Effect of LPS on In Vitro Embryo Development in Bubalus bubalis : A Model for Studying Inflammatory Pathways

Reproductive efficiency and female fertility are crucial for productive and sustainable reproductive outcomes in buffalo. Endotoxin lipopolysaccharide (LPS) produced by Gram-negative bacteria initiates a series of inflammatory cascade events which affect ovarian steroid production, impact oocyte and ultimately hamper the developmental competence of the embryo. The objective of the current study was designed taking endometritis as an in vitro model to elucidate the molecular pathway through which LPS affects embryo competence. To achieve this objective, buffalo ovaries were procured from a nearby slaughterhouse and oocytes were aspirated via the follicular aspiration method with an 18-gauge needle fitted with a 10 mL syringe. After searching and washing, good quality (A and B grade) oocytes were subjected to IVM, IVF and subsequently IVC, with media supplemented with 5 μg/mL of LPS based on previous laboratory standardisation. Our results demonstrate that LPS exposure reduced cleavage rate, blastocyst formation rate, total cell count, mitochondrial membrane potential (MMP), trophectoderm: inner cell mass (TE: ICM) ratio while significantly (p < 0.05) increasing reactive oxygen species (ROS) levels and apoptotic rate in blastocysts. Furthermore, LPS exposure significantly (p < 0.05) upregulated the relative mRNA expression of inflammatory (TLR4, CD14, RPS27A) and apoptotic gene (Caspase 3) whereas it downregulated the expression of antioxidant (GPX1) and pluripotent gene (Oct4) in blastocysts. Based on these findings, we concluded that although a concentration of 5 μg/mL LPS is minimally harmful to the oocytes, its presence during IVC adversely affects embryonic development.