¹Key Laboratory for Animal Genetics & Molecular Breeding of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, CHINA DOI : 10.9775/kvfd.2021.26676 In order to explore the most suitable temperature for the preservation of Hu ram semen at room temperature, the semen samples were placed at 15°C, 20°C, and 25°C respectively. Th e energy metabolism of enzyme activity and triphosphate adenosine (ATP) was detected in the process of preservation. Diff erent parameters such as functional integrity, survival time, reactive oxygen species (ROS) content, superoxide dismutase (SOD) activity, and energy metabolism of adenosine triphosphate (ATP) were detected. Th e results showed that within 2 days of storage, the sperm progressive motility stored at 25°C was the highest. Within 3~5 days of storage, sperm stored at 20°C have the highest progressive motility. Within 6~9 days of storage, the sperm progressive motility stored at 15°C was signifi cantly higher than that of the other groups (P<0.05). Th e eff ective survival time and total survival time of sperm stored at 15°C reached 5.67 d and 18.73 d, respectively, which were signifi cantly higher than those of other groups (P<0.05). On the 1st day of storage, the sperm membrane integrity was signifi cantly higher than that of the other groups (P<0.05). Within 3~5 days of storage, the membrane integrity and acrosome integrity of sperm stored at 15°C and 20°C were signifi cantly higher than the sperm membrane integrity and acrosome integrity stored at 25°C (P<0.05). On the 7th day of storage, the membrane integrity of sperm stored at 15°C was signifi cantly higher than that of other groups (P<0.05). On the 5th day of storage, the level of sperm ROS stored at 15°C was signifi cantly lower than that of group 25°C (P<0.05), SOD activity, Catalase enzyme (CAT) activity and ATP content were signifi cantly higher than that of the other groups (P<0.05). Th erefore, compared with 20°C and 25°C, 15ºC was the most suitable temperature for the preservation of Hu ram semen at room temperature. Keywords : Temperature, Sperm, Motility parameters, Physiological characteristics, Oxidative stress