Effects Of Proanthocyanidins On The Development Of Bovine Oocytes In Vitro

In vitro maturation of bovine oocytes and in vitro development of embryos are important components of in vitro embryo production and embryo engineering.However,due to the low maturation rate of oocytes and blastocyst rate,in vitro production of embryos and development of animal husbandry are seriously restricted.Studies have shown that oxidative stress is the main factor affecting embryo development.Proanthocyanidin is a polyphenol compound with strong antioxidant activity,which can effectively eliminate superoxide anion and hydroxyl free radicals.So far,no studies have been reported on the effect of proanthocyanidins on bovine oocytes and granulocytes.This study aims to explore the effects of PCs on the in vitro maturation of bovine oocytes and the development of parthenogenetic embryos in vitro,as well as the effects on granulocytes under oxidative stress,so as to lay a foundation for the further optimization of bovine embryo development system in vitro.The specific results of this study are as follows:Experiment 1:To investigate the effect of different concentrations of PCs on the in vitro maturation of bovine oocytes.Bovine oocytes were pretreated for 22-24 h with maturation solution(IVM)containing PCs at different concentrations(0,5,10,25,50)μmol/L.The oocyte maturation rate,cleavage rate and blastocyst rate of parthenogenetic embryos were counted,and the optimal PCs addition concentration was selected.The results showed that the expulsion rate of the first polar body in the group treated with PCs supplemented with 25 μmol/L in IVM(71.85%±5.34)was significantly higher than that in the control group(64.82%±2.34)(P<0.05),and there was no significant difference between the other groups and the control group(P>0.05);the results of parthenogenetic embryos showed that the cleavage rate in the group treated with PCs at 25 μmol/L(69.79%±4.39)was significantly higher than that in the control group(63.16%± 4.01),and there was no significant difference between the other groups and the control group(P>0.05);the blastocyst rate in the groups treated with PCs at 10,25,and 50 pmol/L(30.07%±2.85,32.92%±1.12,30.55%±1.51)was significantly higher than that in the control group(25.6%±1.49)(P<0.05),but there was no significant difference between the control group with PCs at 5 μmol/L(P>0.05).Experiment 2:The mechanism of PCs improving bovine oocyte quality was preliminarily investigated.The effects of 25 μmol/L PCs on intracellular reactive oxygen species(ROS)and glutathione species(GSH)content in bovine oocytes were investigated by comparing the PCs treatment groups with each other for 22-24 h.The results showed that compared with the control group,25μmol/L PCs treatment group could significantly reduce the ROS content in oocytes(44.50±8.82 VS 34.51±6.76,P<0.05).PCs treated with 25 μmol/L could significantly reduce the level of GSH in oocytes(62.25±5.25 VS 84.83±10.56,P<0.05).Real-time fluorescence quantification results showed that 25 μmol/L PCs treatment group significantly increased the expression of antioxidant related gene SOD1,embryo development related gene Oct4 and anti-apoptotic gene Bcl-2 mRNA,and down-regulated the expression of pro-apoptotic gene Bax mRNA(P<0.05).Experiment 3:To investigate the protective effect of PCs on oxidative damage of bovine granulosa cells.H2O2 with different concentration gradients(0、100、200、500、1000 μmol/L)were added into the culture medium of granulosa cells and treated for 3 hours.The results showed that the survival rate of cells in the 500 μmol/L H2O2 treatment group was the closest to 50%,which was the optimal treatment concentration.DAPI/TUNEL double staining was used to detect the cell apoptosis in the control group,model group and PCs pretreatment group.The results showed that the apoptosis rate of granulosa cells in model group was significantly higher than that in control group(P<0.05).The apoptosis rate of PCs preconditioning group was significantly lower than that of model group(P<0.05).Experiment 4:The mechanism of PCs action on bovine oxidative stress granulosa cells was preliminarily investigated.The expressions of apoptosis related genes Bax and Bcl-2 and antioxidant related gene SOD]mRNA were detected by real-time quantitative PCR.The results showed as follows:compared with the control group,the expression of anti-apoptotic gene Bcl-2 and antioxidant gene SOD]in model group were significantly decreased,and the expression of apoptotic gene Bax was increased(P<0.05).Compared with model group,PCs+H2O2 group increased the expression of anti-apoptotic gene Bcl-2,antioxidant gene SOD1,and decreased the expression of apoptotic gene Bax(P<0.05),thus increasing the ratio of Bcl-2/Bax.In summary,this study showed that 25 μmol/L PCs could effectively promote bovine oocyte development and inhibit granulosa cell apoptosis,and preliminarily explored the mechanism of PCs on oocytes and granulosa cells under oxidative stress.It lays a foundation for further optimization of bovine oocyte in vitro culture system and industrialized production of bovine in vitro embryos.