Effects of supplementation with freeze-dried Clostridium butyricum powder after replacement of fishmeal with cottonseed protein concentrate on growth performance, immune response, and intestinal microbiota of Litopenaeus vannamei.

The present study was designed to investigate the effects of supplementation with freeze-dried Clostridium butyricum (CB) powder on the growth, immune function and intestinal health of Litopenaeus vannamei after replacing fishmeal in the diet with cottonseed protein concentrate (CPC). Six treatment groups were designed, namely the control group (CON, 25% fish meal) and five alternative groups (CPC replacing 40% fishmeal protein in the control group). Based on the alternative group, 0%, 0.065%, 0.26%, 1.04%, and 4.16% of freeze-dried CB bacterial powder (4.6 × 10⁸ CFU/g) were added, recorded as CB 0, CB 0.065, CB 0.26, CB 1.04, and CB 4.16, respectively. Each treatment had 3 replicates of 40 shrimps (0.29 ± 0.01 g) each and breeding for 8 weeks. After the experiment, serum enzyme activities, muscle amino acids, and intestinal parameters (short-chain fatty acids, digestive enzymes, gene expression, and microbiota) were tested to explore the effects of freeze-dried CB powder in shrimp aquaculture. The results showed that the CB1.04 group had the highest final body weight, weight gain rate, and specific growth ratio (P > 0.05). Freeze-dried CB powder increased the activity of serum superoxide dismutase, glutathione peroxidase, complement 3, and complement 4. Muscle tyrosine, proline, and total essential amino acids were remarkably increased in the CB 1.04 group (P < 0.05). Propionic acid levels were elevated in the CB 1.04 and CB4.16 groups (P < 0.001). The relative expression of Dorsal, Relish, and Target of Rapamycin (TOR) genes was significantly increased in the CB 1.04 group (P < 0.01). Actinobacteria and Demequina abundance was significantly higher in the CB 1.04 group (P < 0.01). The results of the Vibrio parahaemolyticus challenge test showed the highest cumulative mortality rate (43.33%) in the CB0 group and the lowest cumulative mortality rate (20%) in the CB1.04 group. This study confirmed that freeze-dried CB powder alleviated the negative effects of CPC replacement of fish meal protein in Litopenaeus vannamei, and the optimum additive level was 2.11% (9.71 × 10⁹ CFU/kg) as indicated by binary regression analysis of specific growth ratio.