Objective:To study the effect of L-ascorbic acid supplementation on the in vitro development of buffalo embryos and evaluate the relative mRNA abundance of some pro-apoptotic, anti-apoptotic, and embryonic development-related genes. Methods:In experiment 1, we evaluated the effect of the addition of 0 (control), 50, and 100 μM L-ascorbic acid to the in vitro maturation medium on the developmental competence in terms of blastocyst rate and relative mRNA abundance of some pro-apoptotic (BAX, BID), anti-apoptotic (BCL-XL, MCL1), and embryonic development (GDF9, BMP15) related genes. Based on the results, we chose 50 μM as the suitable dose of L-ascorbic acid for the subsequent experiments. We further evaluated the blastocyst rates following the addition of 50 μM L-ascorbic acid to the in vitro culture medium (experiment 2), and in vitro maturation and in vitro culture media (experiment 3). In all three experiments, the maturation and culture media devoid of L-ascorbic acid served as the control group. Results:The blastocyst rate after adding 50 μM L-ascorbic acid to the in vitro maturation medium was significantly higher than the control group (P<0.05), whereas 100 μM L-ascorbic acid exhibited a negative effect on the blastocyst rate. The blastocyst rates for embryos cultured in 50 μM L-ascorbic acid in the in vitro culture medium alone and both in vitro maturation and in vitro culture media were significantly higher than their corresponding control groups (P<0.05). The relative mRNA abundance of BAX significantly decreased in blastocysts produced after the addition of 50 μM L-ascorbic acid as compared with the control group (P<0.05), whereas, for MCL1, it significantly decreased in blastocysts produced after the addition of 100 μM L-ascorbic acid (P<0.05). Conclusions:The supplementation of 50 μM L-ascorbic acid to in vitro maturation and in vitro culture media supports in vitro embryonic development in buffaloes by improving developmental competence and altering the expression of apoptosis-related genes.