Effects of adding different concentrations of melatonin (10 , 10 and 10 M) to maturation (Experiment 1; Control, IVM + 10 , IVM + 10 , IVM + 10 ) and culture media (Experiment 2; Control, IVC + 10 , IVC + 10 , IVC + 10 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10 M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM + 10 , IVC + 10 , IVM/IVC + 10 ). In Experiment 1, maturated oocytes from Control and IVM + 10 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC + 10 (43.5%; 56.7%) and IVC + 10 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC + 10 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC + 10 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC + 10 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC + 10 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM + 10 . Reactive oxygen species production was greater in the IVM/IVC + 10 treatment group. In conclusion, the 10 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.