<b><i>Clostridium butyricum</i></b><b> WF4-2 in Enhancing Growth and Immunity of Red Claw Crayfish (</b><b><i>Cherax quadricarinatus</i></b><b>)</b>

A total of 600 four-week-old C. quadricarinatus juveniles (0.26 ± 0.02 g) were randomly assigned to five groups based on the dietary supplementation levels of lyophilized C. butyricum strain WF4-2: C0 (0 g/kg), C1 (0.002 g/kg), C2 (0.02 g/kg), C3 (0.2 g/kg), and C4 (2 g/kg). Each group included three replicates, with 40 crayfish per replicate, and the feeding trial lasted for eight weeks. Results demonstrated that supplementation with 0.02 g/kg of WF4-2 significantly enhanced both weight gain rate (P = 0.038) and specific growth rate (P = 0.040) in C. quadricarinatus. Broken-line analysis indicated that the optimal supplementation level for growth promotion was 0.02 g/kg. In terms of digestive enzyme activities, trypsin activity was significantly elevated in groups C1 and C2 (P < 0.05), and α-amylase activity increased significantly in groups C2 and C3 (P < 0.05). In serum, catalase (CAT) and glutathione peroxidase (GSH-Px) activities were significantly enhanced in group C2 (P < 0.05), while malondialdehyde (MDA) levels significantly decreased in groups C2 and C3 (P < 0.05). In the hepatopancreas, GSH-Px activity was significantly upregulated in groups C2 and C3 (P < 0.05), and total antioxidant capacity (T-AOC) was significantly increased in all treatment groups (P < 0.05), with MDA content significantly reduced in group C3 (P < 0.05). Transcriptomic analysis revealed a significant upregulation of TRX1 gene expression in groups C2 and C3 (P < 0.05), and GSH-Px gene expression was significantly elevated in all treatment groups (P < 0.05). Microbiota profiling showed a significant decrease in the relative abundance of Proteobacteria (P < 0.05) and a marked increase in Firmicutes (P < 0.05) at the phylum level. At the genus level, the abundance of Hypnocyclicus and Clostridium significantly increased (P < 0.05). Moreover, levels of intestinal metabolites such as crocin-3 and gamma-glutamyl ornithine were significantly higher in treatment groups (P < 0.05). Notably, crayfish in the C2 and C3 groups exhibited enhanced resistance to Aeromonas hydrophila challenge (P < 0.05).