Abstract:The production of juvenile sea cucumber is a vigorous industry in China, with an annual output of 50~60 billion individuals. Antibiotics was used to control the diseases caused by high intensity of seedling in hatchery, which can result in antibiotic residues in adult sea cucumber. Prophylactic administration of probiotics has emerged as a promising alternative strategy to control sea cucumber diseases by strengthening their immunity. Clostridium butyricum, an obligate anaerobic bacterium, is widely used as a feed additive in aquaculture to promote growth, improve immunity, and enhance digestive capacity in aquatic animals. However, its use in sea cucumber has rarely been reported. This work was conducted to evaluate the potential of C. butyricum as a dietary probiotic to improve growth, immunity and digestive capacity in the sea cucumber Apostichopus japonicus. Healthy juvenile sea cucumbers with an average body weight of (5.92±0.11)g were randomly divided into five groups with three replicates in each group, and 50 sea cucumbers in each replicate. The animals were reared in aquaria and fed diets supplemented with C. butyricum at concentrations of 0 CFU/g (control group)(G0), 106 CFU/g (G1), 107 CFU/g (G2), 108 CFU/g (G3), and 109 CFU/g(G4) for 90 days, respectively. During the experiment, water temperature was maintained at 11~23 ℃, salinity at 30~32, dissolved oxygen at a minimum of 6 mg/L, and pH at 7.8~8.2. At the end of the feeding trial, all sea cucumbers in each aquarium were weighed and counted. Five sea cucumbers from each aquarium were sampled, starved for 24h, and their coelomic fluid and intestine were collected for immune and digestive indices analysis, respectively. Coelomocyte counts were determined using a hemocytometer and expressed as cells per ml. Phagocytosis activity was assessed using a modified neutral red method, while intracellular reactive oxygen species (ROS) production was measured using the nitroblue tetrazolium (NBT) method. Phenoloxidase (PO) activity was determined spectrophotometrically using L-3, 4-dihydroxyphenylalanine (L-DOPA) as substrate and trypsin as elicitor. Acid phosphatase (ACP) and alkaline phosphatase (AKP) activity were measured using commercial kits (Nanjing Jiancheng, China). Lysozyme activity was measured by a spectrophotometric method based on the lysis of Micrococcus lysodeikticus. Digestive enzyme (amylase, trypsin, and lipase) activities were determined using enzyme activity assay kits (Nanjing Jiancheng, China) following the manufacturer’s instructions. Results showed that dietary supplementation with C. butyricum at concentrations of 108 CFU/g or higher significantly improved the specific growth rate and weight gain rate of sea cucumbers (P < 0.05). However, C. butyricum had no significant effect on survival rate of the sea cucumber (P＞0.05). Phagocytosis and respiratory burst activity were significantly improved in coelomocytes of sea cucumbers fed with C. butyricum at 109 CFU/g diet (P < 0.05). Additionally, phenoloxidase activity, acid phosphatase activity, and alkaline phosphatase activity in coelomocytes were significantly higher in G3 and G4 groups compared to the other three groups (P < 0.05). Lysozyme activity in coelomocytes was also significantly higher in G3 and G4 groups compared to the G1 and control groups (P < 0.05). Furthermore, C. butyricum also significantly improved the digestive enzyme activity of A. japonicus. Sea cucumbers in the G3 and G4 groups showed significantly higher amylase, trypsin and lipase activities compared to those in the G0 and G1 groups. The above results showed that C. butyricum as a feed additive can promote growth, activate the immune system, and enhance digestive ability in sea cucumber. The present study confirmed the potential beneficial effects of C. butyricum as dietary probiotic in juvenile A. japonicus, and the optimal supplemental level of C. butyricum is 108 CFU/g. These findings provide valuable insights for developing sustainable and antibiotic-free strategies in the sea cucumber industry.
