The Selection And Application Of High-effective Immunostimulants For Sea Cucumber (Apostichopus Japonicus Selenka)

Six feeding trials were conducted in indoor circulating system to investigate the effects of dietary vitamin C,vitamin E,β-glucan, Bacillus subtilis, fructooligosaccharides (FOS), mannooligosaccharides (MOS), selenium yeast (Se-yeast) and yeast culture (Diamond V XP Yeast Culture, XP) on growth, immunity and disease resistance in sea cucumber (Apostichopus japonicus Selenka). The results are summarized as follows:1.A 9-week feeding experiment was conducted to investigate the effect of vitamin C andβ-glucan on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Five hundred and forty individuals (initial body weight:5.08±0.08) g, mean±S.E) were fed with nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of vitamin C (0,500,2000 mg kg-1 diet), and each crossed with 0,625 or 1250 mg kg-1β-glucan. After 9 weeks, five sea cucumbers per tank were sampled for immune indices determination. Then all the sea cucumbers left were challenged by Vibrio splendidus. The results showed that dietary vitamin C andβ-glucan had significant effects on specific growth rate (SGR) of A. japonicus (P<0.05).SGR of the seacucumber with the combination of 2000 mg kg-1 vitamin C and 1250 mg kg-1β-glucan was significantly higher than the control group (P<0.05).Activities of phagocytosis, respiratory burst (RB), phenoloxidase (PO) and acid phosphatase (ACP) were significantly influenced by dietary vitamin C andβ-glucan (P<0.05).In the absence of dietary vitamin C, activities of RB,PO and ACP significantly increased with increasingβ-glucan (P<0.05). In the groups with 500 mg kg-1 vitamin C, sea cucumbers fed with 625 mg kg-1β-glucan had significant higher phagocytosis and PO activity than the control group (P<0.05),and those fed with 1250 mg kg-1β-glucan had significant higher RB activity and ACP activity (P<0.05).In the groups with 2000 mg vitamin C kg-1 diet,625 mg kg-1β-glucan supplementation remarkably increased PO activity and ACP activity (P<0.05), while 1250 mg kg-1β-glucan supplementation significantly enhanced phagocytosis, RB activity, PO activity and ACP activity (P<0.05). Moreover, dietaryβ-glucan had significant effect on cumulative mortality of sea cucumbers after 14 days following V. splendidus exposure (P<0.05).Cumulative mortality was significantly affected by dietaryβ-glucan (P<0.05), and cumulative mortality decreased with the increasing doses ofβ-glucan at 500 and 2000 mg kg-1 vitamin C level.The group fed diets supplemented with 500 mg kg-1 vitamin C and 1250 mg kg-1β-glucan had notably lower cumulative mortality compared to the control (P<0.05).Under the experimental conditions, dietary vitamin C andβ-glucan had a synergistic effect on enhancing phagocytosis and RB activity of sea cucumber(P<0.05).2.A 9-week feeding experiment was conducted to investigate the effects of vitamin C and vitamin E on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Five hundred and forty individuals (initial body weight:5.06±0.10 g, mean±S.E) were fed with nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of vitamin C (0, 500,2000 mg kg-1 diet), and each crossed with 0,80 or 250 mg kg-1 vitamin E. After 9 weeks,five sea cucumbers per tank were sampled for immune indices determination. Then all the sea cucumbers left were challenged by Vibrio splendidus.The results showed that specific growth rate (SGR) of A. japonicus was significantly affected by both vitamin C and vitamin E (P<0.05), and SGR of the groups with 250 mg kg-1 vitamin E alone and 250 mg kg-1 vitamin E at 2000 mg kg-1 vitamin C level were significant higher than the control group (P<0.05). Dietary vitamin C and vitamin E had significant effects on phagocytosis, phenoloxidase (PO) activity and acid phosphatase (ACP) activity (P<0.05). In the groups with vitamin C alone, phagocytosis increased with the increasing doses of vitamin C and was significantly enhanced by 2000 mg kg-1 vitamin C(P<0.05).In the groups without vitamin C supplementation, phagocytosis of sea cucumbers fed with 250 mg kg-1 vitamin E was significantly higher than the control group(P<0.05).In the groups with 500 mg kg-1 vitamin C, phagocytosis, respiratory burst (RB) activity and PO activity showed increasing tendency with the increasing administration doses of vitamin E (P<0.05), and PO activity of the group fed with 250 mg kg-1 vitamin E significantly increased compared with the control (P<0.05).In the groups with 2000 mg kg-1 supplementation of vitamin C, RB activity and PO activity and