Exogenous Glutathione Supplementation In Culture Medium Improves The Bovine Embryo Development After In Vitro Fertilization

Redox balance is important for the development of embryos and exogenous addition of antioxidants in embryo culture medium would scavenge the reactive oxygen species(ROS). It was showed that glutathione(GSH) can neutralizes harmful free radicals and maintain the redox balance in cells. This study aims at the effect and mechanisms of exogenous GSH during the in vitro culture(IVC) on the embryonic development.Different concentrations of GSH were added in the bovine IVF embryo culture medium during IVC to study its effect on embryo development and quality. The cleavage rate, 4- cell rate, 8-cell rate, 16-cell rate, morula rate and blastocyst rate were statistics to determine the best addition concentration. Meanwhile, compared with several blastocyst staining methods, an economical efficient and accurate immunofluorescence triple-staining assay was established to evaluate the quality of bovine blastocysts. Also, the method was used to detect the total cell number(TCN), inner cell mass(ICM) rate and apoptotic cells rate(ACR) for evaluating the effect of GSH on blastocyst quality. The results showed that coincubation of two first/second antibody could greatly shorten the staining time and improve the efficiency. In addition, the trophocyte(TE), ICM and apoptotic cells could be distinguished in the same blastocyst, which improved the accuracy of the blastocyst quality assessment. Compared with the control, the 3m M GSH treatment significantly increased the blastocyst rate and embryo quality. And the highest blastocyst rate(51%) and the best embryo quality appeared in the 3m M GSH treatment. For these reasons, 3m M GSH was the best concentration for bovine IVF embryos.In the condition of 3m M GSH treatment, intracellular GSH and ROS contents in bovine IVF embryos were detected. Additionally, the real-time PCR was used to determine the expression level of embryo development genes(Oct4, Sox2, and Klf4), GSH synthetize genes(Gclm, Gclc, and Gss) and GSH metabolic genes(Gstp, Gstm, and Gpx). The results showed that intracellular content of GSH of embryos at 2- to 4- cell stage significantly increased and the ROS level decreased accordingly with 3m M GSH treatment. However no significant difference was found between 4- to 8- cell stage embryos and blastocysts. QPCR results showed that there was no significant difference in those genes expression level between the two groups. Therefore the exogenous has no significant effect on the intracellular GSH synthesis and metabolism.The transcriptomics and lnc RNA expression between control and GSH treatment in 8- to 16- cell stage embryos and morale were compared. The RNA-seq results showed that the GSH complicated the development process from 8- to 16-cell stage to morula stage of bovine IVF embryos. In addition, the GSH metabolism pathway was active, which maybe the key factors of the changes induced by GSH. Screening the lnc RNAs, we forecasted 4,275 lnc RNAs among which five lnc RNAs were highly expressed. The lnc RNA CUFF.21976.3 may impact the nearby genes such as ATP6V1C2、RRM2、GRHL1、KLF11、ODC1 and HPCAL1, which matter the cell developmental and cell biological processes. In brief, exogenous GSH can promote anti-oxidative effects significantly in bovine IVF embryo and improve the efficiency of embryo IVC.