Effects Of Maturation Time And NMN On In Vitro Maturation Of Porcine Oocytes

Porcine oocytes that mature in vitro usually come from small and mediumsized follicles(2～6mm),The problem of non-synchronous nuclear and cytoplasmic maturation is prominent.How to prolong the culture time of in vitro maturation and compensate for the cytoplasmic maturation of oocytes during the further development of follicles is an important strategy to improve the quality of oocytes in vitro maturation.Therefore,this study intends to alleviate the DNA damage and senescence of oocytes which induced during in vitro culture by adding β-nicotinamide mononucleotide(β-Nicotinamidemononucleotide,NMN,an antioxidant),and to explore the feasibility of prolonging in vitro maturation time and improving the quality of cytoplasmic maturation,so as to improve and optimize the in vitro maturation system of porcine oocytes.The main results of this study are as follows:1.Effect of prolonging culture time on in vitro maturation of porcine oocytesIn order to improve the cytoplasmic maturity,the in vitro culture time of oocytes was prolonged from 44 h to 47 h,50 h and 53 h,respectively.The results showed that the first polar body excretion rate of oocytes cultured in vitro for 47 h,50 h and 53 h was higher than that of 44 h(P > 0.05),but not significantly different.Prolonging the oocyte culture time to 47 h or 50 h,the blastocyst rate of oocytes after parthenogenetic activation was significantly increased(P <0.05),and the blastocyst rate of 47 h group was the highest.Therefore,the oocytes cultured in vitro for 44 h and 47 h were selected to explore the underlying difference of maturation quality.2.Comparative study on maturation quality of oocytes cultured in vitro for 44 h and 47 hCompared with oocytes cultured in vitro for 44 h,the content of mitochondria,the normal distribution rate of mitochondria,the membrane potential of mitochondria and the normal distribution rate of cortical granules of oocytes cultured in vitro for 47 h were significantly increased(P < 0.05).However,the ROS level of oocytes cultured in vitro for 47 h was significantly increased,while the m RNA expression levels of DNA damage repair,antioxidation and anti-apoptosis genes were significantly decreased.The results showed that the cytoplasmic maturity of oocytes cultured in vitro for 47 h was higher than that of oocytes cultured in vitro for 44 h,but there were potential senescence and damage3.Effect of NMN on maturation quality of oocytes cultured in vitro for 47 hWhen 100 μM NMN was added to the maturation medium,the maturation rate of oocytes and the development rate of activated blastocysts were slightly increased,the total number of blastocyst cells and the expression level of blastocyst pluripotency related genes were significantly increased.In addition,the ROS level of oocytes decreased significantly,while the expression levels of DNA damage repair,anti-oxidation,anti-apoptosis genes,oocyte maternal factors and SIRT family genes were increased significantly.Meanwhile,there was no significant difference in mitochondrial content,normal mitochondrial distribution rate and mitochondrial membrane potential of oocytes(P > 0.05).Therefore,NMN has protective effect on oocytes cultured in vitro.These results suggest that prolonging the in vitro culture time to 47 h and adding NMN in the maturation medium can improve the in vitro maturation quality of oocytes and the embryo development rate after parthenogenetic activation.This study not only laid a theoretical and experimental basis for improving the efficiency of in vitro maturation of porcine oocytes,but also provided a new idea for in vitro oocytes maturation and embryo production of large domestic animals.