Effect Of Asiatic Acid On In Vitro Development Of Porcine Oocytes And Early Embryos

The porcine oocytes IVM and the early embryos IVC is the important content of the livestock embryos development research and the operation of embryo engineering.It is a major problem that the poor quality of oocytes and embryos obtained in vitro compared with in vivo.IVM of oocytes and IVC of embryos are easily affected by many factors such as free radicals.Asiatic acid is a pentacyclic triterpene that exists in Centella asiatic.Its main functional groups include three hydroxyl groups,an unsaturated bond and a carboxyl group.Previous studies have suggested that asiatic acid also has a variety of pharmacological activities,including anti-apoptosis,anti-aging,antiinflammation and anti-oxidation,etc.However,the effect of asiatic acid on oocyte maturation and early embryonic development has not been reported.The purpose of the current study was to explore the effects of asiatic acid on porcine oocytes IVM and early-stage embryos IVC and the potential mechanisms.The details are as follows:Experiment 1:The effects of asiatic acid on oocyte maturation were studied by supplementting asiatic acid at IVM stage.IVM of the GV stage oocytes after 42-44h that from the ovary.And the maturation rate was counted of M?period oocytes.The result showed that the maturation rate has slight improved but there was no significant difference compared with control group.However,the embryo development have significantly improved of PA and IVF early embryos supplementation asiatic acid during IVM period and the blastocyst quality also have enhanced.The oocytes'antioxidant capacity also was tested.The results showed that supplementation with asiatic acid prominently decreased the intracellular ROS levels and increased the GSH levels.The asiatic acid alleviate mitochondrial dysfunction caused by ROS and increased intracellular ATP level.Meanwhile,that asiatic acid effectively alleviated the senescence process of oocytes by?-galactosidase fluorescent staining.In addition,except oocytes,the status of granulosa cells was also detected that the closely related oocyte.The result showed that supplement with asiatic acid increased the cumulus cells diffusion area after co-cultured with oocytes.Moreover,the flow cytometry result showed that the antioxidant capacity increased and the apoptosis rate decreased of granulosa cells.Experiment 2:The effects of asiatic acid on early embryos development werestudied by supplementting asiatic acid at IVC stage.The results showed that 10?M asiatic acid supplementation during the IVC period dramatically improved developmental competence in porcine embryos derived from PA,SCNT and IVF.Further analysis revealed that asiatic acid attenuated H₂O₂-induced intracellular ROS generation.Notably,asiatic acid not only enhanced intracellular GSH levels but also attenuated mitochondrial dysfunction.Gene expression analysis revealed that asiatic acid upregulated expression of the antioxidant-related gene Sod-1 and the blastocyst formation related gene Cox-2,while downregulating expression of the apoptosis-related gene Caspase-9 in SCNT blastocysts.In summary,the results showed that supplementation asiatic acid during oocyte IVM stage can effectively improve the antioxidant capacity of oocytes,improve oocyte quality and promote the development ability of early embryos.Moreover,supplementation asiatic acid during embryos IVC stage can improve the development potential of early embryo and protect early embryo from oxidative stress.