Studies Related To The Effect And Mechanism Of Betaine On Porcine Oocyte In Vitro Maturation

Compared to oocytes matured in vivo,oocytes matured in vitro(In Vitro Maturition,IVM)are susceptible to stimulation by oxidative stress,which affects the quality of oocyte maturation.Betaine(Bet)is found in red beet and has good antioxidant properties.However,the role of Bet in regulating the maturation of porcine oocytes and improving the reprod uctive capacity of livestock has been rarely reported.In this study,we investigated the effect of Bet on the in vitro maturation of porcine oocytes and analysed its potential molecular mechanism,with a view to providing theoretical support for optimising the in vitro maturation system of porcine oocytes.The main research elements and results are as follows:1.Effects of Bet on in vitro maturation of porcine oocytes and subsequent embryonic developmentFirstly,we investigated the effect of adding different concentrations of Bet(0 mmol/L,8 mmol/L,16 mmol/L and 32 mmol/L)to the maturati on medium on the in vitro maturation of porcine cumulus-oocyte complexes(COCs),the results showed that after 44 h of in vitro culture,the first polar body expulsion rate of porcine oocytes in the 16 mmol/L Bettreated group was significantly higher than that in the 0 mmol/L control,4 mmol/L,8 mmol/L and 32 mmol/L-treated groups(P<0.05).Subsequently,each group of MII stage porcine oocytes was subjected to parthenogenetic activation(PA),and the development of PA embryos was observed and counted,the results showed that the cleavage rate and blastocystr ate of PA embryos in the 16 mmol/L Bet-treated group were significantl-y higher than those in the control group(P<0.05).Therefore,16 mmol/L was selected as the optimum concentration of Bet.Second,the effect of Bet on the distribution of cortical granules in porcine oocytes was explored and the results were not significant(P>0.05).2.Studies on the mechanism of Bet in influencing the in vitro maturation of porcine oocytesIn order to investigate the mechanism of Bet in promoting the in vitro maturation of porcine oocytes,the antioxidant capacity of porcine oocytes was first detected and analyzed by immunofluorescence staining and qRT-PCR.The results showed that the expression of antioxidant genes CAT,GSH-Px and SOD1 were significantly increased(P<0.05)and the reactive oxygen species level was significantly decreased(P<0.05)in the Bet-treated porcine oocytes compared to the control group.Secondly,Bet treatment was found to be effective in increasing mitochondrial content and enhancing mitochondrial function by measuring mitochondrial co-ntent,membrane potential levels,ATP and expression of the fusion-fission genes MFN2,OPA1 and DRP1.Subsequently,the effect of Bet on the meiotic spindle and chromosome morphology of porcine oocytes was assessed.The results revealed that Bet treatment significantly reduced the rate of spindle and chromosome morphological abnormalities(P<0.05).In addition,to further investigate whether Bet enhances the antioxidant capacity and mitochondrial function of porcine oocytes via the AMPK pathway,the AMPK-specific inhibitor Compound C was added to the maturation fluid.The results showed that Bet treatment rescued the decrease in expression of key AMPK pathway genes,reduced expression of an tioxidant genes,excessive accumulation of ROS and abnormal mitochondrial content and function caused by Compound C compared to the control group.The above findings suggest that the addition of Bet(16 mmol/L)to the in vitro maturation medium significantly improves the in vitro maturation efficiency and early embryonic development potential of porcine oocytes,possibly by activating the AMPK pathway to enhance the antioxidant capacity and mitochondrial function of oocytes,thereby promoting the in vitro maturation and subsequent embryonic development of porcine oocytes.