| In this study,the effects of soybean meal replace of fishmeal on the growth performance,intestinal microbiota and intestinal health of the northern snakehead(Channa argus Cantor,1842)were investigated.Firstly,the dominant community in the intestinal tract was determined by detecting the microbiota structure in the intestinal tract of wild C.argus.Secondly,the aerobic bacteria in the intestine of the C.argus were cultured and the protease-producing ability was measured to evaluate the effects of soybean meal on the composition and protease production of aerobic bacteria in C.argus.Finally,the effects of soybean meal on the intestinal microbiota and intestinal health of C.argus was studied to studying the relationship between soybean meal,intestinal microbiota and intestinal health in dietary.Experiment 1:Microbial communities in different gastrointestinal regions(stomach,foregut,midgut,and hindgut)of C.argus were compared by polymerase chain reaction and partial 16S rDNA sequencing.A total of 194,140,212,and 122 OTUs were detected in the stomach,foregut,midgut,and hindgut,respectively.Significant differences were found in the Sobs,ACE,Shannon,and Simpson indices among samples(P<0.05).The gastrointestinal microbial community of C.argus consisted predominantly of Proteobacteria with either Halomonas,Shewanella,Plesiomonas or Sphingomonas.Fusobacteria,Firmicutes,and Bacteroidetes also existed in the gastrointestinal tracts.However,significant differences were found in the compositions of microbial community among the four regions(P<0.05).Cyanobacteria and Spirochetes were significantly higher in the midgut and hindgut(P<0.05).Fusobacteria and Firmicutes were dominant in the hindgut and foregut,respectively(P<0.05).Proteobacteria was the lowest in the hindgut(P<0.05).At genus level,Cetobacterium and Plesiomonas were significantly higher in the hindgut than in the other three samples(P<0.05).Clostridium and Prevotella were the highest in the midgut(P<0.05).Halomonas,Shewanella and Sphingomonas were the highest in the foregut(P<0.05).Paracoccus and Vibrio were the highest in the stomach.Several genera were only detected in certain regions,as follows:stomach,Paracoccus and Vibrio;foregut,Halomonas,Shewanella and Sphingomonas;midgut,Clostridium and Prevotella;and hindgut,Cetobacterium and Plesiomonas(P<0.05).At the species level,Acinetobacter rhizosphaerae was only detected in the stomach.Prevotella copri and Clostridium perfring were not detected in the foregut and midgut,respectively,whereas Prevotella copri and Faecalibacterium pra were not detected in the hindgut.These findings provide valuable information on the microbial community in each gastrointestinal region of C.argus.Moreover,this study indicated that microbial community was not only related to rearing environment but also to the physico-chemical characteristics of each gastrointestinal region.Experiment 2:In order to investigate the effects of replacement of fish meal by soybean meal on composition and protease-producing activity of protease-producing aerobic bacteria in intestinal tract of C.argus,the protease producing bacteria in intestinal tract of C.argus were isolated and cultured.The selected strains were further quantitatively assayed for the protease-producing activity.One control group(D1 group)diet was formulated with defatted fishmeal as the main protein source,and the defatted fishmeal addition in this diet was 55%.Next then,two experimental group diets were formulated using soybean meal to replace different ratio of fish meal in control group diet,and the soybean meal in those two diets was 35%(D2 group)and 75%(D3 group),respectively.Each diet was assigned to three replicates of 28 C.argus with the average body weight of(10.50±0.84)g for 8 weeks in a re-circulated water system indoor.The results showed as follows:The final weight,weight gain rate and specific growth rate was significantly decreased with the replacement ratio of fish meal by soybean meal increasing(P<0.05).A total of 21 protease-producing aerobic bacteria were isolated from the intestine of C.argus,which was 16 from D1 group,19 from D2 group and 20 from D3 group.The replacement of fish meal by soybean meal significantly decreased the hydrolysis spot diameter(R)/strain diameter(r)value of strains P1004 and P1018(P<0.05),which was used to express the protease-producing activity,but significantly increased the R/r value of strains P1009 and P1012(P<0.05).With the replacement ratio of fish meal by soybean meal increasing,the R/r value of strain P1018 was significantly decreased(P<0.05).Physiological and biochemical characteristics identification and 16S rDNA sequence analysis revealed that the strain P1009,whose protease-producing activity was the highest in the present study,was Pseudomonas aeruginosa.In summary,the results indicate that there exist a great variety of protease-producing aerobic bacteria in intestinal tract of C.argus,and the replacement of fish meal by soybean meal can affect both the quantity and protease-producing activity of protease-producing bacteria.Strain P1009 which from the intestinal tract of C.argus has the highest protease-producing activity,and it can be as a potential source of probiotics.Experiment 3:A 63-day feeding trial was conducted in C.argus to observe the effects of a dietary soybean meal substitution on the microbiota community,morphology and inflammatory cytokine gene expression in the intestine.Four isonitrogenous and isoenergetic diets containing increasing levels of soybean meal were used to replace 0%,25%,50%and 75%of the defatted fishmeal(diets are referred to G1,G2,G3 and G4,respectively).Different dietary soybean meal substitutions significantly affected the intestinal microbiota composition.At the phylum level,Firmicutes abundance was the lowest in the G4 group,in contrast with Proteobacteria,Bacteroidetes and Planctomycetes.At the genus level,significantly lower abundance of Lactococcus,Geobacillus,Pseudomonas,Streptococcus,Bacillus and Acinetobacter,but higher abundance of Cetobacterium,Planctomyces,Shewanella,Thermomonas,Rubrivivax and Carnobacterium was observed in fish fed the G4 diet.With increased dietary soybean meal,the thickness of the muscularis,the height of the fold and the height of the microvillus in the distal intestine decreased,but the relative expression of IL-1?,IL-10 and IL-17F was significantly upregulated.In conclusion,more emphasis should be placed on the functionality of intestinal microbiota and the pathogenesis of mucosal inflammation to assess the effects of diet and fish intestinal health through intestinal microbiota profiling. |
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