The Effect Of Dietary Supplementation With Clostridium Butyricum And Enterococcus Faecalis On The Growth Performance,Immunity,Intestinal Microbiota And Disease Resistance Of Tilapia (Oreochromis Niloticus)

Fish diseases become more and more serious,and have resulted in serious economic losses with the increase of intensive cultivation.Antibiotics have been extensively used to prevent and control bacterial diseases.However,the extensive usage of antibiotics may cause some negative effects,such as affecting the health and immunity of fish,the emergence of antibiotic-resistant bacteria and antibiotic residues,affecting food safety and human health.Therefore,the reduction of antibiotic use and the application of antibiotic substitutes have been widely concerned.Probiotics,Chinese herbal medicine and immune enhancers are potential substitutes with good application prospects.Clostridium butyricum(CB)and Enterococcus faecalis(EF)are beneficial bacteria in human and animal intestines.They have been widely used in animal husbandry and shown good effects,but the research in aquaculture was relatively limited.In this study,we investigated the effect of CB and EF on the growth performance,immune function,intestinal microbiota and disease resistance of tilapia(Oreochromis niloticus),and the inhibition of pathogenic bacteria in fish.Our research included the following three aspects:Part ? The effect of dietary supplementation with CB and EF on the growth performance and health status of tilapiaTwo single factor experiments were conducted to study the effects of dietary CB or EF on growth performance and health status of tilapia.In each single factor experiment,300 healthy tilapias weighing 56.16±0.82g were selected and divided into 5 treatments with 3 replicates in each treatment.Five diets with CB concentration of 0,1×10⁴,1×10⁵,1×10⁶ and 1×10⁷ CFU/g were formulated,and denoted as CGc,CB1,CB2,CB3 and CB4groups respectively.Five diets with EF concentration of 0,1×10⁵,1×10⁶,1×10⁷ and1×10⁸CFU/g were formulated,and denoted as CGe,EF1,EF2,EF3 and EF4 groups respectively.A 56 day feeding experiment was conducted in each experiment to investigate the production performance,body composition and blood index of tilapia.After feeding experiment,tilapia were challenged with Streptococcus agalactiae to evaluate the effect of dietary CB or EF on the disease resistance of tilapia.The results showed that:(1)Compare to the CGc group,the final weight,weight gain rate(WGR)and specific growth rate(SGR)were significantly improved in CB2 group(P<0.05).Dietary CB had no significant effect on condition factor(CF),hepatopancreas index(PHI),visceral index(VSI)and the moisture(H₂O),crude protein(CP)and crude ash(Ash)of whole fish,the activity of lysozyme(LZM)and alkaline phosphatase(ALP),D-lactate and endotoxin(P>0.05).Dietary CB significantly reduced the serum total cholesterol(TC)level,malondialdehyde(MDA)concentration,serum aspartate transaminase(AST)and diamine oxidase(DAO)activity(P<0.05),increased total antioxidant capacity(T-AOC)(P<0.05).The serum alanine aminotransferase(ALT)activity was significantly decreased in CB1 and CB2 groups(P<0.05);Dietary CB significantly decreased the serum glucose(GLU)at levels of 1×10⁵and 1×10⁶ CFU/g diet(P<0.05).The serum triglyceride(TG)was significantly decreased,the serum superoxide dismutase(SOD)activity,complement 3(C3)and complement 4(C4)concentration were significantly improved by dietary CB at levels of 1×10⁵,1×10⁶ and1×10⁷CFU/g diet(P<0.05).Dietary CB significantly increased the lipid content(Fat)of whole fish at levels of 1×10⁶and 1×10⁷ CFU/g diet(P<0.05).Dietary CB significantly decreased the cumulative mortality(CM)of tilapia at levels of 1 x 10⁵,1 x 10⁶ and 1 x10⁷ CFU/g diet(P<0.05).(2)Compare to the CGe group,the final weight,WGR and SGR were significantly improved,and feed conversion ratio(FCR)was significantly decreased in EF3 group(P<0.05).Dietary EF had no significant effect on PHI,VSI,H₂O and Ash of whole fish,serum endotoxin,T-AOC,SOD and LZM activity(P>0.05).Dietary EF significantly reduced the serum ALT activity and D-lactate level,improved the CP of whole fish(P<0.05).Dietary EF significantly decreased serum AST activity and increased ALP activity at levels of 1 x 10⁵,1 x 10⁶ and 1 x 10⁷ CFU/g diet(P<0.05),significantly improved the Fat of whole fish at level of 1 x 10⁷ CFU/g diet(P<0.05).The serum C3 and C4 concentration were significantly increased by dietary EF at levels of 1 x10⁶,1 x 10⁷and 1 x 10⁸ CFU/g diet(P<0.05).The CF was significantly improved,and serum TC,TG,GLU content and DAO activity significantly decreased by dietary EF at levels of 1 x 10⁷and 1 x 10⁸ CFU/g diet(P<0.05).Dietary EF significantly decreased the serum MDA concentration at level of 1 x 10⁸ CFU/g diet(P<0.05).Dietary EF significantly decreased the CM of tilapia at levels of 1 x 10⁶,1 x 10⁷ and 1 x 10⁸ CFU/g diet(P<0.05).The results revealed that dietary CB or EF could improve metabolism,fat deposition,liver and intestinal health,antioxidant capacity and humoral immunity,growth performance and disease resistance of tilapiaPart ? The effects of dietary CB and EF on intestinal related gene expression of tilapiaThe experimental design and experimental diet were the same as the second chapter.At the end of the 56 day feeding experiment,6 fish were taken from each treatment and their intestines were separated.The gene expression of tight junction protein(TJP)and immune related genes in intestines was detected by real-time fluorescent quantitative PCR.The results showed that:(1)Dietary CB significantly increased the expression of IRAK-4(P<0.05).When dietary CB concentrations were 1×10⁵,1×10⁶ and 1×10⁷CFU/g,the gene expressions of IL-8,TNF-a and My D88 were significantly up-regulated(P<0.05).When dietary CB concentrations were 1×10⁶ and 1×10⁷CFU/g,TLR2 gene expression was significantly up-regulated(P<0.05).Dietary CB had no significant effect on the gene expression of IL-10,Claudin-1 and Occludin(P>0.05).There was a significant positive correlation between the gene expression of TNF-?and TLR2,My D88 or IRAK-4,and between IL-8 and TLR2,My D88 or IRAK-4(P<0.05).