Inhibition of miR-34a Expression in GIFT by Injection of miRNA Antagomir

A total of 360 GIFT juveniles (9.6 g ± 0.5) were randomly placed into nine 600-L tanks, each of which had 40 fish. Each treatment had three replicates. The synthesized miR-34a antagomir and NC fragments were each injected into the tail of GIFT at a dose of 50 mg/kg body weight (20 μL reagent per fish) (Yuan et al., 2019). At 12 h after injection, GIFT were placed into a hypoxic environment (DO of 1.1 ± 0.04 mg/L). Real−time readings with a DO meter (LDO101 probe, range 0.1–20.0 mg/L, Hach, Loveland, CO, United States) were used monitor the DO content in water, and the DO content was adjusted by altering the nitrogen and air charge. At the beginning of the experiment, the DO level in the experimental tanks was rapidly reduced over 1 h by pumping nitrogen (100 m³/h) into water from a nitrogen gas cylinder (Wuxi Guangming Special Gas Co., Ltd., Wuxi, China). Four fish were randomly selected from each tank at 0, 12, 24, 48, and 96 h after injection. After rapid deep anesthesia, blood was drawn from the tail vein using a 1.5-ml syringe. Blood samples were kept at 4°C for 2 h, then centrifuged at 3000 × g at 4°C for 10 min. The serum was collected and placed stored at −80°C until analysis of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. At the same time, liver tissues were sampled, rapidly frozen in liquid nitrogen, and then stored at −80°C until analyses of the miR-34a regulatory response mechanism.