In order to study the response and survival mechanism of spermatozoa in the external environment, the contents of energy substances, the activities of key glycolipid metabolizing enzymes in the spermatophore, and sperm motility after a 36-h preservation were examined under different temperatures (16℃, 20℃, 24℃) and dissolved oxygen levels (1 mg/L, 3 mg/L, 6 mg/L). The results showed that the glycogen and triglyceride contents in the spermatophore after the 36-h treatment were significantly lower than those before the 36-h treatment, but the protein content showed no significant differences before and after the 36-h treatment. When the water temperature increased from 16℃ to 24℃, the glycogen content decreased, while the triglyceride content first decreased and then stabilized. The activities of hexokinase (HK), lactate dehydrogenase (LDH), pyruvate dehydrogenase (PDH), and succinate dehydrogenase (SDH) increased when the water temperature increased, while the activity of lipase (LIP) first increased and then stabilized. The initial velocity and immotile rate after activation had no significant changes, but the progressive motility rate was first stable and then decreased when the water temperature increased. When the dissolved oxygen content increased from 1 mg/L to 6 mg/L, the glycogen content decreased, while the triglyceride content was first stable and then decreased. The activities of HK, PDH, and SDH increased, while the activity of LDH first increased and then decreased when the dissolved oxygen content increased. The activity of LIP was first stable and then increased, while the initial velocity and progressive motility rate after activation first increased and then stabilized, while the immotile rate first decreased and then stabilized. These results suggested that glycogen and triglycerides in the spermatophore were the main energy substances of the spermatozoa. Low energy substance consumption was the foundation for the long-term survival of the sperm. Low temperature and hypoxia, which reduced the energy consumption of the spermatozoa, were beneficial for its long-term survival.