Simple SummarySperm has oxidative stress during freezing in boar semen. Oxidative stress causes reactive oxygen species to occur and produces and nitric oxide. The viability and motility of sperm are damaged by oxidative stress. We investigated the effects of melatonin and silymarin on reactive oxygen species, nitric oxide production, sperm viability, and motility in frozen–thawed boar semen. As a result, melatonin and silymarin elevated the viability and motility of sperm, and also reactive oxygen species and nitric oxide production decreased. Therefore, we suggested that melatonin and silymarin are beneficial for antioxidants during freezing sperm in pigs.AbstractSperm during the freezing and thawing process is damaged by oxidative stress. Thus, its antioxidant scavenger is essential for sperm survival and death in frozen–thawed semen. We used melatonin and silymarin in experiments after the dose-dependent experiment. Our study aimed to identify the effect of melatonin and silymarin on the motility and viability of sperm, reactive oxygen species (ROS), and nitric oxide (NO) production in frozen–thawed boar semen. Melatonin and silymarin were treated alone and cotreated in the fresh boar semen. Boar semen was collected using the gloved-hand method from ten crossbred pigs, and samples were used in the experiments. We evaluated sperm viability using SYBR-14 and PI kit, and ROS and NO production were detected by DCF-DA and DAF-2, respectively. The sperm motility was not significantly different between non-treatment and treatment. ROS and NO production in frozen–thawed sperm were decreased by melatonin and silymarin. Moreover, silymarin significantly reduced NO production more than melatonin. Melatonin and silymarin enhanced the viability of sperm. We suggest that melatonin and silymarin are essential antioxidants in semen cryopreservation for protecting sperm damage and maintaining sperm viability. Melatonin and silymarin may be useful antioxidants in freezing boar sperm.