A019 2 1

Supernatants of CXCR4^lo and CXCR4^hi neutrophils were collected after 6-hour cell culture, and lactate was assessed via a lactate assay kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. For glucose uptake detection, isolated neutrophils were incubated with 100 mM 2-NBDG (N13195, Invitrogen) for 2 h and then stained with PE-Cy7 conjugated anti-human CD15 (301924, 1:100, BioLegend) and PE conjugated anti-human CXCR4 (306506, 1:100, BioLegend) at 4 °C for 30 min before measuring fluorescence by flow cytometry.

The LPS concentration in the serum was quantified by using fish LPS enzyme-linked immunosorbent assays (ELISA) kits (HB794-QT, Shanghai Hengyuan Biotechnology Co., Ltd., China). The levels of lactate in intestinal contents were measured using the commercial kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instruction.

Biochemical parameters including plasma glucose, triglycerides (TG), cortisol, and lactic acid levels were determined using commercial kits (A154-1-1, A110-1-1, H094, and A019-2-1; Jiancheng Bioengineering Institute, China). Liver tissues were accurately weighed and ground in sodium chloride buffer (0.9%) to prepare a 10% homogenate (w/v). Then, supernatants were obtained after centrifugation at 4°C for enzymatic analysis. Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) activities were determined using commercial kits according to the manufacturer’s recommendations (A015-2, A001-3-2, A007-1-1, and A003-1-2, Jiancheng Bioengineering Institute, China).

A lactic acid kit (A019-2-1, Nanjing Jiancheng, China) was used to measure lactate production. The enzyme working solution and chromogenic agent were prepared based on the instructions of the kit, and 1 ml of enzyme working solution and 0.2 ml of chromogenic reagent were then added successively to 0.02 ml of double distilled water (blank tube), 0.02 ml of 3 mmol/l standard (standard tube), or 0.02 ml of the sample to be tested (determination tube). After mixing, the samples were reacted in a water bath at 37 °C for 10 min. Finally, 2 ml of termination solution was added to each tube. After mixing, the reaction solution was extracted and added to a 96-well plate (200 µl per well). Triplicate wells were established for each group. The OD value of each well at 530 nm was measured in an enzyme labeling instrument. Lactate production (mmol/l) = (determination tube OD value − blank tube OD value)/(standard tube OD value − blank tube OD value) × (standard concentration/sample protein concentration). The experiment was repeated three times.

24 h, 48 h and 72 h biofilms were incubated on specimen disks. Six disks were measured for each group at each time period. The disks were then washed with PBS and immersed in 1.5 mL of buffered peptone water (BPW) supplemented with 1.0% sucrose and incubated at 37 °C in 5% CO₂ for 4 h. The lactic acid production was then measured using a lactic acid assay kit (A019-2-1, Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions, and the absorbance at 530 nm was measured.