Background: Artificial insemination is one of the important management tools to control reproductive activities and increase the productivity of the livestock industry. This technique can play an important role in genetic improvement even in small herds. Sperm preparation is of particular importance for reproductive techniques, such as artificial insemination, but many harmful factors, such as temperature shock and oxidative stress, reduce sperm quality and directly affect sperm fertility during sperm preparation and storage. Therefore, the addition of protective substances and antioxidants in the diluent is considered essential in maintaining sperm quality during the cooling and freezing stages. Curcumin is a yellow substance obtained from the root of turmeric, and its biochemical structure contains a phenolic ring and a beta-di- ketone part in its structure. It can neutralize free radicals, but it has low solubility in hydrophilic environments. To solve this problem, the nanoliposome method has been proposed. Using this method can solve the problem of curcumin's low solubility in aqueous solutions and, additionally, curcumin nanoparticles can be guided to exert their effects in a completely targeted manner inside the target cell. On the other hand, sucrose protects sperm from cold shock by first rinsing sperm cells and maintaining the osmotic pressure of the diluting medium. This study aimed to investigate the effects of separate and mixed use of different levels of curcumin nanoparticles and sucrose sugar in ram epididymal sperm diluent during storage at 5 ˚C at different cooling times. Methods: The present study was conducted in the Animal Physiology Laboratory of the Department of Animal and Poultry Sciences, Aburihan Agricultural Campus, the University of Tehran. The testicular tissues were prepared from a slaughterhouse on the day of the experiment and transported to the laboratory with special flux in less than an hour. Sperm samples were collected from the epididymal tail of the ram testicular tissue in the laboratory. Sperm was sampled after initial evaluation, with progressive motility of more than 75% and abnormality of less than 10%, and were added to the diluent at 37 °C. In this study, the diluent consisted of Tris (27.1 g/L), citric acid (14 g/L), fructose (10 g/L), egg yolk (20%), and glycerol (7%), with a pH of around 6.2-7.8. The osmolarity was set at about 310-320 milliosmoles. Sucrose sugar was obtained from the Sigma Aldridge Company, and curcumin nanoparticles were prepared from pure curcumin powder (Sigma Aldridge) by the nanoliposome method and using rotary devices, a homogenizer, and a prop sonicator. A DLS device was used to examine the particle size of curcumin nanoparticles (less than 80 nanograms). The experimental groups included 25 and 50 μM of nanoparticles of curcumin (NC), sucrose (S) at 100 and 150 mM separately, combined levels (25NC100S, 25NC150S, 50NC100S, and 50NC150S), and a group without these additives (control), which were added to the diluent at 37 °C. The diluents containing different levels of the treatments were refrigerated in 15 ml falcones and inside a water container at the same temperature, which gradually reached a temperature of 5 ˚C in about 2 hours. After stabilization at this temperature, total motility parameters, progressive motility using the KASA system, the viability with the eosin-nigrosin method (16.7 g of eosin, 100 g of nigrosin, and 29 g of citrate buffer in 1 L of double-distilled water), membrane integrity by the HOST solution method (9 g of fructose and 4.9 g of sodium citrate in 1 L of double-distilled water with an osmolarity of 100 milliosmoles), and the percentage of sperm abnormalities using Hancock's solution (5 62/L of 37% formalin, 150 ml of saline solution, 150 ml of a buffer solution, and 500 ml of double distilled water) were evaluated at 6, 12, 24, and 48 hours. The experimental results were analyzed in a completely random design by SAS software (9.2) and the GLM procedure at a significance level of P < 0.05. The means of the treatments were compared with Tukey's test. Results: The use of 25NC100S in the diluent improved the parameters of total motility, progressive motility, viability, and integrity of ram epididymal sperm membrane in all evaluated times compared to the other groups, especially the control group (p < 0.05). No significant differences were observed between the treatments in the percentage of sperm abnormalities at 6 and 12 hours (p > 0.05). At 24 and 48 hours, however, the 25NC100S concentration significantly reduced the percentages of sperm abnormalities compared to the other concentrations (p < 0.05). Compared to the control group, adding different levels of curcumin nanoparticles and sucrose sugar separately significantly improved the evaluated parameters, but the best performance was observed in the combined treatment, indicating the synergistic role of these two compounds. Conclusion: The results of the present study demonstrate that using a mixture of curcumin nanoparticles with sucrose in the diluent can protect sperm against oxidative damage and cold shock. The antioxidant effects of curcumin nanoparticles and the protective effects of sucrose sugar can result in positive effects on the quality of ram epididymal sperm during cooling. Therefore, it is recommended to use the 25NC100S level in ram sperm diluents. [ABSTRACT FROM AUTHOR]