| The present study evaluated the effect of dietary administration of viable and heat-inactivated autochthonous probiotic Bucillus claussi(DE5), Bacillus pumilus(SE5) and Psychrobacter sp.(SE6) on the growth performance, intestinal microbiota, immune-related genes expression in the gut and head-kidney of grouper(Epinephelus coioides). 525 juvenile fish with an average of initial weight of(14.7±0.2) g were randomly divided into 7 groups(group T0, T1, T2, T3, T4, T5 and T6) with 3 replicate per group and 25 fish per replicate. The fish in each group were fed respectively for 60 d with basical diet containing control group(T0), viable B. claussi DE5(group T1), heat-inactivated B. claussi DE5(group T2), viable Bucillus pumilus SE5(group T3), heat-inactivated Bucillus pumilus SE5(group T3), viable Psychrobacter sp. SE6(group T5) and heat-inactivated Psychrobacter sp. SE6(group T6), and the level of probiotic bacterium in each group was 1.0×108 cfu/g. The general results in the trial were showed as follows:Trial 1: During the early period(0 to day 30), final average body weight(FBW) and specific growth ratio(SGR) of group T5 were significantly higher than those of control group(P<0.05),but other exprimental groups had no significant difference(P>0.05). The weight gain rate(WGR) of groups T3, T4, T5 and T6 were significantly higher than that in the control group(P<0.05), however, feed coversation ratio(FCR) of experimental groups were lower than that of control group, and groups T4 and T5 showed significantly a decrease(P<0.05). During the later period(31 to day 60), FBW of groups T4, T5 and T6 were significantly higher than that in the control group(P<0.05). The specific growth ratio(SGR) of T4 was significantly higher than the control(P<0.05), while that in other groups were increased slightly with no significant difference(P>0.05). Feed coversation ratio(FCR) of groups T2 and T4 were significantly lower than that of T0 group(P<0.05), but other groups showed no significant difference(P>0.05). During the whole period(0 to day 60), dietary administrations of heat-inactivated B. pumilus SE5, viable and heat-inactivated Psychrobacter sp. significantly improved the growth performance of grouper, as the AWG, SGR and WGR in groups T4, T5 and T6 were significantly higher than those in the control group(P<0.05). The FCR of each experimental group was lower than that of control group, and FCR in groups T1, T2, T3, T4 and T5 decreased significantly(P<0.05).Trial 2: The serum lysozyme activity, serum total superoxide dismutase actvity(T-SOD), complement C3 and IgM levels in each experimental group had no significant difference compared with those in the group T0 at day 30(P>0.05). The serum lysozyme activity in groups T1, T3, T4 and T6, and T-SOD level in groups T3, T4 and T6 were higher than those of control group, respectively. Besides, complement C3 and IgM levels in each group were higher than the control group. At day 60, serum lysozyme activity in groups T1, T2, T5 and T6 were higher than that of the control(P>0.05), meanwhile, T-SOD of each experimental group was higher than that of control group(P>0.05). Compared with the control group, IgM level in each experimental group increased in different degrees, meanwhile, those in groups T4, T5 and T6 showed significant significance(P<0.05). Complement C3 levels in experimental groups were higher than the control group except group T1, and that in groups T4 and T5 had significant difference(P<0.05). In conclusion, dietary administration of viable and heat-inactivated autochthonous probiotic B. claussi(DE5), B. pumilus(SE5) and Psychrobacter sp.(SE6) improved the serum immunity function of grouper(E. coioides) in varying degrees.Trial 3: In the present study, real-time quantitative PCR technique was used to investigate the effects of viable and heat-inactivated probiotics on the immune related genes expression in the gut and head-kidney of grouper. The viable B. claussi DE5(group T1) had no significant effect on genes expression compared with control group(P<0.05), and only expression of TLR2, IL-8 and IgM in the gut and head-kidney were slightly up-regulated(P>0.05). Group T2 had significantly augmented expressions of TLR5, MyD88, IL-8, IL-1β and TGF-β1 in the gut and expressions of TLR5, IL-8 and IL-1β in the HK compared with control group(P<0.05). Group T3 had significantly increased expression of TLR1 in the gut and expression of MyD88 in the HK compared with the control group(P<0.05). Meanwhile, expression of TLR2, MyD88, IL-8, IL-1β, Epinecidin-1 and IgM in the gut and expression of TLR2, IL-1β, TGF-β1 and IgM in the HK of group T3 were slightly up-regulated compared with the control group(P>0.05). Group T4 showed a significantly up-regulated expression of TGF-β1 in the gut(P<0.05) and Group T5 showed significantly up-regulated expression of TLR2, TLR5, TGF-β1 and Epinecidin-1 in the gut and expressions of TLR1, TLR5 and IL-8 in the HK compared with the control group(P<0.05); Group T4 had also significantly up-regulated expression of Epinecidin-1 in the gut and expressions of TLR1 and TGF-β1 in the HK compared with the control group(P<0.05).Trial 4: PCR-DGGE techniques were used to investigate the intestinal microbiota of orange-spotted grouper after dietary administration of viable and heat-inactivated probiotics for 30 days and 60 days. Compared with the control group, bacteria such as uncultured Escherichia sp. and Shigella dysenteriae strain were present in the group T1 at day 30, and uncultured Escherichia sp. appeared in the gut of the groups T1 and T2 at day 30, however, the uncultured Escherichia sp. bacteria disappeared but uncultured bacterium, Vibrio ruber appeared in the gut of group T2 at day 60. The uncultured proteobacterium was colonized in the intestine of group T2 at day 30. Compared wth the control group in each period respectively, some bacteria such as Photobacterium sp., uncultured bacterium and uncutured marine bacterioplankton were present in group T3, however, three bacteria appeared and uncultured proteobacterium disappeared in group T4 on day 30. The uncultured Escherichia sp. appeared, while Psychroserpens burtonensis subsp., uncutured bacterium and uncultured Escherichia sp. disappeared in group T4 at day 60. Compared with the control group, uncultured gamma proteobacterium was appeared in group T5 and two uncultured bacterium appeared in group T6 at day 30; Three uncultured bacterium disappeared between group T5 and group T6 at day 60. |
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