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The present study aimed to evaluate the influence of myoinositol (MYO), ferulic acid (FA), and melatonin (MEL) in equine cooled semen. Ejaculates were collected and distributed into the following four treatments: MYO, FA, MEL, and control. A skim milk–based extender was used. Samples were cooled at 5°C and evaluated at 0, 4, and 8 hours after storage for motility, plasma and acrosomal membranes integrity, mitochondrial potential, and production of reactive oxygen species (ROS). Motility characteristics were not affected by treatment, except for the amplitude of lateral head displacement, which was higher in MYO (8.3 ± 0.2) compared with the control group (7.8 ± 0.2). No difference was observed among treatments for intact plasma membrane (%). However, the percentage of cells with intact plasma and acrosomal membranes and high mitochondrial potential was greater in the MEL (78.1 ± 2.0) and FA groups (78.8 ± 1.7) compared with the control group (73.8 ± 2.0). The high mitochondrial potential (%) was also greater in groups treated with MEL (80.1 ± 1.9) and FA (81.0 ± 1.5) compared with the control group (76.6 ± 2.0). In addition, percentage of cells with intact acrosome membrane was greater in MEL group (99.7 ± 0.1) compared with all other treatments. ROS production was not affected by treatments. In conclusion, FA and MEL provided the best protection to mitochondria, acrosome, and plasma membranes, suggesting that the addition of these antioxidants to equine semen extender can improve sperm quality.Ferulic acid and melatonin provided the best protection to all membranes.Melatonin increases the percentage of cells with intact acrosome.No difference was observed among treatments for intact plasma membrane (%).Motility characteristics were not affected, except for the amplitude of lateral head displacement (higher in myoinositol).Reactive oxygen species production was not affected by treatments.