| This thesis was studied the effect of sietary supplementation of Grobiotic?-A and raffinose as immunostimulants on growth performance, non-specific immune response, antioxidant performance, resisting bacteria challenge and intestinal flora of Japanese sea bass. The aim of this study was to find the optimal supplemental dosage of Grobiotic?-A and raffinose, and to compare the function of two additives as prebiotics. The study included five independent experiments. The results were summarized as follows:1,In the experiment 1, effects of Grobiotic?-A on growth performance, immunity, anti-stress and survival of Japanese sea bass (Lateolabrax japonicus P.) was studied. The basal diet was formulated to contain 43% crude protein, 11.34% crude lipid and 17.2 MJ/kg gross energy. A total of five treatments, supplements of 0, 0.2, 0.4, 0.8 and 2% Grobiotic?-A were singularly added to the basal diet, and named as G0, G2, G4, G8 and G20, respectively. Each diet was fed to three replicate groups of Japanese sea bass (initial body weight: averaging 3g), and each replicate had 30 fish.The experiment was lasted 16w. At 16 weeks of weighing and taking blood, and indicator of immunology and antioxidant were measured. In addition, separately anti-stress and Aeromonas hydrophila were carried out.Growth performance index showed that highest survival and weight gain rate (WGR) were showed in G8 group and significantly higher than G4 (P < 0.05). Innate immune responses as follows: MDA of fish of G4 were significantly lower thanG20 and fish of G4 showed lowest SOD (P < 0.05). G4 and G8 Grobiotic?-A inclusion increased non-specific immune response of Japanese seabass. Survivals of experimental fish of Grobiotic?-A inclusion groups were significantly higher than that of the fish in control group after a challenge by Aeromonas hydrophila. So, 4~8g/kg Grobiotic?-A will be recommended to utilized in Japanese seabass diets.2,In the experiment 2, A 16w growth trial was conducted to study the effects of raffinose on growth performance, non-specific immunity, stress response and survival of Japanese sea bass (Lateolabrax japonicus) challenged with pathogenic bacteria, Aeromonas hydrophila in a recirculation system. Five experimental diets with 0, 200, 400, 800 and 2000 mg/kg of raffinose supplements were prepared, and named as R0, R200, R400, R800 and R2000, respectively. Each diet was fed to triplicates groups of Japanese sea bass (initial body weight: averaging 3g), with 30 fish in each tank. At the end of trial, fish were weighed and serum sample were collected. Besides, a 30s air-exposure stress test and a challenge test with A. hydrophila were carried out.Survival and feed intake of fish did not show significant difference among all groups ( P > 0.05 ). Weight gain rate (WGR) of R800 group was significantly higher than that of control, R400 and R2000 groups (P < 0.05). Feed Conversion Ratio (FCR) and protein efficiency rate (PER) of R800 treatment were significantly lower and higher than R400 treatment, respectively (P < 0.05). Condition factor (CF) of control, R200 and R2000 treatments were significantly higher than R400 and R800 treatments. There were no difference on serum lysozyme, superoxide dismutase and malondialdehyde, and glucose and cortisol level of serum after 30s air stress were no difference, either. However, accumulative survival of control group significantly lower than that of groups with various level of raffinose after being challenged by Aeromonas hydrophila. 800mg/kg of raffinose was recommended to use in diet of Japanese sea bass.3,In the experiment 3, using dilution spread plate method, Japanese sea bass intestinal bacteria were isolated and identification by dilution spread plate method, and then the numbers of Japanese sea bass intestinal bacteria were counted and the diversities of intestinal bacteria were checked, moreover, the diversities of the same group intestinal bacteria at different stages were studied. The numbers of Japanese sea bass intestinal bacteria were 104cfu/g at the initial, 108cfu/g at 8 week and 1011cfu/g at 16 week. In Grobiotic?-A test, the numbers of intestinal bacteria of all groups at 16 week were higher than the numbers of all groups at 8 week (p < 0.05), and there was no difference between the control and all test groups at the same stage. In Grobiotic?-A test, 41 strains were isolated from the intestine of Japanese sea bass. By 16S rDNA analysis, these strains belonged to Sporosarcina, Rahnella, Enterobacter, Pseudomonas, Staphylococcus, Citrobacter, Arthrobacter, Bacillus, Brevundimonas and Aeromonas. Pseudomonas was the dominant bacteria of the control; 8 week, Enterobacter was the dominant bacteria of Grobiotic?-A test groups; 16 week, Pseudomonas and Bacillus were the dominant bacteria of Grobiotic?-A test groups. In raffinose test, 35 strains were isolated from the intestine of Japanese sea bass. These strains belonged to Sporosarcina, Rahnella, Enterobacter, Pseudomonas, Acinetobacter, Microbacterium, Arthrobacter, Bacillus and Aeromonas. At 8 week, Acinetobacter was the dominant bacteria of raffinose test groups; at 16 week, Pseudomonas and Enterobacter were the dominant bacteria of raffinose test groups.4,In the experiment 4, Analysis of the diversity of Japanese sea bass intestinal samples at the initials, 8 and 16 weeks was by means of PCR and denaturing gradient electrophoresis(PCR-DGGE)technique. After analysis of the intestinal bacteria 16S rDNA V3 region genes by DGGE, the results showed the intestines of Japanese sea bass were rich in the diversity of the bacteria. The results as follows: (1) At 8 week, Pseudomonas was colonized in the intestine of all test groups, but it disappeared from the intestine of all treatments at 16 week. (2) Except G2 group at 8 week, Enterobacter existed in the intestine of other treatments at 8 week, and it could exist in the intestine of all treatments at 16 week. (3) Stenotrophomonas was colonized at the G8 and G20 intestine at the initial, 8 and 16 week. (4) Klebsiella appeared in the intestine of G8 and G20 at 16 week. (5) At 8 week, Pseudoalteromonas appeared in the intestine of all treatments, but it only appeared in the intestine of all test treatments at 16 week, so we concluded Grobiotic?-A can promote the colonization of Pseudoalteromonas in the intestine.5,In the experiment 5, analysis of the diversity of Japanese sea bass intestinal samples at the initials, 8 and 16 weeks was by means of PCR and denaturing gradient electrophoresis(PCR-DGGE)technique. After analysis of the intestinal bacteria 16S rDNA V3 region genes by DGGE, the results showed the intestines of Japanese sea bass were rich in the diversity of the bacteria. The results as follows: (1) Except R800 group at 8 week, Pseudoalteromonas lived in the intestine of other treatments at 8 week, and it could exist in the intestine of all treatments at 16 week, so raffinose could promote the colonization of Pseudoalteromonas in the intestine. (2) At 8 week, Stenotrophomonas and Pseudomonas were colonized in the intestine of all raffinose treatments, but they disappeared at 16 week. (3) At 16 week, Enterobacter and Methylobacterium appeared in all treatments. The intestinal bacteria of R800 did not exist Flavobacteria and Actinobacteridae, so 800mg/kg of raffinose was recommended to use in diet of Japanese sea bass.
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