REPLACING SOYBEAN-DERIVED PROTEINS WITH NOVEL PROTEIN SOURCES ALTERS INTESTINAL HEALTH AND MICROBIOTA COMPOSITION OF LARGEMOUTH BASS (MICROPTERUS SALMOIDES)

To investigate the effects of replacing soybean-derived proteins with cottonseed protein concentrate (CPC) and Clostridium autoethanogenum protein (CAP) on intestinal health and microbiota composition of largemouth bass (Micropterus salmoides), eight isonitrogenous (48%) and isolipidic (8%) experimental diets were formulated. CPC and CAP were used to replace soybean meal (10%) and soy protein concentrate (13%) with equal proportions of 0, 15%, 45%, and 100%, respectively. Largemouth bass with an initial body weight of 9.40 g were fed the experimental diets for 60 days before sampling. In the CPC experiment, compared with the CPC0 group, the CPC100 group significantly increased intestinal villus height and width (P<0.05), along with upregulated expression of the tight junction protein gene occludin and promoted intestinal barrier function. For CAP, the 15% substitution level significantly increased intestinal villus width and muscle thickness, with no significant effects on the expression of intestinal barrier-related genes (zo-1, occludin, claudin) (P>0.05). However, when the substitution level reached 45% or higher, muscle thickness declined and the expression of the anti-inflammatory factor tgfb1a was significantly downregulated. Intestinal microbiota analysis showed that replacing soybean-derived proteins with CPC or CAP did not significantly affect alpha diversity, although microbial richness tended to decrease when CAP substitution reached 45% or higher. Both protein sources significantly altered the intestinal microbial community structure. With increasing CPC inclusion, the abundance of opportunistic pathogens such as Aeromonas decreased continuously, whereas beneficial genera including Romboutsia and Cetobacterium became more abundant. CAP inclusion similarly reduced the abundance of potential pathogens such as Stenotrophomonas, and mainly enriched Clostridium, but at high inclusion levels, it caused the intestinal microbiota to become simplified. LEfSe analysis further identified distinct microbial biomarkers, with the CPC100 group characterized by commensal genera such as Romboutsia and the CAP15 group dominated by protein degradation-related taxa such as Clostridium. In summary, complete replacement of soybean-derived proteins with CPC can sustainably improve the intestinal structure of largemouth bass, demonstrating its application potential in aquaculture. In contrast, CAP is suitable for low-level inclusion, while high inclusion levels may compromise intestinal health and microbial stability.