Effect Of Probiotics On Growth Performance, Digestive Enzymes Activities, Immunity And Gut Microbiota Of Epiniphelus Coioides

In this study, probiotics with the ability of inhibition on potential pathogens bacterium and tolerance to mimic gastrointestinal environments were screened from the gut of grouper (Epinephlus coioides). And the effect of probiotics on growth performance, digestive enzyme activities, immunity and gastrointestinal flora of grouper were discussed. The study includes four trials and the general results are presented as follow:Trial 1:Biological characteristics of five strains (DE5, Bacillus clausii; SE5, Bacillus pumilus, SE6 Psychrobacter sp., MM1 Lactococcus lactis, and MM4, Enterococcus faecium) isolated from the gut of grouper, such as inhibition on potential pathogens, tolerance to mimic gastrointestinal environments and the growth curves were studied in vitro. The results showed that five strains could selectively inhibit potential pathogens (Vibrio parahaemolyticus, Vibrio harveyi, Vibrio metschnikovi, and Staphylococcus aureus) of grouper Epinephlus coioides. The strains SE6, MM1, and MM4 could inhibit the four pathogens strains; while DE5 could inhibit Vibrio harveyi, Vibrio metschnikovi and Staphylococcus aureus, SE5 could inhibit Vibrio parahaemolyticus and Staphylococcus aureus. The growth curves study showed that SE5 entered logarithmic growth phase after 4 hours of incubation, while others took for 8 to 12 hours. The results also showed that the ability of mimic gastrointestinal environments tolerant depended on different strains; DE5 exhibited the best tolerance to artificial gastric juice, followed by SE6, SE5, MM4 and MM1; while DE5 exhibited the best tolerance to artificial intestinal juice, followed by SE5, DE5, MM4 and MM1. Therefore, the five strains have the potential to be probiotics as these strains have a good performance on growth, tolerance to mimic gastrointestinal environment and antagonistic activities to potential pathogens.Trial 2:In this study,252 fishes of uniform size (40±5 g) were randomly distributed in 6 groups (T～T5), each with 3 replicates. The fishes in control group (T0) were fed based diet, while the treatment groups T1 to T5 were fed with probiotics diets by adding DE5, SE5, SE6, MM1 and MM4 (1×108 cells/g) respectively for 60 days. The results showed that during the period of 0 to 30 days, there were no significant effect on growth performance of five treatment groups; however the final average body weight (FAW), average weight gain (AWG), weight gain rate (WGR) and special growth ration(SGR) of groups T3 and T4 were higher than that of group T0(P> 0.05), the feed conversion ratio (FCR) were lower than that of group T0(P> 0.05). During the period of 31 to 60 days, there were no significant effect on FAW of all treatment groups, but AWG of group T2 were significantly higher than that of group T0(P< 0.05); WGR and SGR of groups T2, T5 were significantly higher than that of group T0(P< 0.05); FCR of groups T1, T2, T3 and T5 were significantly lower than that of group T0(P< 0.05), while the FCR of group T4 was lower than that of group T0(P> 0.05). During the period of 0 to 60 days, there were no significant effects on growth performance of five treatment groups; however, the FCR of treatment groups were significantly lower than that of group TO except group T5(P<0.05).The results showed that the activities of digestive enzyme depended on different strains on different times. The trypsin and amylase activity in hepatopancreas of group T1 were significantly higher(P<0.05) than that of the of group TO at 30 days, and lipase activity was higher(P>0.05) than that of the of group T0; However there were no significant effects on digestive enzyme activities of hepatopancreas at 60 days. The trypsin and amylase activity of hepatopancreas of group T2 were higher(P>0.05) than that of the of group T0 at 30 and 60 days. There were no significant effects on digestive enzyme activities of hepatopancreas of group T3 and T4, at 30 or 60 days. The trypsin activity of hepatopancreas in group T5 was significantly higher(P<0.05) than that of the of group T0 at 60 days; while there were no significant effects on trypsin activity of hepatopancreas at 30 days, and no effect on amylase and lipase activity of hepatopancreas at 30 or 60 days. The study indicated there were no significant effects on digestive enzyme activities of intestinal in all treatment groups at 30 or 60 days.Trial 3:The results showed that serum lysozyme activity of grouper in group T1 increased significantly (P<0.05) compared with the group T0 at 60 days, and serum lysozyme activity increased by 42.85%(P>0.05) at 30 days. While there were no significant effect on serum T-SOD, IgM, complement C3 and C4 levels of group T1 compared with the control group (TO) at 30 or 60 days. However, compared to group T0, the serum T-SOD in group T1 increased by 11.43%(P> 