Effects of Eucalyptus Biochar on Intestinal Health and Function in Largemouth Bass (Micropterus salmoides)

Intestinal health is critical for nutrient absorption and disease resistance in cultured fish. Yet, the effects of dietary Eucalyptus-derived biochar on the gut of largemouth bass (<i>Micropterus salmoides</i>) remain largely unexplored. This study evaluated whether supplementing diets with Eucalyptus biochar c profiles. In a 56-day feeding trial, <i>M. salmoides</i> were offered a standard diet containing either 0% (control) or graded levels of biochar. Juvenile fish (initial body weight 13.34 g) were randomly distributed into six groups with three replicates each (30 fish per replicate). Six extruded diets were formulated with 0, 2.5, 5.0, 10.0, 20.0, or 40.0 g kg^-1 of biochar, designated G0 through G5. Biochar had no significant effects on villus length, muscle layer thickness, villus width, or the activities of trypsin, amylase, and lipase, though goblet cell number was significantly higher in G5. mRNA expression of <i>Claudin-3</i> and <i>IL-10</i> was significantly upregulated in G1-G4, while IL-1β was significantly downregulated in G4 and G5, and <i>TNF-α</i> expression was reduced in G2 and G3. 16S rDNA sequencing showed increasing trends in the relative abundance of Firmicutes (43% to 49.17%) and <i>Lactococcus</i> (0% to 1.10%) in G3, accompanied by decreases in <i>Proteobacteria</i> and <i>Klebsiella</i>. Metabolomic analysis indicated significant upregulation of taurochenodeoxycholic acid-7-sulfate, apigenin, genistein, baicalein, taurocholic acid-3-sulfate, taurochenodeoxycholic acid-3-sulfate, and arginylmethionine in G3, whereas etoxazole and soyasaponin were significantly reduced. Dietary inclusion of 10 g kg^-1 Eucalyptus biochar improved intestinal health in largemouth bass by shaping the gut microbiota, promoting isoflavone biosynthesis and bile acid and amino acid metabolism, inhibiting the NF-κB pathway, and reinforcing the intestinal barrier.