ACP activity increased with the doses of vitamin E, and all the immune indices in the group fed with 250 mg kg-1 vitamin E was significant higher than the control group (P<0.05).Moreover, dietary vitamin E had significant effect on cumulative mortality of sea cucumbers after 14 days following V. splendidus exposure (P<0.05). Cumulative mortality decreased with the increasing doses of vitamin E at each vitamin C level.The group fed with diets supplemented with 2000 mg kg-1 vitamin C and 250 mg kg-1 vitamin E had notably lower cumulative mortality compared to the control (P<0.05).Under the experimental conditions, dietary vitamin C and vitamin E had a synergistic effect on enhancing phagocytosis of sea cucumber (P<0.05).3.An 8-week feeding experiment was conducted to investigate the interaction of probiotic Bacillus subtilis and prebiotic fructooligosaccharide (FOS) on the growth performance, immunity, intestinal microflora and disease resistance of sea cucumber (A.japonicus Selenka).Five hundred and forty individuals (initial body weight:5.09±0.03 g, mean±S.E) were fed nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of B. subtilis (0,0.2 or 0.5%),crossed with 0,0.25% or 0.50% FOS.After 8 weeks, five sea cucumbers per tank were sampled for bacterial quantification and immune indices determination. Then all remaining sea cucumbers were challenged by Vibrio splendidus.The results showed that dietary B. subtilis significantly increased the specific growth rate (SGR), phagocytosis and acid phosphatase (ACP) activity of sea cucumbers, the counts of total viable bacteria (TBC) and disease resistance to V.splendidus (P<0.05),whereas the counts of Vibrio (VBC) decreased (P<0.05).However, dietary B. subtilis had no significant effect on phenoloxidase (PO) activity (P>0.05).The SGR, PO activity, TBC and VBC were significantly affected by dietary FOS.In the groups with B. subtilis alone, SGR and phagocytosis of 0.2% B. subtilis administration significantly increased compared to the control group (P<0.05), while cumulative mortality of sea cucumbers significantly decreased (P<0.05).In the groups without B. subtilis, SGR, phagocytosis and PO activity increased with the doses of FOS,and phagocytosis, PO activity and ACP activity of the group fed with 0.5% FOS significantly enhanced compared the control (P<0.05). In the groups with 0.2% B. subtilis,0.5% FOS supplementation significantly increased the SGR, phagocytosis and PO activity of sea cucumbers, while notably decreased VBC and cumulative mortality (P<0.05).In the groups fed with 0.5% B. subtilis,0.25% FOS supplementation significantly increased the PO activity and ACP activity of sea cucumbers, while significantly decreased cumulative mortality. Under the experimental conditions, dietary B. subtilis and FOS had a synergistic effect on enhancing phagocytosis, PO activity and disease resistance of sea cucumber (P<0.05).4.An 8-week feeding experiment was conducted to evaluate the effects of FOS and MOS on growth, non-specific immunity of sea cucumber (A. japonicus Selenka) as well as its resistance against V.splendidus.FOS and MOS were administered to sea cucumbers through the diets at three levels (0,0.25% and 0.5%, respectively). The basal diet without FOS or MOS was used as the control.Each diet was randomly allocated to triplicate groups of sea cucumbers in indoor 50 1 tanks with circulating seawater and constant aeration. And each tank was stocked with 22 sea cucumbers (initial average weight 3.04±0.06 g).The results showed that:Ⅰ. FOS supplemented at 0.5% significantly enhanced the specific growth rate (SGR) of sea cucumbers (P<0.05).However, no significant difference in SGR was observed among the group containing 0.25% FOS and the control groups (P>0.05).Vibrio bacteria counts (VBC) instead of total bacteria counts (TBC) were significantly affected by FOS administration (P<0.05). Phagocytosis, RB activity, PO activity and ACP activity significantly increased with the increasing doses of FOS, and all of these four immune indices were remarkably enhanced by 0.5% FOS supplementation (P<0.05).Ⅱ.Sea cucumbers fed with 0.25% and 0.5% MOS supplementation had no significant difference in SGR (P>0.05).The groups with two levels of MOS had higher TBC than the control group (P<0.05), and 0.5% MOS had lower VBC compared with the control (P<0.05). Phagocytosis and RB activity in sea cucumbers fed with 0.25% MOS was significant high when compared with the control and 0.5% MOS supplementation group (P<0.05).PO activity and ACP activity increased with the increasing doses of MOS, but there were no significant differences among the three treatments (P>0.05). The challenge experiment showed that sea cucumbers fed the diet with 0.25% MOS had significantly lower cumulative morbidity compared with the control group (P<0.05).