Screening Immunostimulants For Sea Cucumber (apostichopus Japonicus) In Vitro And Verification In Vivo

In vitro and in vivo trials were conducted to investigate the effects of different immunostimulants on sea cucumber A. japonicus. The results are summarized as follows:1. The in vitro effects of P-glucan, peptidoglycan, chitosan, mannan oligosaccharides (MOS), CpG oligodeoxynucleotide (CpG ODN), lactoferrin, vitamin C and vitamin E on the non-specific immune response of sea cucumber Apostichopus japonicus coelomocytes were studied. Coelomocytes were cultured in L-15 medium containing different concentrations of P-glucan (0,5,25 and 100μg ml-1), peptidoglycan (0,2,10 and 50μg ml-1), chitocan (0,80,100 and 400μg ml-1), CpG ODN (0,0.5,2.5 and 5μM), MOS (0,40,80 and 120μg ml-1), lactoferrin (0, 5,25 and 100μg ml-1), vitamin C (0,25,100 and 250μg ml-1) and vitamin E (0, 20,40 and 100μg ml-1). Coelomocytes were incubated with the above immunostimulants for 1 h,3 h,6 h,12 h or 24 h except vitamin C and vitamin E for 3 h,6 h,12 h,24 h or 48 h. Coelomocytes incubated in medium without immunostimulants were took as controls at each incubation time. After exposed to these substances, coelomocytes were sampled and immune parameters viz., phagocytosis, superoxide anion production, superoxide dismutase (SOD) activity and total nitric oxide synthase (T-NOS) activity were determined. All immune parameters were significantly (P<0.05) enhanced by P-glucan, peptidoglycan, MOS and CpG ODN. Lactoferrin induced significant (P<0.05) increase in superoxide anion production and SOD activity of coelomocytes but did not affect the phagocytosis and T-NOS activity. Vitamin C and vitamin E significantly (P<0.05) enhanced the SOD and T-NOS activities of coelomocytes while no significant effects were observed in phagocytosis and superoxide anion production. Chitosan did not affect any immune parameters of sea cucumber coelomocytes. The results suggest thatβ-glucan, peptidoglycan, MOS, CpG ODN, lactoferrin, vitamin C and vitamin E can enhance the non-specific immune response of sea cucumber in vitro.2. The immune responses of sea cucumber Apostichopus japonicus coelomocytes were evaluated after incubated withβ-glucan, mannan oligosaccharide (MOS) and their combinations in vitro. Coelomocytes were incubated in L-15 medium containing 0,10 and 25μg/mlβ-glucan alone or in combination with 0,40 or 80μg/ml MOS respectively. Coelomocytes were sampled at 1 h,3 h,6 h,12 h and 24 h, and immune indices including phagocytosis, superoxide anion production, superoxide dismutase (SOD) activity and total nitric oxide synthase (T-NOS) activity were determined. The results showed that all the four immune indices of coelomoytes incubated withβ-glucan alone and combined with MOS were significantly (P<0.05) higher than those of their respective control. MOS could increase phagocytosis, superoxide anion production and SOD activity of coelomoytes significantly (P<0.05), while no difference was observed in T-NOS activity. Synergistic effect ofβ-glucan and MOS was observed. Combination of 25μg/mlβ-glucan and 40μg/ml MOS showed the best effects on increasing the immune indices.In order to confirm the results in the in vitro experiment, a feeding trial was conducted. Sea cucumbers were fed with basal diets, supplemented 0,0.075 and 0.15%β-glucan alone or in combination with 0,0.1 or 0.2% MOS respectively. After 4-week feeding, sea cucumbers were sampled. Growth performance and resistance against Vibrio splendidus were determined. During the experimental period, immune indices including total coelomocytes count (TCC), phagocytosis, superoxide anion production, SOD and T-NOS activities were measured on Day 7, 11,15,18,25 and 29. The growth, TCC, phagocytosis, superoxide anion production, SOD activity and infection resistance of sea cucumbers fed withβ-glucan, MOS and their combinations were significantly (P<0.05) higher than those of their respective control. Only 0.15%β-glucan and combinations of P-glucan and MOS could significantly (P<0.05) increase the T-NOS activity. Furthermore, P-glucan and MOS showed synergistic effect on growth performance, immunity and infection resistance of sea cucumbers. Combined use ofβ-glucan and MOS also prolonged the high levels of immune indices compared withβ-glucan or MOS supplementation separately. Combination of 0.15%β-glucan and 0.1% MOS had the best effects on increasing the immune indices.In conclusion, continuous feeding ofβ-glucan and MOS, especially their combinations to sea cucumber for 4 weeks showed considerable improvement in growth, immunity and infection resistance of sea cucumbers.The results of the in vivo experiment are similar to those of the in vitro experiment, which indicate that in vitro experiment could be used as an effective and reliable method to value whether a substance has the potential as an immunostimulant.