| The intestine of broiler is a home to complex and dynamic microbial community.The microbiota living the intestine has a symbiotic relationship with the host,which are vital to host intestinal health and growth performance.The establishment of broiler gut microbes mainly starts after hatching,and the initially colonized microbes can affect the development of subsequent gut microbes.Therefore,the period after hatching is a window of opportunity for modulatting broiler gut microbes.In this study,the cecal fermentation broth of broiler was prepared by a fermentation system in vitro,and then the fermentation broth was used to inoculate the chicks to explore its effect on broiler gut microbial colonization and intestinal barrier function.The main research contents and results are represented as follows:1 Construction of in vitro fermentation system for cecal microbiota of broilersThe purpose of this experiment was to construct cecal microbiota fermentation system in vitro and prepare cecal fermentation broth of broilers.Five chickens were selected as the donor,and their cecum contents were taken as the inoculation source for the fermentation.16S rRNA high-throughput sequencing was performed on the cecal contents of donor chickens and fermentation broth to investigate the succession rule of microbiota during the fermentation process.The results showed that the number of OTUs and the Shannon index were higher in No.2 and No.3 donor chickens.With the increase of fermentation days,the number of OTUs and the Shannon index in the fermentation broth gradually decreased,and the fluctuation began to decrease on the 7th day of fermentation.Principal co-ordinates analysis(PCoA)and unweighted pair-group method with arithmetic mean analysis(UPGMA)can cluster 5 donor chickens samples into a cluster,and the corresponding fermentation broth of donor chicken was clustered separately.The dominant bacteria in the cecum of donor chicken and fermentation broth were Firmicutes,Bacteroidetes and Proteobacteria.No.4 and No.5 donor groups detected more Enterobacteriaceae bacteria,while the relative abundance of Enterobacteriaceae in No.3 was lower.The results indicated that the microbiota in the fermentation broth reached a dynamic equilibrium state after continuous fermentation for 7 days.The fermentation process of No.3 donor chicken was the most stable,and the microbiota in the fermentation broth had a high similarity with that in the cecum contents.2 Effects of fermentation broth intervention on intestinal microbiota colonization and metabolic function of broilersIn this study,we used cecal fermentation broth prepared by in vitro continuous fermentation system to inoculate chicks to study the effects of early intervention of fermentation broth on intestinal microbiota colonization and metabolic function of broilers.One hundred and twenty newly hatched Arbor Acres(AA)broilers were randomly divided into two groups,the control group(C group)and the fermentation broth group(F group).Each broiler in the F group was given 0.5 mL of fermentation broth within 2h after hatching,while the C group was treated with the same amount of PBS solution.Eight broilers were randomly selected from each group at 1,3,7,14 and 28 days of age and then slaughtered and sampled for the determination of relevant indicators.The results are mainly divided into the following three aspects:1)Effects of fermentation broth intervention on growth performance and intestinal morphology of broilersAt 14 and 28 days of age,the body weight of broilers in the F group was significantly higher than that in the C group(P<0.05).The intestinal morphology analysis showed that the intervention of fermentation broth significantly increased the villus height of jejunum at 14 and 28 days of age(P<0.05),and the villus height of duodenum and ileum at 28 days of age(P<0.05).At 14 days of age,the crypt depth of ileum in the F group was significantly decreased(P<0.01).Except ileum at 14 days of age,the ratio of villus height to crypt depth(V/C)of duodenum,jejunum and ileum of broilers in the F group was significantly higher than that in the C group(P<0.05).The results of intestinal tight junction protein-related genes showed that early intervention with fermentation broth significantly increased the mRNA expression of ZO1 in the ileum at 14 and 28 days of age(P<0.05),and CLDN1 mRNA expression at 28 days of age(P<0.05).On the contrary,early intervention with fermentation broth significantly reduced the mRNA expression of CLDN2 in the ileum of broilers at 14(P<0.05)and 28(P<0.01)days of age.These results indicated that early intervention with cecal fermentation broth can increase the growth performance,improve the intestinal morphology,and increase the intestinal barrier function of broilers.2)Effects of fermentation broth intervention on intestinal microbiota colonization and development of broilersEarly intervention of fermentation broth significantly decreased the Chaol index of cecal microbiota of broilers at 3 and 7 days of age(P<0.01)and increased the Shannon index at 1 day of age(P<0.01).The ?