This study’s goal was to reveal the effects of antioxidant supplementation on motility, motion characters, morphology, DNA integrity, and fertilizing potential of cryopreserved bovine sperm. The ejaculates were collected from three Holstein bulls. At least ten
ejaculates were collected per bull. The ejaculates were immediately separated into five aliquots, diluted in the Cryobos (Magapor Co. Ltd., Zaragoza, Spain) commercial extender, including 2.5 mM taurine, 2.5 mM cysteine, 2.5 mM methionine, 2.5 mM glutamine, and no additives were used. Afterward, they were frozen in 0.25 mL French straws, and liquid nitrogen was used for the storage of semen. The supplementation of methionine resulted in a higher subjective motility rate in comparison with the other groups. Taurine led to the lowest post-thawed CASA motility rate (p < 0.05). The addition of antioxidants did not cause any improvements in sperm motion characteristics when compared to the controls (p > 0.05). Cysteine led to a higher protection of acrosome abnormality, in comparison with the other groups (p < 0.05). For the comet test, the minimum percentage of sperm with damaged DNA was obtained in the groups with cysteine and glutamine (p < 0.05). There was not any significant difference among the groups in terms of pregnancy rates (p > 0.05). In conclusion, cysteine supplementation to the semen extender prior to freezing ameliorated the post-thawed semen quality.