| Reactive oxygen species(ROS)produced by long-term consumption of highenergy and high-protein diets tend to cause delayed sexual maturity,less ovulation,low pregnancy rate and low fecundity of sows,which restrict the healthy development of pig husbandry in China.The addition of antioxidants to the diet can effectively alleviate the above situation.Carnosic acid(CA)has the characteristics of strong antioxidant capacity,stable structure,and low production cost.In recent years,it has been used in feed production as an additive.However,relatively few evaluations have been made regarding its effects on the female reproductive system,especially oocyte maturation and early embryonic development.Therefore,in this study,pig oocytes and parthenogenetically activated embryos cultured in vitro were used as the research objects,and the effects of CA on oocyte maturation and early embryos were discussed from the perspectives of cellular antioxidant capacity and mitochondrial function by adding CA to the culture medium,which can explore the technical measures to improve the fertility of sows through nutrition and reproduction regulation.The details are as follows:In order to clarify the effects of CA on the maturation of porcine oocytes,10 μM CA was added to the in vitro maturation medium of oocytes based on previous research results.Real-time quantitative PCR(RT-q PCR)and other methods detected changes in the degree of cumulus cell proliferation and the rate of oocyte first polar body excretion.Subsequently,in this study,fluorescence staining,chemiluminescence,RT-q PCR,and other methods were used in this study to detect the total ROS levels,glutathione(GSH),mitochondrial membrane potential(MMP),and ATP production,respectively.The results showed that the rate of the first polar body excretion of oocytes in the CA supplemented group was significantly higher than that in the control group(p < 0.05),the expansion area of cumulus cells increased 1.51 ± 0.18 times that of the control group(p < 0.05),and the cumulus cells proliferation-related genes HAS2(p < 0.01)and PTX3(p < 0.05)were significantly up-regulated,but the expressions of AREG and COX2 had no significant effects(p > 0.05).At the same time,the level of ROS in oocytes in the CA supplemented group was decreased by0.76 ± 0.03 times that of the control group(p < 0.05),GSH levels,mitochondrial MMP levels,and mitochondrial ATP production were 1.3 ± 0.05-fold(p < 0.05),1.26± 0.46-fold(p < 0.05),and 1.40 ± 0.09-fold(p < 0.01)higher than those in the control group,respectively.These results indicate that 10 μM of CA can significantly improve oocyte quality and its maturation rate.After confirming that the addition of CA can improve the quality of oocytes in vitro cultured,in order to clarify the effects of CA on the early embryonic development of pigs,different concentrations of CA(0 μM,5 μM,10 μM,20 μM,50μM)were added to in vitro culture(IVC)medium in this study.Firstly,the changes of cleavage rate,blastocyst rate,and hatching rate of embryos at different stages were detected.Then,the changes of total cell number,apoptosis rate,ROS level,GSH level,and MMP level of early parthenogenetic embryos were detected by fluorescent staining method.Finally,the changes of expression levels of early embryo-related functional genes were detected by RT-q PCR.The results showed that compared with the control group,the cleavage rate,blastocyst rate,and hatching rate of the parthenogenetically activated embryos in the 10 μM CA-treated group were significantly increased(p < 0.05 or p< 0.01).Day 7,the total number of cells at the blastocyst stage of the parthenogenetically activated embryos increased significantly,59.25 ± 21.36 in the control group and 72.34 ± 23.05 in the CA-treated group(p <0.01);and decreased the number of apoptotic cells,the cells apoptotic rate was 6.71 ±2.85% in the control group and 3.10 ± 1.71% in the CA-added group(p < 0.05).However,at the 4-cell stage during the critical period of zygotic genome activation in pig embryos,the level of ROS in the CA supplemented group decreased to 0.81 ±0.23 times(p < 0.01)that of the control group,and the levels of GSH and MMP increased to 1.41 ± 0.24 times(p < 0.01)and 1.34 ± 0.48 times(p < 0.01)that of the control group,respectively.In addition,the addition of CA up-regulated the expression levels of NANOG,SOX2,GATA4,COX2,ITGA5,and RICTOR(p < 0.05 or p < 0.01)and down-regulated the expression levels of pro-apoptosis-related genes BIRC5(p < 0.05)and Caspase 3(p < 0.01)in blastocysts.These results suggest that10 μM CA has the effects of promoting parthenogenetic embryos development by enhancing antioxidant capacity.In conclusion,CA promotes oocyte maturation and early embryonic development by improving intracellular antioxidant capacity in vitro.Importantly,CA can enhance mitochondrial function and regulate embryonic pluripotency gene expression.However,the corresponding results also showed that excess CA(above 25 μM)inhibited embryonic development.The research results provide a theoretical basis for in-depth understanding of the biological roles of CA,analysis of its molecular regulation mechanism,and its development and utilization as a feed additive. |
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