly reducing their maturation and development potential.