Supplementation of sperm cooling medium with helpful additives is a reasonable method to conserve sperm fertility potential during cooling storage process. This study was aimed to determine the effect of sperm cooling medium supplementation with Zinc and Zinc oxide nanoparticles (NZn and NZnO) on rooster semen quality and fertility efficiency during storage periods. Semen samples were diluted in the Lake medium and assigned into five equal aliquots. The first was Control group and the other groups received 50 μg/ml NZn, 50 μg/ml NZnO, 100 μg/ml NZn and 100 μg/ml NZnO. Then, the samples were cooled at 5 °C and conserved up to 45 h. Total motility, progressive motility, mitochondrial activity, viability, membrane integrity and lipid peroxidation of samples were analyzed during 0, 22 and 45 h post-cooling. Artificial insemination was also performed using 22- hrs cooled semen. No difference was found among groups during quality evaluations at 0 h storage. Extender supplementation with 100 μg/ml NZn and NZnO presented higher (P ≤ 0.05) total motility, progressive motility, mitochondrial activity, viability, membrane integrity and lower lipid peroxidation compared to other groups during 22 and 45 h cooling storage. Fertility rate of 22-h cooled-stored semen samples was higher (P ≤ 0.05) in groups contained 100 μg/ml NZn and NZnO compared to the Control group. In conclusion, addition of 100 μg/ml NZn and NZnO to the sperm storage medium could be introduced as an effective method to preserve rooster semen quality during cooling storage period.