Effect Of Laminarin And Keamperfol On Aging Oocytes And Early Embryo Development Of Pig

In vitro culture of pig oocytes and embryos is widely used to study the mechanism of embryo development and animal reproductive technology.Oxidative stress is an important factor affecting the quality of oocytes and embryonic development,so reducing oxidative stress in oocytes and embryos is of great significance in studying the mechanism of embryonic development and animal reproductive technology.The purpose of this study was to investigate the effects of two antioxidants,laminarin(LAM)and kaempferol(KAE),on the aging and early embryonic development of porcine oocytes.Which are mainly divided into the following four experiments:Experiment 1:Effect of laminarin on aging oocytesLaminarin(LAM),a β-glucan mixture with good biocompatibility,improves the growth performance and immune response when used as food additives and nutraceuticals.We found that LAM delayed the aging process and improved the quality of aged oocytes.LAM supplementation enhanced the subsequent developmental competence of aged oocytes during the in vitro aging process.The blastocyst formation rate was significantly increased in aged oocytes treated with LAM compared to non-treated aged oocytes(P<0.01).The mRNA levels of apoptosis-related genes,B cell lymphoma-2-associated X protein(Bax)and Caspase-3,were significantly lower in blastocysts derived from the LAM-treated aged oocytes during the in vitro aging process(P<0.01).Furthermore,the level of intracellular reactive oxygen species was significantly decreased(P<0.01)and that of glutathione was significantly increased in aged oocytes following LAM treatment(P<0.01).Mitochondrial membrane potential was increased(P<0.01),and the activities of caspase-3 and cathepsin B were significantly reduced in the LAM-treated aged oocytes compared with the non-treated aged oocytes(P<0.01).Taken together,these results suggest that LAM is beneficial for delaying the aging process in porcine oocytes.Experiment 2:Effect of laminarin on porcine embryo developmentThe aim of the present research was to explore the effects of LAM on porcine early stage embryo development,as well as the underlying mechanisms.The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LAM supplementation during the in vitro culture period.The presence of 20 μg/mL LAM during the in vitro culture period significantly improved cleavage rates(P<0.01),blastocyst formation rates(P<0.01),hatching rates(P<0.05),and total cell number in the blastocyst compared to that in the control group(P<0.01).Notably,LAM attenuated the intracellular reactive oxygen species generation induced by H202(P<0.01).Furthermore,LAM not only increased intracellular glutathione levels(P<0.01),but also ameliorated mitochondrial membrane potential(P<0.01).In addition,the expression of a zygotic genome activation related gene Yap1,pluripotency-related genes(Oct4,Nanog,and Sox2),and hatching-related genes(Cox2,Gata4,and Itga5)were up-regulated following LAM supplementation during porcine early stage embryo development(P<0.05).Experiment 3:Effect of Kaempferol on aged oocytesKaempferol(3,4’,5,7-tetrahydroxyflavone,KAE)is one of the most commonly occurring dietary flavonols.The biological and pharmacological effects of kaempferol depend upon whether it acts as an antioxidant,anti-inflammatory,or anticancer agent.The present study investigated the influence and underlying mechanisms of KAE supplementation in aged porcine oocytes in vitro.The results show that KAE treatment can alleviate the ageing-related reduction of developmental competence.We observed that the blastocyst rate of 0.1 μM KAE-treated aged oocytes was significantly higher than that of untreated aged oocytes(P<0.05).The KAE treated aged oocytes had a significantly reduced level of reactive oxygen species.Furthermore,the mRNA levels of embryonic pluripotency related genes,Oct4,Nanog,and Itga5 were significantly increased in blastocysts derived from KAE treated oocytes(P<0.05).KAE treatment during excessive oocyte culture maintained the mitochondrial membrane potential(P<0.05)and reduced the apoptosis(P<0.05);however,this was not observed in untreated aged oocytes.In conclusion,our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.Experiment 4:Effect of Kaempferol on porcine embryo developmentThe present study explored the influence of KAE supplementation on in vitro damage to porcine embryo and its underlying mechanisms.Different concentrations of KAE were added to porcine zygote medium 5 during in vitro culture.The results showed that supplementation with 0.1 μM KAE significantly increased the blastocyst formation rate(P<0.05).Blastocyst formation and quality were significantly increased in the 200 μM H2O2 treatment group following addition of 0.1 μM KAE(P<0.05).KAE prevented the H2O2-induced compromise of mitochondrial membrane potential and reactive oxygen species generation(P<0.05).Furthermore,the extent of autophagy(P<0.05)and DNA damage in the blastocysts was attenuated by supplementation with KAE in the H2O2 induced oxidative injury group compared to in controls(P<0.05).These results suggest that KAE has beneficial effects on the development of porcine parthenotes by attenuating oxidative stress and increasing mitochondrial function.In summary,this study shows that both KAE and LAM can effectively improve the in vitro development rate of porcine embryos,maintain the quality of aging oocytes,enhance the development potential of aging oocytes,and delay the aging of oocytes.