Ⅲ.These results suggested that feeding FOS at a dose of 0.5% or MOS at a dose of 0.25% could enhance growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber.5.An 8-week feeding experiment was conducted to investigate the effect of selenium yeast (Se-yeast) and vitamin E on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Six hundred individuals (initial body weight:3.52±0.08 g, mean±S.E) were fed eight practical diets according to a 2×4 factorial design: the basal diet as the control diet supplemented with two levels of vitamin E (0,250 mg kg-1 diet), crossed with 0,100,300 or 600 mg kg-1 selenium yeast (Se-yeast). The results showed that dietary Se-yeast and vitamin E had significant influence on specific growth rate (SGR) (P<0.05).And SGR of the sea cucumbers fed with the combination of 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E was significantly increased when compared with the control group (P<0.05). Significant higher phagocytosis,respiratory burst (RB) activity, phenoloxidase (PO) activity and acid phosphatase (ACP) activity were observed in the group with 600 mg kg-1 Se-yeast alone compared with the control group (P<0.05). In the groups with 250 mg kg-1 vitamin E, sea cucumbers fed with 600 mg kg-1 Se-yeast had significant higher phagocytosis, RB activity and PO activity when compared with the control group (P<0.05).Dietary vitamin E had significant effect on catalase (CAT) activity,glutathione peroxidase (GSH-Px) activity and total superoxide dismutase (SOD) activity (P<0.05), but Se-yeast significantly influenced the activities of only GSH-Px and SOD (P<0.05), and there were significantly interactions between Se-yeast and vitamin E on GSH-Px activity and SOD activity (P<0.05). The challenge trial showed that dietary Se-yeast and vitamin E had significant effects on cumulative mortality after 14 days following V. splendidus exposure (P<0.05).In the absent of vitamin E, cumulative mortality showed increasing tendency with increasing Se-yeast, but there were no significant difference compared to the control (P>0.05).In the groups with 250 mg kg-1 vitamin E, sea cucumbers fed each Se-yeast levels had significant lower cumulative mortality compared with other groups and the control group (P<0.05). It could be concluded that the group with 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E could significantly enhance the SGR and immunity, keep the antioxidant enzymes in a balanced level and decrease cumulative mortality of sea cucumbers. Therefore, combination of 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E was the optimal group in the present study.6.An 8-week feeding experiment was conducted to investigate the effect of yeast culture (Diamond V XP Yeast Culture, XP) and Bacillus subtilis on the growth performance, intestinal microflora, immunity and disease resistance of sea cucumber (A.japonicus Selenka).Six hundred individuals (initial body weight:3.49±0.06 g, mean±S.E) were fed eight practical diets according to a 2×4 factorial design:the basal diet as the control diet supplemented with two levels of Bacillus subtilis (0,0.2%), crossed with 0,0.1%, 0.2% or 0.4% yeast culture (XP).After 8 weeks, five sea cucumbers per tank were sampled for immune indices determination. Then all remaining sea cucumbers were challenged by Vibrio splendidus.The results showed that dietary XP had significant influence on specific growth rate (SGR), total bacteria counts (TBC) and Vibrio bacteria counts (VBC), activities of phagocytosis, respiratory burst (RB) activity, phenoloxidase (PO) activity and acid phosphatase (ACP) activity and cumulative mortality, but Bacillus subtilis significant influenced only SGR, counts of TBC and VBC, cumulative mortality (P<0.05).Evident increases of all the immune enzymes tested in the present study were noticed in sea cucumbers fed with o.4% XP in the groups without Bacillus subtilis (P<0.05). In the groups with 0.2% Bacillus subtilis, SGR, TBC, RB, ACP of each level of XP administration significantly increased compared to the group (P<0.05), while cumulative mortality of sea cucumbers significantly decreased (P<0.05).Under the experimental conditions, dietary vitamin XP and Bacillus subtilis had a synergistic effect on enhancing TBA, phagocytosis, RB activity, PO activity and ACP activity of sea cucumber (P<0.05).