title: Influence of oligochitosan on hepatic immune-metabolic homeostasis in hybrid grouper infected with Vibrio harveyi
design: Influence of oligochitosan on hepatic immune-metabolic homeostasis in hybrid grouper infected with Vibrio harveyi
library_construction_protocol: Healthy hybrid groupers with an average body weight of 41.4 ± 2.9 g were selected for the experiment. All experimental fish were obtained from the Guangdong Provincial Marine Fisheries Research Institute. Before the experiment, fish were acclimated for 14 days in a temperature-controlled recirculating aquaculture system to ensure full adaptation to the laboratory environment. Throughout the acclimation and experimental periods, water temperature was strictly maintained between 25 and 28 °C. Salinity was regulated at approximately 12‰ by the addition of marine salt, pH was controlled within the range of 7.8-8.3, dissolved oxygen levels were kept above 6.0 mg/L, ammonia concentrations were maintained below 0.08 mg/L, and nitrite levels remained under 0.1 mg/L.  A commercial basal diet provided by Guangdong Yuequn Marine Biotechnology Co., Ltd. was used as the base feed. Oligochitosan (degree of deacetylation ≥ 90%) produced by Shandong Aokang Biotechnology Co., Ltd. was used as the dietary additive. Based on our previous findings (Chen et al. 2022, Shi et al. 2022), oligochitosan was supplemented at an optimal level of 800 mg/kg by weighing, mixing with the diet, and grinding into powder before feeding.  V. harveyi preserved in our laboratory was cultured on Luria-Bertani (LB) agar plates supplemented with ampicillin (prevent cross-contamination) at 28 °C for 24 h. The bacterial concentration was determined by measuring the optical density at 600 nm (OD₆₀₀) using a spectrophotometer. According to our previous study, the median lethal concentration (LD₅₀) of V. harveyi was determined to be 1.01 × 10⁶ CFU/g (Shi et al. 2022). The bacterial suspension was harvested at a concentration of 1.0 × 10⁷ CFU/mL and washed twice with PBS. The final inoculum was adjusted to 4.5 × 10⁴ CFU/g of fish, calculated according to the mean body weight.  Liver RNA from hybrid groupers was extracted using TRIzol reagent (Life Technologies, USA) following the manufacturer's procedure. cDNA libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit and sequenced on the Illumina HiSeq X Ten platform (OE Biotech Co., Ltd., Shanghai, China) (Ma et al. 2026).  