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This study examined the effects of skim-milk based extenders supplemented with proline and glutamine on motility, lipid peroxidation and enzymatic antioxidant status of cooled-stored equine sperm and determined the role of seminal plasma as well. The semen was collected with artificial vagina. In experiment 1, native semen was diluted in skim-milk based extender containing 5mM glutamine and 3mM proline, stored at 5°C and analyzed at 4, 24 and 48 hours storage for motion parameters. In experiment 2, semen was centrifugated, sperm pellet resuspended in the extenders and stored at 5°C for 4 hours to determine motion parameters. The level of catalase, glutathione peroxidase activity and malondialdehyde formation was determined for all samples at 4 hours. Glutamine and proline signifi cantly preserved the percentage of motile sperm (76.5 ± 2.7 and 79.4 ± 1 vs 69 ± 1.4), increased the progressive motility of cold-stored semen (66.1 ± 2.5 and 73.7 ± 2.9 vs 56.2 ± 1.4), increased catalase activity, and decreased malondialdehyde. However these effects were disappeared after seminal plasma removal. We conclude that glutamine and proline would amplify the antioxidant activity of equine cold-stored semen and preserve its motility. This effect seems to be related to interactions with seminal plasma.