Optimal feeding levels to enhance growth performance and gut microbiota balance in red swamp crayfish (Procambarus clarkii)

This study examined the impact of various feeding levels on the growth performance and intestinal microbiota composition of Procambarus clarkii. A total of 540 juvenile crayfish (initial weight: 5.95 +/- 0.25 g) were allocated to six experimental groups (1 %, 2 %, 3 %, 4 %, 5 %, and 6 % of body weight), with three replicates per group (30 crayfish per replicate), and cultured for 45 days. Growth parameters, physiological indices, gene expression, and intestinal microbiota were assessed to identity the optimal feeding level. The results revealed that feeding level significantly influenced growth outcomes. The FL4 group (4.48 % feeding level) demonstrated the highest flesh content (FC), survival rate (SR), and overall growth performance. Both excessively high and low feeding levels impaired antioxidant capacity (SOD, CAT), immune function (ACP, AKP, LZM), and induced liver damage (increased AST and ALT) and oxidative stress (elevated MDA). Additionally, feeding level modulated liver digestive enzyme activity, with FL4 exhibiting the most favorable biochemical profile. Gene expression analysis highlighted that TGF-beta and Mstn were inversely correlated with growth, with the lowest expression observed in FL4. Conversely, growth-related genes (ACTL, TNT, MCL, MHC) were most highly expressed in FL4. The molt- related gene MIH showed a negative correlation with feeding level, whereas E75, EcR, and RXR were optimally expressed in FL4. Intestinal microbiota analysis indicated that feeding level significantly impacted microbial diversity and abundance. Higher feeding levels promoted an increase in Bacteroidota, while Firmicutes and the Firmicutes/Bacteroidetes ratio were negatively correlated with feeding levels. Lower feeding levels favored the growth of potential pathogens, such as Aeromonas, Shewanella, and Tyzzerella, which were negatively correlated with both growth and molt-related gene expression. In conclusion, a feeding level of 4.48 % optimized growth performance andmaintained a balanced intestinal microbiota.