2.2. Experimental Fish and Sample Collection

Silver carp (53.64 ± 4.84 g) in this study were taken from Yaowan Experimental Farm, Yangtze River Fishery Research Institute, Chinese Academy of Fisheries Sciences. The silver carp were temporarily cultured in a tank for a week. Before the experiment, the experimental fish were transferred to the 52.5 L tank for 24 h, and the normal aeration was carried out to maintain the oxygen concentration in the water. A total of 120 healthy silver carp were selected and divided into a normoxia group (T0) and three hypoxia stress groups (T1, T2, T3). They were placed in white water tank (50 cm × 35 cm × 30 cm), and 10 fish were placed in each tank, with 3 replicates in each group. Normal aeration was maintained in the normoxic group (all the fish breathed normally, recorded as T0, dissolved oxygen 6.45 mg/L); hypoxia stress experimental group stopped aeration, sealed water tank with plastic film, respectively. Silver carp gasping for air period (most fish tried to breathe directly through their mouths, recorded as T1, 4 h after the beginning of the experiment, dissolved oxygen 0.76 mg/L), semi-asphyxia period (half of fish lost balance, recorded as T2, 5 h after the beginning of the experiment, dissolved oxygen 0.58 mg/L), asphyxiation period (half of fish sank without the rhythmical opening and closing of the gill flaps, recorded as T3, 6 h after the beginning of the experiment, dissolved oxygen 0.21 mg/L). The fish were sampled, and three silver carp were collected in each water tank at each experiment point after MS-222 (100 mg/L) anesthesia. The heart, liver, brain and gill were kept in 2 mL cryopreservation tube and put into liquid nitrogen, and then stored at −80 °C.