Effects Of Shikimic Acid On Porcine Oocyte In Vitro Maturation And Parthenogenetic Embryo Development

Reducing the level of oxidative stress can effectively improve the efficiency of porcine in vitro embryo production(IVP)including oocyte in vitro maturation(IVM)and in vitro embryo culture(IVC).Therefore,optimizing the embryo culture system,improving the antioxidant capacity of oocytes and embryos is meaningful for improving the efficiency of IVM and IVC.Shikimic acid(SA)is widely present in a variety of plants and microorganisms.It has various pharmacological activities such as inhibition of platelet aggregation,venous and cerebral thrombosis,anti-inflammatory,analgesic,antioxidant,and antibacterial activities.However,the effects of shikimic acid on animal reproduction systems,especially oocyte maturation and early embryonic development are still unclear.Therefore,in this study,SA was added to the culture medium to clarify the effects and potential mechanism of SA on porcine oocyte maturation in vitro and early embryonic development.We first added SA with different concentrations(0 μM,20 μM,40 μM,60 μM)to the porcine IVM system and calculated the first polar body extrusion rate of oocytes to explore the most suitable concentration for enhancing the oocyte maturation.The results showed that the addition of 40 μM SA could significantly increase the maturation rate of oocytes and the cleavage rate,and blastocyst rate of the subsequent parthenogenetic activated embryo.Next,we used fluorescent staining,chemiluminescence absorption,immunofluorescence,and quantitative RT-PCR(q RTPCR)methods to explore the effects of SA on ROS level,GSH level,SOD level,total anti oxidation capacity(T-AOC),mitochondrial membrane potential(MMP)and distribution,cortical granule distribution,β-galactosidase,DNA double-strand breaks in MII stage oocytes,as well as m RNA levels of oocyte quality-related genes.The results showed that the GSH level,SOD level,T-AOC level,normal cortical granule distribution rate,normal mitochondrial distribution rate,and the level of MMP were significantly higher in the SA-addition group.At the same time,the levels of oocyte quality-related genes MOS,CCNB1,GDF9,and BMP15 were significantly higher than those in the control group;while the ROS levels,β-galactosidase level,and DNA double-strand breaks-positive MII stage oocytes were lower than that in the control group.Subsequently,we added SA with different concentrations(0 μM,20 μM,40 μM,60 μM)to the porcine IVC system.The results showed that the addition of 40 μM SA could increase the cleavage rate,blastocyst rate,blastocyst diameter and total cell number in the blastocyst.Next,we used fluorescence staining,TUNEL,and q RT-PCR to clarify the ROS and GSH level in the 4-cell stage parthenogenetic activated embryos at the critical stage of porcine zygotic genome activation with or without 40μM SA,as well as MMP,apoptosis rate of blastomeres,and m RNA levels of blastocyst quality-related genes.The results showed that the addition of SA significantly increased the levels of GSH and MMP in the 4-cell stage parthenogenetic embryos and effectively reduced the ROS accumulation.In addition,the addition of SA significantly reduced the rate of apoptotic cells in the blastocyst and increased expression level of Nanog,Sirt-1,Sox-2,and Cox-2 while reducing the level of Caspase-3.In summary,this study confirmed that SA(40 μM)addition to the porcine IVM system could increase the antioxidant capacity of oocytes,improve mitochondrial function,and enhance the subsequent development potential of oocytes.The addition of SA(40 μM)to the IVC system could reduce the apoptosis,improve embryonic mitochondrial function,antioxidant capacity,and potentials of embryonic development.These results provide a new theoretical basis for analyzing the biological effects of SA and optimizing the porcine embryo IVP systems.