Comparative effects of bile salts and free bile acids on intestinal health of grouper (Epinephelus spp.) fed a high cottonseed protein concentrate diet.

The physiological impacts of bile acids (BA) conjugation states remain underexplored in aquatic species. This study investigated the effects of supplementing conjugated BA (bile salts [BS]) or non-conjugated BA (free bile acids [FBA]) on the intestinal health of pearl gentian grouper (Epinephelus fuscoguttatus ♂ × Epinephelus lanceolatus ♀) fed a high plant-protein diet for 8 weeks. A total of 300 healthy juveniles (approximately 90-d-old; initial body weight 15.21 ± 0.04 g) were randomly distributed into four dietary groups in triplicate (25 fish per tank): CD group (a fishmeal-based control diet [CD]), PD group (a plant protein-based diet [PD], with 50% fishmeal replaced by cottonseed protein concentrate), BS group (PD supplemented with 0.09% BS), and FBA group (PD supplemented with 0.09% FBA). Compared to the CD group, the PD group significantly impaired growth performance (final body weight and weight gain rate), gut morphology (muscle thickness, plica height, and width), and induced inflammatory response, oxidative stress, endoplasmic reticulum stress, cell apoptosis, and dysregulated amino acid metabolism (P < 0.05). The BS supplementation reversed these adverse effects: it improved final body weight, intestinal morphology, enhanced amino acid transport gene expression, and reduced pro-inflammatory and pro-apoptotic signals (P < 0.05). Transcriptomic analysis revealed that BS shifted the intestinal gene expression profile towards that of the CD group. In contrast, while FBA supplementation showed negligible benefits on most physiological parameters, it did significantly enrich the pathways of "G-protein coupled receptor binding" and "regulation of actin cytoskeleton" (P < 0.05). In conclusion, BS, but not FBA, effectively attenuates intestinal damage and promotes health in groupers fed high plant-protein diets, highlighting the critical importance of BA conjugation in nutritional interventions.