Effects Of Epigallocatechin-3-gallate On Oocyte Matured In Vitro And Frozen Semen Quality Of Bovine

Reactive oxygen species(ROS)play an important role in maintaining the normal physiological function of female and male gametes in mammals,but excessive ROS could lead to oxidative stress(OS)which results in damages to both female and male gametes in mammals.Epigallocatechin-3-gallate(EGCG)is a major component of catechins in green tea,and it is considered one of the most promising bioactive substances for its strong antioxidant properties,and it can scavenge ROS and improve the quality of female and male gametes in return.In this study,different concentrations of EGCG were added to bovine oocyte maturation medium and freeze-thawed semen to investigate its effects on oocytes in vitro maturation and frozen semen quality of bovine.The results were as follows:1.Experiment I: Different concentrations of EGCG(0,25,50 100,and 200 ?M)were supplemented to the maturation medium during in vitro maturation of bovine oocytes,and the nuclear and cytoplasmic maturation,cumulus cell expansion,intracellular ROS levels,total antioxidant capacity,the early apoptosis and the developmental competence of in vitro fertilized embryos were measured.The mRNA abundances of antioxidant genes(NRF2,SOD1,CAT,and GPX4)in matured bovine oocytes were also quantified.The results showed that(1)Compared with the control group,the first polar body extrusion rate,the percentage of oocytes with peripheral and cortical distribution of cortical granules,and the percentage of oocytes with homogeneous mitochondrial were significantly improved in the 50 ?M EGCG treatment group(P<0.05).(2)The cumulus cell expansion index(CEI)and mRNA levels of cumulus cell expansion-related genes(HAS2,TNFAIP6,PTX3,and PTGS2)were significantly increased in 50 ?M EGCG treatment group compared with the control group(P<0.05).(3)In comparison with the control group,both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the 50 ?M EGCG treatment group,and the total antioxidant ability was markedly enhanced(P<0.05).(4)Both the cleavage and blastocyst rates were significantly higher in the 50 ?M EGCG-treated oocytes after in vitro fertilization than those in the control group(P<0.05).(5)The mRNA abundance of NRF2,SOD1,CAT,and GPX4 were significantly increased in the 50 ?M EGCG-treated oocytes when compared with the control group(P<0.05).2.Experiment II: The sperm after thawing was incubated with EGCG of different concentrations(0,5,10,and 20 ?M)for 1 h at 38.5?,then sperm kinetic parameters,the structural integrity,the activities of related enzymes and the in vitro fertilization ability were measured.The results revealed that(1)Supplementation of 10 ?M EGCG could significantly improve the percentage of total motile sperm(TM,%)and the beat cross frequency(BCF,Hz,P<0.05)compared with the control group.(2)In comparison with the control group,the plasma membrane integrity and acrosome integrity of the sperms were significantly increased in 5 ?M,10 ?M and 20 ?M EGCG treatment groups(P<0.05),and the integrity of mitochondrial membrane was significantly higher in 5 ?M and 10 ?M EGCG treatment groups(P<0.05).(3)Compared with the control group,the activities of SOD and CAT were significantly increased while the activity of LDH and production of MDA were significantly decreased(P<0.05),but the activity of AST was not affected in bovine frozen-thawed sperm after incubation with 10 ?M EGCG.Although the supplementation of 20 ?M EGCG could significantly enhanced the activities of SOD and CAT and decreased the activity of AST(P<0.05),the production of MDA was significantly elevated(P<0.05).(4)The pretreatment of frozen-thawed sperm with 10 ?M and 20 ?M EGCG could significantly increase the cleavage and blastocyst rates of early embryos in comparison with the control group(P<0.05).In this study,it was found that 50 ?M EGCG could promote the in vitro maturation of bovine oocytes and 10 ?M EGCG could improve the quality of bovine frozen semen and the mechanism may be related with the expression of antioxidant enzymes(genes),which providing theoretical basis for improving the efficiency of in vitro production of bovine embryo.