Oocyte in vitro maturation (IVM) is crucial for subsequent in vitro embryo production. It involves acquisition of competence for fertilization and embryo development. Therefore, its optimization could have a direct impact on in vitro embryo development. Dimethyl sulfoxide (DMSO) is commonly used as solvent or vehicle, but also increases the membrane permeability and behaves as a scavenger of cytotoxic free radicals. The aim of this study was to evaluate the effect of DMSO supplementation during bovine oocyte maturation on subsequent in vitro embryo development and to determine the optimal usage dose with no toxic effect. To this aim, cumulus-oocyte complexes were collected from slaughterhouse ovaries and IVM in TCM 199 containing 10% fetal bovine serum, 10 µg/mL of FSH, 0.3 mm sodium pyruvate, 100 mm cysteamine, and 2% antibiotic-antimycotic. The oocytes were incubated for 24 h at 6.5% CO₂ in humidified air at 38.5°C. For Experiment 1, IVM medium was supplemented with DMSO at concentrations of 0, 0.1, 0.5, 1, or 10% (vol/vol) DMSO (n = 241, 195, 42, 192, 172 oocytes) and IVM rate was determined by presence of the first polar body. For Experiment 2, 0, 0.1, 0.25, 0.5, 0.75, 1, or 10% (vol/vol) DMSO (n = 446, 322, 65, 194, 77, 250, 39 oocytes) was supplemented to IVM medium and cleavage and blastocyst rates were determined to establish the optimal usage dose. In vitro fertilization was performed according to Brackett and Oliphant (1975), with 16 × 10⁶ spermatozoa/mL for 5 h. Afterwards, presumptive zygotes were cultured in SOF for 7 days at 38.5°C and 5% O₂. Cleavage and blastocyst rates were determined on Days 2 and 7, respectively. Results were statistically analysed using Fisher’s exact test by GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Also, the percentage of blastocyst was adjusted to DMSO concentration using the R software quadratic regression model. The optimum usage dose was determined by calculating the maximum of the estimated predictive equation. In vitro maturation in 10% DMSO resulted in significantly lower first polar body extrusion rates (0% = 74%^a, 0.1% = 73%^a, 0.5% = 83%^a, 1% = 66%^a, and 10% = 8%^b; different letters indicate statistical differences) and lower cleavage rates (0% = 75%^a, 0.1% = 77%^a, 0.25% = 80%^a, 0.5% = 79%^a, 0.75% = 78%^a, 1% = 77%^a, and 10% = 3%^b) than the other treatments. Furthermore, blastocyst production was higher for the 0.25 and 0.5% (vol/vol) supplemented DMSO groups (0% = 26%^b, 0.1% = 37%^ab, 0.25% = 40%^a, 0.5% = 41%^a, 0.75% = 34%^ab, 1% = 23%^b, and 10% = 0%^c). The predictive equation results indicate that the maximum percentage of blastocysts is obtained with a concentration of 0.458% (vol/vol) of DMSO. In conclusion, DMSO supplementation during IVM of bovine oocytes had a positive effect on in vitro development. Further studies will be carried out to elucidate its mechanism of action.