3 aminobenzoic acid ethyl ester methanesulfonate

Tg(pvalb3b:β-actin-mCherry) zebrafish injected with DNA plasmid, pMT/pV3b/tmc2b-mGFP, at 150 ng/μl and Tol2 RNA at 25 ng/μl^{39 (link)} were anesthetized in 650 mM 3-aminobenzoic acid ethyl ester methanesulfonate (Sigma-Aldrich) in fish water at 4 dpf. Immobilized fish were imaged by confocal microscopy with a 40× objective (Leica SP8; Leica). 488 nm and 594 nm wavelengths were used to excite mGFP and mCherry, respectively.

We collected the C. guichenoti used in this experiment from the Fisheries Institute, Sichuan Academy of Agricultural Sciences in Sichuan, China. Their body length and weight were 5.71 ± 0.44 g and 1.26 ± 0.27 cm, respectively. Before the start of the formal experiment, the fish were temporarily kept in the laboratory for 7 days, and a pre-experiment was conducted to with 4°C (low temperature, LT), 22°C (normal temperature, NT), and 30°C (high temperature, HT). According to the pre-experimental results, we used LT, NT, and HT for 24 h in the main experiment. Each treatment group contained 30 fish.
The fish were euthanized by a high dose of 3-Aminobenzoic acid ethyl ester methanesulfonate (Sigma-Aldrich, St. Louis, MO, United States) before dissection. We removed brain and gill tissues using sterilized scalpels and scissors. Tissues were quickly placed in liquid nitrogen for quick freezing and then stored in a −80°C freezer until used for analysis.

At the termination of the trial, fish were fasted for 24 h and then anesthetized with 3-aminobenzoic acid ethyl ester methanesulfonate (40 mg/L, Sigma, Oakland, CA, USA) before sampling. Fish per tank were counted and weighed to analyze the SR, FBW, WGR, SGR, and FC. Feed intake (FI) was determined as the gravimetric difference between the feed offered and orts. Six fish in each tank were randomly selected for analysis of CF, VSI, his, and ISI.
Blood was collected from the caudal veins of six fish in each tank, kept at 25 °C for 30 min, and centrifuged at 8000× g for 10 min. Serum was stored at −80 °C for subsequent analysis of serum antioxidant and immune indexes.
The livers of three fish per tank were taken to determine antioxidant and immune indexes.
The intestines of three fish per tank were sampled for trypsin analysis (Ultraviolet colorimetry), lipase (colorimetry), and amylase (colorimetry) activities using commercial kits supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The intestines of three other fish per tank were randomly collected for intestinal histological examination [42 (link)].