0.05) at 60 days, IgM increased by 38.89%(P>0.05) at 30 days, serum C3 of group T1 increased by 11.99% and 15.9% respectively at 30 and 60 days, serum C4 increased by 6.37% at 30 days. The phagocytic percentage (PP) and phagocytosis activity index (PI) of leukocyte of group T1 were significantly higher(P<0.05) than that of group TO at 60 days. Compared to group TO, serum lysozyme of group T2 increased by 4.49%(P> 0.05) at 30 days, T-SOD increased by 18.49%(P>0.05) at 60 days, IgM increased by 50%(P> 0.05) at 30 days, serum C3 and C4 increased by 11.17% and 11.43%(P> 0.05) respectively at 30 days; and the PP and PI of leukocyte of group T2 were higher(P> 0.05) than that of group TO at 60 days. Serum lysozyme, T-SOD, IgM, C3 and C4 in group T3 showed no significant difference compared with the group TO at 30 or 60 days; while T-SOD increased by 6.92% at 60 days, IgM increased by 61.11% and 12.12% respectively at 30 and 60 days, C3 increased by 15.31% and 33.58% respectively at 30 and 60 days, C4 increased by 13.77% at 60 days, and the PP and PI of group T2 were higher than (P> 0.05) that of group TO at 60 days. In group T4, the serum lysozyme increased by 59.18% and 25.84%(P> 0.05), T-SOD were increased by 19.77% and 6.92%(P> 0.05), IgM increased by 77.78% and 33.33%(P> 0.05), C3 increased by 11.72% and 33.58%(P> 0.05), C4 increased by 41.54% and 3.58%(P> 0.05) respectively compared with the group TO at 30 and 60 days. However, dietary administration of strain MM1 (group T4) did not affect the PP and PI of leukocyte compared with the group TO at 60 days. There were no effects on serum lysozyme of dietary administration of stain MM4(group T5) compared with the group TO at 30 and 60 days; while T-SOD increased by 2.97%(P> 0.05) at 60 days, IgM increased by 61.11% and 24.24%(P> 0.05) respectively at 30 and 60 days, C3 increased by 2.54% and 37.56%(P> 0.05) respectively at 30 and 60 days, C4 increased by 14.57%(P>0.05) at 60 days. The PP and PI of group T5 were higher(P>0.05) than that of group TO at 60 days.The results indicated that the five probiotic stains had some positive effect on immunity of grouper.Trial 4:The purpose of this study was to use PCR-DGGE and sequencing to evaluate the autochthonous microbiota in three parts of the digestive tract (stomach, proximal and distal intestine) and effect of dietary probiotics on gastrointestinal flora of juvenile grouper. The result showed that a total of 14 common bands were successfully sequenced from the DGGE gel of 16S rDNA V3 regions in three parts of the digestive tract. And similarities between the PCR-DGGE banding patterns were analyzed using the Pearson correlation coefficient. The result showed that high similarity of band patterns in different sections along the gastrointestinal tract were detected, while there are some differences between different individuals; and the common dominant autochthonous bacteria in different sections along the gastrointestinal tract belonged to Pseudomonas sp, Uncultured gamma proteobacterium, Bacillus sp., Nitratireductor sp., Methylobacterium sp., Dietzia sp. Microbacterium sp., and some uncultured bacterium.The results also showed that add probiotics had some effect on gastrointestinal flora of grouper. Compared with group T0, the colonization of Staphylococcus sp. disappeared in the stomach of group T1, while Uncultured Actinomycetales bacterium seemed to increase, Photobacterium sp. appeared in the stomach. And Photobacterium sp. and Bacillus sp. in the proximal intestine tended to increase; Enterococcus sp. and Vibrio sp. appeared in the intestine. Compared with group TO, colonization of Staphylococcus sp. and uncultured Bacillus sp. in the stomach of group T2 seemed to increase, while uncultured Bacillus sp. in the intestine seemed to increase. In group T3, colonization of Micrococcus sp., uncultured alpha proteobacterium, uncultured beta proteobacterium and some uncultured bacterium appeared in the stomach. Colonization of Bacillus sp. in the intestine seemed to decrease, however Alcanivorax dieselolei and uncultured alpha proteobacterium appeared in the intestine. Compared with group T0, colonization of Staphylococcus sp. and Bacillus sp. disappeared in the stomach of group T4, while uncultured Sphingobacteriaceae sp., Shewanella sp., Vibrio sp. and uncultured Comamonadaceae sp. appeared in the stomach. Colonization of Staphylococcus sp., Flavobacteria sp., Photobacterium sp. and some uncultured bacterium appeared in the proximal intestine; Vibrio sp. and Flavobacteria sp. in the distal intestine seemed to increase; some uncultured bacterium (band 15) decreased in the stomach and intestine. Compared to group T0, colonization of alpha proteobacterium, Comamonas sp. and some uncultured bacterium appeared in the stomach in group T5; Dietzia sp. appeared in intestine; Dietzia sp., Comamonas sp. and some uncultured bacterium in the distal intestine seemed to increase.