Effects Of Probiotics On Growth Performance And Immune Response Of The Sea Cucumber (Apostichopus Japonicus)

The objective of this work was to select probiotic bacterial strains for sea cucumber Apostichopus japonicus. Three strains isolated from the intestines of A. japonicus were identified by 16S rDNA sequence genotyping as Bacillus GSC-1, Bacillus GSC-2 and Enterococcus GSC-3. To screen safe potential probiotics for A. japonicus, both the sensitivity to antibiotics and the toxicity to juvenile A. japonicus of these potential probiotics were evaluated. The determination of their sensitivity to antibiotics showed that all three potential probiotics were sensitive to more than 50 percent antibiotics tested. Juvenile A. japonicus were immersed in sea water with GSC-1, GSC-2, GSC-3 or Vibrio splendidus at 100,103,105 or 107 CFU/ml respectively for 7 days. Results showed that GSC-1 and GSC-3 treatments were safe under the designed concentrations based on the mortality rates. However, GSC-2 caused significantly higher mortality at 107 CFU/ml than the blank control (P<0.05).A 20-day feeding trial was conducted to investigate the effects of GSC-1 and GSC-3 on the growth performance and innate immune response of juvenile A. japonicus. Feeds were algae (Sargassum thunbergii) powder supplemented with GSC-1 or GSC-3 at 0 (control) and 109 CFU/g respectively. The specific growth rate and survival rate were significantly increased in treatment supplemented with GSC-1. Comparatively, survival rate and discoloration rate were remarkably improved in treatment of GSC-3 (P<0.05). Moreover, GSC-1 increased total bacteria counts and decreased Vibrio spp. counts significantly (P<0.05). Phenoloxidase and lysozyme activities in body tissue of the juvenile A. japonicus were significantly higher in the group treated with GSC-1 or GSC-3 than the control (P<0.05) while acid phosphatase and superoxide dismutase activities were remarkably higher than control in GSC-1 group only (P<0.05). Both GSC-1 and GSC-3 showed positive effects on disease resistance of juvenile A. japonicus, especially GSC-3 significantly improved disease resistance to V. splendidus exposure (P<0.05). In conclusion, both Bacillus GSC-1 and Enterococcus GSC-3 show probiotic effects, therefore they can be used as probiotics in the farming of juvenile sea cucumber A. japonicus.An 8-week feeding experiment was conducted to investigate the effect of Bacillus GSC-1 on A. japonicus (average weight 4.120±0.120g) based on growth, microflora of intestine, immunity and disease resistance. GSC-3 was supplemented in the basic diet either by different forms (live or heat-inactived) or by different concentrations (107 CFU/g or 109 CFU/g). Results showed that 109 CFU/g treatments (live and heat-inactived) showed significantly higher specific growth rate than the control (P<0.05), but no significant differences were found between the 107 CFU/g treatments and the control (P>0.05). The remarkably increased total bacteria counts and the obviously decreased Vibrio spp. counts were found in the live GSC-1 treatments only (P<0.05). Furthermore, the treatment of 109 CFU/g live GSC-1 gained the highest total bacteria counts and the lowest Vibrio spp.counts. GSC-1 showed positive effects both on the immunity and disease resistance, while the activities of immune enzymes and the survival rate after Vibrio splendidus infection of live GSC-1 treatments were significantly higher than the heat-inactived GSC-1 treatments. These results indicate that Bacillus GSC-1 treatment is promising as an alternative method to antibiotics for disease prevention in aquaculture, and the viability of probiotic is a key factor to induce more potential effect of probiotics used for A. japonicus production. The advisable concentration is more than 109 CFU/g.Another 8-week feeding experiment was conducted to investigate the effect of Enterococcus sp. GSC-3 on A. japonicus (average weight 4.094±0.057 g) based on growth, microflora of intestine, immunity and disease resistance. GSC-3 was supplemented in the basic diet either by different forms (live or heat-inactived) or by different concentrations (107 CFU/g or 109 CFU/g). Results showed that 109 CFU/g treatments (live and heat-inactived) showed significantly higher specific growth rate than the control (P<0.05). The total bacteria counts in intestine of A. japonicus were remarkably increased in all probiotic treatments (P<0.05) while only live GSC-3 obviously decreased Vibrio spp. counts (P<0.05). GSC-3 showed positive effects both on the immunity and disease resistance, while the activity of immune enzyme and the survival rate after Vibrio splendidus infection of heat-inactived GSC-3 treatments were higher than the live GSC-3 treatments. These results indicate that Enterococcus sp.GSC-3 treatment is promising as an alternative method to antibiotics for disease prevention in A. japonicus culture. Although heat-inactived GSC-3 treatments can't decrease the Vibrio spp. counts in intestine of A. japonicus as live treatments, the immune response and disease resistance prove that heat-inactived GSC-3 is superior to live GSC-3. The advisable concentration is 109 CFU/g.In conclusion, our results suggest that Bacillus GSC-1 and Enterococcus GSC-3 show positive effects both on the growth and disease resistance of A. japonicus, therefore they are effective probiotics for A. japonicus. This study settles the theoretical and applied basement of development and manufacture of probiotics for A. japonicus culture.