-diversity analysis of the cecal microbiota found that samples of the two groups were clearly separated at each sampling age,and the samples of 1-day-old were far apart from the other days.Bacteroidetes were the most abundant phyla in the cecal microbiota of the intervention broilers at all ages,with an average relative abundance of 41.58?66.38%,which was significantly higher than that of the C group.However,the abundances of Proteobacteria and Escherichia-Shigella in the cecum of the intervention broilers were lower than that of the C group.The measurement of short-chain fatty acids(SCFAs)showed that early intervention with fermentation broth increased the concentration of acetic acid in the cecum of broilers at 28 days of age(P<0.05),the concentrations of butyric acid and isovalerate at 1 and 14 days of age(P<0.05),and the concentration of propionic acid at each sampling age(P<0.05).These results indicate that early intervention with cecal fermentation broth can modulate the colonization of intestinal microbiota of broilers,increase the abundance of beneficial bacteria while reduce the abundance of pathogenic microorganisms,and increase the content of SCFAs in the cecum of broilers.3)Effects of fermentation broth intervention on intestinal metabolic function of broilersFrom the results of principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA),early intervention with fermentation broth significantly changed the metabolic performance of the caecal microbiota of broilers.The two groups screened out 19 and 48 qualitative metabolites at 7 and 14 days of age,respectively,among which 1,3-propanediamine was the only metabolite that was continuously upregulated in the cecum of the intervention broilers.The enrichment pathway analysis of differential metabolites found that 18 and 9 metabolic pathways were identified at 7 and 14 days of age,respectively.These results suggest that 1,3-diaminopropane,a differential metabolite,could be used as a potential biomarker for early intervention with fermentation broth.Amino acid metabolism and unsaturated fatty acid synthesis metabolism are the main metabolic pathways affected by early intervention with fermentation broth.3 Protective effect of fermentation broth on broilers infected with Escherichia coli 1)Establishment of a broiler colibacillosis model80 1-day-old AA broilers were randomly divided into four groups.At the age of 4 days,chicks in the three groups were orally gavaged with 0.3 mL of Escherichia coli(E.coli)suspension,and the viable bacteria counts were 1×106 CFU/mL,1×107 CFU/mL,1×108 CFU/mL,respectively,followed by low-dose group(L group),medium-dose group(M group)and high-dose group(H group).The remaining group was the control group(C group),chicks in the C group was gavaged with the same amount of sterile normal saline.Seven days after the challenge,6 birds were randomly selected from each group,weighed,slaughtered for sampling,and related indicators were determined.The body weight and thymus index of broilers in the H group were significantly lower than those in the C group(P<0.05),and spleen index was significantly higher than that in the C group(P<0.05).The H group significantly increased the concentration of endotoxin in serum of broilers(P<0.05).Compared with the C group,the crypt depth of jejunum in H group was significantly deepened and the villus area was significantly decreased(P<0.05).The ratio of V/C in the M and H groups was significantly decreased(P<0.05).These results indicate that the pathological model of chicken E.coli was successfully established after oral administration of 0.3 mL 1×108 CFU/mL of E.coli suspension at 4 days of age,causing growth retardation and intestinal mucosal damage in broilers.2)Therapeutic effect of fermentation broth intervention on colibacillosis of broilersA total of 180 newly hatched AA broilers were purchased and randomly divided into three groups.Broilers in control group were given 0.3 mL sterile normal saline per day at 1 to 4 days of age.Broilers in challenge group were given 0.3 mL 1 ×108 CFU/mL E.coli suspension at 1 day of age,and 0.3 mL sterile normal saline per chicken at 2 to 4 days of age per day.Broilers in the treatment group were gavaged 0.3 mL 1×108 CFU/mL of E.coli suspension at the age of 1 day,and 0.3 mL of fermentation broth was gavaged per chicken at the age of 2 to 4 days.At 7,10,14 and 21 days of age,8 broilers were randomly selected from each group for weighing and sampling,and the corresponding indexes were determined.The body weight of broilers in