Study On Allicin Alleviated Lipopolysaccharide-induced In Vitro Maturation Damage Of Porcine Oocytes

Lipopolysaccharide(LPS)is a virulence factor released by Gramnegative bacteria and is one of the important pathogenic factors that cause reproductive diseases in livestock.Multiple studies have reported that LPS exposure can leads to oocyte maturation damage and retarded embryonic development.Allicin,is a bioactive component extracted from the bulb of garlic,has a variety of pharmacological activities and is beneficial to for oocyte maturation process.However,whether allicin can alleviate the oocyte maturation injury induced by LPS exposure and its possible mechanism have not been elucidated.In this experiment,porcine oocytes were used as the research object,and various methods were used to do the detection such as in vitro oocyte culture,drug treatment,parthenogenetic activation,immunofluorescence staining,laser confocal,and real-time quantitative fluorescence PCR et al.The aim of this study was to preliminarily explore the effect of allicin on alleviating the decline in the quality of porcine oocyte maturation caused by LPS exposure and promoting the development of parthenogenetic embryos subsequently.This study will provide a theoretical basis for the alleviation of reproductive damage caused by LPS exposure.This research can be mainly divided into three parts:(1)Effect of allicin alleviates the porcine oocyte nuclear maturation damage by LPS exposure.We added different concentrations of LPS(0,5,15,25 μg/m L)to the oocyte culture medium TCM-199,to construct a porcine oocyte LPS injury model,and examined and counted the first polar body expulsion rate of oocytes in each group.The results showed that the polar body expulsion rates of the 15 μg/m L and 25 μg/m L LPS treatment groups were significantly reduced compared with the control group.To ensure the experimental effect,15 μg/m L LPS were chosen as the optimal treatment concentration in the follow-up study.Then,various concentrations of allicin(0.1,1,10 μM)were added to the porcine oocyte TCM-199 maturation medium containing 15 μg/m L LPS.The results showed that 0.1μM and 1 μM allicin addition could significantly increase the expulsion rate of its polar bodies,compared with the LPS group,and the concentration of allicin at 1μM had better effect.Therefore,to better investigate the relieving effect of allicin,we chose 1 μM for follow-up experiments.Subsequently,we further found that LPS treatment disrupted the normal assembly of spindle,resulting in disordered chromosome arrangement and impaired the assembly of microfilament construction.Detecting the oocytes under the combined action of allicin and LPS,we found that the above abnormal phenomenon has been significantly alleviated.The above results confirmed that allicin could alleviate the damage of LPSinduced porcine oocyte nuclear maturation.(2)Effect of allicin alleviates the cytoplasmic maturation injury of porcine oocytes by LPS exposureCompared with the control group,LPS-treated pig oocytes showed the abnormal distribution of cortical granules,and the significantly decreased of level ATP,the level of autophagy was also inhibited and,the relative expression of autophagy-related genes was significantly down-regulated,and the level of reactive oxygen species in oocytes was significantly reduced.The relative expression of antioxidant genes was significantly down-regulated,time,the early apoptosis rate of oocytes was also significantly increased.However,all of the above resulting abnormalities were significantly improved after we administered allicin supplementation to LPS-treated porcine oocytes.Therefore,the results of this experiment indicated that allicin could alleviate the cytoplasmic maturation damage of porcine oocytes caused by LPS exposure.(3)Effect of allicin alleviates reduction of parthenogenetic embryonic development ability of porcine oocytes by LPS exposure.In this study,we selected mature porcine oocytes from control group and treatment groups for pulsed electrical activation to produce parthenogenetic embryos for culture.The results showed that LPS treatment had no significant effect on the cleavage rate and blastocyst cell activity of parthenogenetic embryos,but the blastocyst rate and the total number of cells in blastocysts were significantly reduced,and the blastocyst rate and the total number of cells in blastocysts of parthenogenetic activated embryos were significantly improved by supplementation with allicin.The results of this experiment indicated that LPSexposed porcine oocytes treated with allicin could promote their subsequent parthenogenetic embryonic development.In conclusion,all these results demonstrate that allicin can effectively alleviate the maturation damage of porcine oocytes caused by LPS exposure and improve the subsequent development of parthenogenetic embryos.This study has important theoretical and practical value for the optimization of porcine oocyte in vitro maturation system using plant-derived active substances.