Effects of leonurine supplementation during in vitro culture on oxidative stress, cell proliferation, apoptosis, and autophagy in bovine embryos

A critical factor contributing to the reduced production and development of embryos during in vitro production is oxidative stress, which arises due to excessive accumulation of reactive oxygen species (ROS). In order to reduce or prevent ROS levels in the culture medium and embryos, the use of antioxidant agents is commonplace. The present study sets out to evaluate the effects of leonurine, a compound with antioxidant properties, when supplemented into the in vitro culture medium, on embryo development and quality, cell proliferation, apoptosis, autophagy, ROS and GSH levels. Blastocyst quality was evaluated by performing differential staining, ROS, GSH, TUNEL, EdU, and LC3B staining on the developing embryos. The results demonstrated that the supplementation of the culture medium with 20 µM leonurine (Leo20) resulted in a significant enhancement of blastocyst development, as evidenced by increased numbers of ICM, TE, and TCC in embryos when compared to the control group. Furthermore, the number of apoptotic cells and the apoptotic index were found to be lower in the Leo20 group than in the control group. Furthermore, the levels of ROS and LC3B were reduced, while GSH levels were elevated in the leonurine-treated group. With regard to cell proliferation, the number of EdU-positive cells and the proliferation rate were found to be significantly higher in the Leo20 group than in the control group. Gene expression analysis revealed upregulation of antioxidant-related genes (SOD, GPX4, and CAT) and pluripotency-related genes (OCT4, NANOG, and SOX2), while the expression of apoptosis-related genes (BAX and CASP3) and autophagy-related genes (ATG12 and LC3B) was downregulated. It has been demonstrated that the supplementation of leonurine to an in vitro culture medium has the potential to enhance the efficiency of blastocyst formation and to improve the viability of embryos, a phenomenon that may be attributed to the ability of leonurine to reduce oxidative stress. Consequently, the reduction of oxidative stress in embryos is hypothesized to result in increased cell proliferation and decreased rates of apoptosis and autophagy.