Cooling method was proposed to maintain the sperm quality for several days. Nevertheless, during this procedure, sperm is encountered to “cold shock”, and its quality decreases time-dependently. This study was designed to improve the in vitro sperm preservation methods. Thirty normal semen samples were examined in Shiraz, Iran, 2017. Fifteen samples were incubated at 22-27 °C and 15 samples were cooled moderately to 4 °C. Each sample was divided into five subgroups; control, solvent, 200 μM Trolox, 40 μM Coenzyme Q10, and 10 mM ATP. ATP was added only 15 minutes before the analysis. Assessments of motility parameters and sperm viability were done every 24 hours. Statistical analysis was performed using SPSS 16 software. The differences between two main groups and subgroups were compared by t test and one-way ANOVA, respectively. The effect of time was analyzed by repeated measurement test. Sperm motility and viability were the same in both groups until 24 hours, except the straight line velocity was greater in the cold group. Even after 48 hours, progressive motility and sperm velocity, but not viability, were still the same. The greatest reduction in progressive motility occurred on the second day; and after 72 hours, sperm quality was better preserved in 22-27 °C. Treatment with Trolox, coenzyme-Q10, and extracellular ATP did not have effect on sperm quality. Cold temperature is recommended for in-vitro sperm preservation up to 24 hours, and 22-27 °C is preferred for longer time storage. The sperm does not need antioxidant therapy for quality maintenance, but the extender media must be supplied with nutrients and antibiotics.