challenge and treatment groups was significantly lower than that in control group(P<0.05).The spleen index of broilers at 10,14 and 21 days of age in challenge group was significantly higher than that in control group,and the spleen index of broilers at 14 days of age in treatment group was also significantly higher than that in control group(P<0.05).Thymus index of challenge group at 10 days of age was significantly lower than that of control group,bursal index of challenge group at 10 and 14 days of age was significantly lower than that of control group(P<0.05).However,there was no significant difference between treatment group and control group.The diamine oxidase activity(DO A)in serum of broilers in challenge group and treatment group at 7 and 21 days of age was significantly higher than that in control group(P<0.05),and the DOA in serum of broilers in treatment group at 14 days of age was significantly higher than that in control group(P<0.05).At 21 days of age,the concentration of sIgA in the ileum of broilers in challenge group and treatment group was significantly lower than that in control group(P<0.05).The ratio of V/C of duodenum,jejunum and ileum of broilers in challenge group and treatment group was significantly lower than that in control group(P<0.05).These results indicate that cecal fermentation broth treatment cannot effectively alleviate the damage caused by E.coli infection on growth performance,immune organ development and intestinal barrier function of broilers.3)Preventive effect of fermentation broth intervention on colibacillosis of broilersA total of 180 newly hatched AA broilers were randomly divided into three groups.Broilers in the control group were given 0.3 mL sterile normal saline per day at 1 to 4 days of age.Broilers in challenge group were given 0.3 mL sterile normal saline per day at 1 to 3 days of age,and 0.3 mL 1×108 CFU/mL E.coli suspension at 4 days of age.Broilers in the prevention group were given 0.3 mL of fermentation broth per day at 1 to 3 days of age,and 0.3 mL of 1×108 CFU/mL of E.coli suspension at 4 days of age.At 7,10,14 and 21 days of age,8 broilers were randomly selected from each group for weighing and sampling,and the corresponding indexes were determined.The body weight and bursal index of broilers in challenge group were significantly lower than those in control group(P<0.05),and the spleen index was significantly higher than that in control group(P<0.05).The contents of D-lactic acid and endotoxin and the DOA in serum of broilers in challenge group were significantly higher than those in control group(P<0.05),but there were no significant differences between prevention group and control group.At 21 days of age,the content of sIgA in the ileum of broilers in challenge group was significantly lower than that in control group and prevention group(P<0.05).The ratio of V/C of j ejunum and ileum of broilers in challenge group was significantly lower than that in control group(P<0.05),but there was no significant difference between prevention group and control group.The mRNA expression levels of ZO1,ZO2 and CLDN3 in the ileum of challenge group were significantly lower than that of control group(P<0.05),and there was no significant difference between prevention group and control group.The Chaol and Shannon indexes of cecal microbiota of broilers in challenge group were significantly decreased(P<0.05).Escherichia-Shigella was significantly enriched in the cecum of broilers in the challenge group,and Bacteroides,Parabacteroides and Megasphaera were obviously greater in the cecum of broilers in the prevention group.The number of functional genes in cecum of broilers in the challenge group was significantly decreased(P<0.05),and the metabolic performance of functional genes in the prevention group was the strongest.These results indicate that cecal fermentation broth prophylaxis can effectively prevent the colonization of pathogenic microbiota in the intestine of broilers and alleviate the damage caused by E.coli.In conclusion,the early intervention with fermentation broth can modulate the colonization and development of intestinal microbiota in broilers,change the metabolic characteristics of intestinal microorganisms.The preventive effect of fermentation broth on broilers' colibacillosis is better than the therapeutic effect.Early inoculation of fermentation broth can reduce the colonization of pathogenic microorganisms in the intestinal tract,alleviate the damage of E.coli infection on the growth performance,immune organ development and intestinal barrier function of broilers.The concentration of propionic acid in cecum of broilers could be significantly increased by fermentation broth intervention under normal conditions or E.coli infection. |
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