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The objective of this study was to find out the effect of l-carnitine on oocyte maturation and subsequent embryo development, with l-carnitine-mediated alteration if any in transcript level of antioxidant enzymes (GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2) in oocytes and developing sheep embryos produced in vitro. Different concentrations of l-carnitine (0 mm, 2.5 mm, 5 mm, 7.5 mm and 10 mm) were used in maturation medium. Oocytes matured with 10 mm l-carnitine showed significantly (p < 0.05) higher cleavage (66.80% vs 39.66, 41.76, 44.64, 64.31%), morula (48.50% vs 20.88, 26.01, 26.99, 44.72%) and blastocyst (33.22% vs 7.66, 9.19, 10.71, 28.57%) percentage as compared to lower concentrations (0 mm, 2.5 mm, 5 mm and 7.5 mm). Cleavage percentage between 10 mm and 7.5 mm l-carnitine were not significantly different. Maturation rate was not influenced by supplementation of any experimental concentration of l-carnitine. There was a significant (p < 0.05) decrease in intracellular ROS and increase in intracellular GSH in 10 mm l-carnitine-treated oocytes and embryos than control group. Antioxidant effect of l-carnitine was proved by culturing oocytes and embryos with H2O2 in the presence of l-carnitine which could be able to protect oocytes and embryos from H2O2-induced oxidative damage. l-carnitine supplementation significantly (p < 0.05) upregulated the expression of GPx and downregulated the expression of SOD2 genes, whereas the expression pattern of SOD1 and GAPDH (housekeeping gene) genes was unaffected in oocytes and embryos. It was concluded from the study that l-carnitine supplementation during in vitro maturation reduces oxidative stress-induced embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH that in turn improved developmental potential of oocytes and embryos and alters transcript level of antioxidant enzymes.