Potassium oxalate

Before the start of the experiment, an initial sample of three fish was taken and kept in the freezer at −20 °C. At the end of the experiment, all fish were starved for 24 h. Three fish per tank were then collected and kept at −20 °C to analyze the final whole-body composition. Sixteen fish per treatment (four fish per tank) were anesthetized with chlorobutanol (300 mg/mL) for sampling, including measurements of body length, body weight, viscera, and liver weight. The remaining fish of each tank were counted and batch-weighted to analyze growth performance. Blood samples were drawn from the tail vein of the fish using 2% sodium fluoride (SINOPHARM, Beijing, China) and 4% potassium oxalate (SINOPHARM, Beijing, China) as anticoagulation agents, and then immediately centrifuged at 4 °C (4000 rpm, 10 min). Plasma samples were kept in the −40 °C freezer. Two liver samples were collected near the bile duct. One was put into a 1.5 mL RNase-free tube (Axygen, Waltham CA, USA), immediately frozen in liquid nitrogen, and kept in a −80 °C freezer pending mRNA extraction and enzymatic analysis. The other (0.5 mm length) was fixed with 4% paraformaldehyde solution for histological examination.

Standard chemicals, namely, danofloxacin, erythromycin, sulfadimidine, benzylpenicillin potassium, chloramphenicol, lincomycin, tetracycline, spectinomycin, thiamphenicol, dexamethasone, kanamycin, melamine, trimethoprim, zearanol, aflatoxin M₁, flunixin meglumine, and florfenicol, were purchased from Sigma (St. Louis, MO, USA). All the chemicals used were of analytical grade. Lead acetate, potassium oxalate, disodium hydrogen phosphate, trichloroacetic acid, sodium hydroxide, and dichloromethane were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). High-purity water was obtained from a Milli-Q water system (Millipore, Billerica, MA, USA). Pure milk was purchased from a local supermarket. Commercialized semiquantitative ICA strip and an ICA strip reader were supplied by Beijing Meizheng Bio-Tech Co., Ltd. (Beijing, China)