The present study aims to compare different antioxidant treatments added to semen at different freezing lengths, and their impact on thawed and refrozen semen. In the study, we conducted three experiments. For experiments, a total of 10 pairs of ram testes were obtained from a local slaughterhouse. In experiment 1, the treatments consist of basic Tris-diluent supplemented with 5% basal medium eagle (BME) and 1 mM vitamin E (VE) alone or together in aliquots during different short-term storage (1.5, 3, 6, and 9 h at 5°C). The control contained no additives. Freezing of cooled and refreezing of frozen-thawed sperm cells were experiments 2 and 3, respectively. Sperm characteristics including motility, progressive motility, viability, acrosome integrity, membrane integrity, and recovery rate between initial evaluation and after cooling were examined in all three experiments. Statistical analysis of data was performed using Proc GLM of SAS in a randomized block design. In the first experiment, the addition of BME alone to extenders significantly improved (P<0.05) the rates of all assessed parameters except acrosome integrity. Moreover, with increasing cooling length, all the assessed characteristics decreased significantly. In addition, with increasing cooling length, all evaluated sperm characteristics were significantly decreased (P<0.05). The second experiment showed that the extender supplemented with BME significantly improved (P<0.05) all assessed parameters, except the recovery rate. In frozen-thawed sperm with increasing cooling length up to 9 h, the assessed parameters decreased significantly (P<0.05). Finally, in the third experiment, the extender supplemented with BME alone was significantly the best diluent. Our study indicates that the extender containing BME was significantly the best diluent, and the increasing of cooling lengths up to 9 h before the freezing process negatively affects the quality of epididymis sperm cells
