Chitooligosaccharides promote intestinal adhesion of Clostridium butyricum and enhance gut health, immunity, and antioxidant capacity in largemouth bass

Prebiotic polysaccharides and oligosaccharides serve as critical modulators of probiotic functionality, influencing gut microbial ecology and host physiological responses. This study aimed to identify a high-efficacy prebiotic for enhancing the proliferation and intestinal adhesion of Clostridium butyricum (CB) among multiple polysaccharides and to evaluate its impact of combined treatment with CB in largemouth bass ( Micropterus salmoides ). Through systematic screening, chitooligosaccharides (COS) were selected due to their superior ability to promote CB growth and adhesion. Although no significant differences in growth performance were observed among groups, dietary supplementation with COS and CB in combination reduced hepatic stress markers (ALT: –75.8%; AST: –83.8%) and significantly improved intestinal antioxidant capacity (GSH-Px: +282.4%; T-AOC: +121.0%; MDA: –34.3%). RT-qRCR analysis revealed profound upregulation of tight junction proteins (Claudin-1, Claudin-4, ZO-1), indicating enhanced barrier integrity. Hepatic cytokine profiling showed strong immunomodulatory effects, with suppression of IL-1β and IL-8 by 75.7% and 73.6%, respectively, and upregulation of IL-10 (2.29-fold) and TCR-α/β (3–4-fold). Gut microbiome restructuring featured a reduction in Pseudomonadota and enrichment of beneficial Bacillota . These results demonstrate that combined COS and CB supplementation improved intestinal barrier function, antioxidant capacity, and immunomodulation, and modulated gut microbiota, suggesting its potential as a sustainable strategy for aquaculture.