(2)Dietary EF significantly upregulated the gene expression of Claudin-1,IL-8,TNF-a,TLR2,My D88 and IRAK-4(P<0.05).When the dietary EF concentrations were 1×10⁵ and 1×10⁷CFU/g,the gene expression of Occludin was significantly up-regulated(P<0.05).When the dietary EF concentration was 1×10⁷CFU/g,the gene expression of IL-10 was significantly up-regulated(P<0.05).The results revealed that dietary EF increased TJP gene expression and improved the intestinal structure and health of tilapia.Dietary CB or EF up-regulated the gene expression of IL-8,TNF-a,TLR2,My D88 and IRAK-4,activated intestinal immunity,and improved the intestinal immune function of tilapia.Part ? The effects of dietary CB and EF on intestinal microflora of tilapiaA single factor experiment was conducted to evaluate the regulatory effect of dietary CB or EF on the intestinal microflora of tilapia.A total of 180 healthy tilapias weighing78.54±0.75g were selected and divided into three treatments with 60 tilapias in each treatment.The three treatments were divided into CG(control group),CB2(CB concentration was 1×10⁵ CFU/g)and EF3(EF concentration was 1×10⁷CFU/g).After 56days of feeding trial,6 fish were randomly selected from each treatment,and the intestinal contents were taken out to determine the microbial composition.The results showed that:(1)the number of intestinal microbiota OUT increased in CB2 and EF3group.(2)The alpha diversity indexes of Sobs,Ace,Chao1 and Shannon Alpha were significantly improved in probiotic groups at the OUT,genus and phylum level(P<0.05).(3)There was higher similarity of intestinal microbial composition in the probiotic groups.(4)Dietary CB and EF significantly changed the intestinal microflora of tilapia.A total of49 phyla were identified,and the relative abundances of 42 phyla were significantly different between control group and probiotic groups(P<0.05).Compared with the CG group,the relative abundances of Bacteroidetes,Firmicutes,candidate division-SR1,Chloroflexi,Chlorobi,Acidobacteria,Spirochaetae,Nitrospirae,Parcubacteria,Planctomycetes and WCHB1-60 were significantly increased,and those of CKC4 and Fusobacteria were significantly decreased in CB2 and EF3 groups(P<0.05).There was no significant difference in Proteobacteria among the three groups(P>0.05).A total of708 genera were identified,and the relative abundances of 368 genera was significantly different between control group and probiotic groups(P<0.05).Compared with the control group,the relative abundances of Cetobacterium,Aeromonas and CKC4 in CB2and EF3 groups were were significantly decreased,and those of Candidate?division?SR1?norank,Bacteroidetes?vadin HA17?Norank,Dechloromonas,Zoogloea,Oligodextrales?noran,Draconibacteriaceae?uncultured,Comamonadaceae?unclassified,Saprospiraceae?uncultured,Bacillus,Nitrospira,WCHB1-69?Norank and Parcubacteria?norank were significantly increased(P<0.05)The results revealed that dietary CB or EF significantly changed the structure and composition of intestinal microbial in tilapia,increased the diversity index and individual similarity of intestinal microbial,and the relative abundances of beneficial microorganisms such as Bacillus,and decreased the relative abundances of conditional pathogens bacteria such as Aeromonas,and reduced the risk of disease in tilapia.Part ? The inhibitory effect of CB or EF on common pathogens in fishThe antibacterial activity of CB and EF on streptococcus agalactiae,Edwardiella tardans or Aeromonas hydrophila were investigated in vitro.The LB agar culture medium was dissolved and cooled to 48-50?,then the pathogen was added,quickly shaken and put into culture dish.After the culture medium solidifies,the Oxford cup was placed in the dish,then the sterile water,enrofloxacin,penicillin,CB or EF culture medium in equal volume was added into the Oxford cup.The culture dish was covered and cultured at37?for 17h.Then,the diameter of the transparent circle was observed.The results showed that CB and EF exhibited significant antibacterial activity on the growth of S.agalactiae and E.tardans,and the antibacterial activity was weaker than that of penicillin,but was equal to or stronger than that of enrofloxacin.CB and EF had significant inhibitory effect on A.hydrophila,and the antibacterial activity was much better than penicillin.The results indicated that CB and EF have a certain inhibitory effect on common pathogenic bacteria of fish,and have a good prospect as alternative antibiotic.In conclusion,CB and EF had significant antibacterial activity on common pathogens of fish.Dietary CB and EF at a suitable dose changed the structure and composition of intestinal microbial,regulated the gene expression of intestinal immune related genes and TJP,enhanced the humoral and intestinal immune functions,and improved the growth performance and disease resistance of tilapia.According to the growth performance,immunity and disease resistance,the optimal dose of dietary CB and EF was 1 x 10⁵ CFU/g and 1 x 10⁷ CFU/g,respectively.