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Broiler chicken is one of the most important source of animal protein associated with meat production for human consumption.The gastrointestinal tract(GIT)of chickens accommodate a large number of microbial communities,the interactions of the gut microbiota affect the physiological,immunological and nutritional status of the host,the gut microbiome can be affected by diet,and different dietary interventions are used by poultry producers to enhance chicken growth reduce the risk of enteric infection and increase the production of safety product to consumers.This study investigated the effects of two Bacillus-based probiotics;Bacillus subtilis(BS),B21strain(2x109CFU/g)and Bacillus licheniformis(BL),B26strain(2x109CFU/g)and antibiotic growth promotor(AGP)Enramycin(5mg/kg)on growth performance,gut microbial composition,nutrient absorption efficiency,fecal noxious gas emissions and intestinal health in broiler chickens challenged with Clostridium perfringens(C.perfringens)induced necrotic enteritis(NE).One-d-old Arbor Acre(n=480)were randomly assigned to four treatments groups with10cages of12birds each used for the entire experiments conducted during this study period(1to day42),the treatment groups are as follows:1)fed basal diet only negative control(NC)group with neither probiotics nor antibiotic;2)basal diet+enramycin(5mg/kg);3)basal diet+Bacillus subtilis2x109CFU/g;3)basal diet+Bacillus licheniformis(2x109CFU/g),except the NC group,all the birds in AGP or probiotics fed groups were challenged with C.perfringens(CPA)strainα-toxin(108CFU/mL)0.5mL at day1of age,and then repeated with1mL(108CFU/mL)from day14to day21of age once a day,for NC birds,the same amount of PBS was orally gavaged at the same time points mentioned above.The entire study results were grouped into four experiments thus;
Experiment1.Effect of dietary treatment on growth performance and growth-related parameters of broiler Challenged with C.perfringens induced NE
The first part of the experiment was to assess the effects of growth performance of broilers challenged with C.perfringens induced NE,broiler performance was measured weekly in terms of average daily weight gain(ADWG),Average daily feed intake(ADFI)and feed conversion ratio(FCR)from day1to42days of age while histomorphology and intestinal lesion scores were measured at21and35days of age,the growth performance,intestinal morphology and intestinal lesion scores,were assessed.In the present experiment,result of the study shown that, the NC and BL fed-groups showed significantly higher(P=0.005)average daily feed intake(ADFI)from d1to d21than in AGP and BS fed-groups,whereas,BS and AGP fed-group showed higher average daily weight gain(ADWG)from d22to d42and from d1to d42of age.FCR was significantly(P=0.047)lower in BS fed-group than in BL and NC at d1to21day of age.Higher mortality rate of(12.5%)and lower(5.5%)was recorded in AGP and NC groups respectively,lesion score was higher in BS and BL than in AGP,and the lesion scores recorded at21days of age were relatively higher than those recorded at day35of age,while no lesion was observed in NC group,results of this study also revealed higher duodenum and jejunum villus height to crypt depth(VH:CD)by NC,AGP and BL in duodenum and by AGP and BL in jejunum compared with other fed group.Based on this result,it indicated that both BS and BL had virtually similar effect with AGP in enhancing growth,improving feed efficiency,intestinal integrity and prevention or partially averting the negative effect of NE infection in broilers.
Experiment2.Effect of dietary probiotics and antibiotic on mucosal barrier tight junction mRNA expression,nutrient transporters mRNA expression and cytokines mRNA genes expression of broilers challenged with C.perfringens
The2nd experiment was conducted to investigate the effects of treatments on the tight junctions gene expression,nutrients transporters/absorption and cytokines mRNA relative genes expression,from the intestinal tissue sample of the intestinal scrapings at7and21day post challenged(PC),In this experiment,supplementation of AGP,BS and BL in broiler challenged with C,perfringens,had resulted to the upregulation of mRNA expression of TJ protein of claudin-1in duodenum and jejunum at7and21d PC and markedly enhanced(P<0.05)the expression of ZO-1in duodenum and ileum,while supplementation of AGP,BS and BL upregulated the mRNA expression of occludens in jejunum and ileum.Similarly,mRNA expression of GLUT-2,and SGLT-1in duodenum and ileum were significantly(P<0.05)up-regulated by BS fed-group at7and21d PC,more so,the mRNA expression of rBAT was significantly(P<0.05)higher in BS fed-group in both duodenum,jejunum and ileum compared to other treatment groups.While the results revealed that the mRNA expression of LAT-2was significantly(P<0.05)upregulated in AGP and BS fed-groups,while CAT-2expression was significantly(P<0.05)upregulated by AGP than in other fed-groups.Nevertheless,the expression of PePT-1was significantly(P<0.05)higher in BS and BL fed-groups,while the expression of proinflammatory cytokines of IL-10and NF-kB were significantly(P<0.05) higher by AGP and BS fed-groups whereas,the expression of SigA was significantly(P<0.05)influenced by BL fed group than in other fed-groups,while low expression level of proinflammatory cytokine of TNF-αwas observed both at7and21d PC,even though the expression of IL-6and IFN-γcould be regulated by AGP and probiotics supplementation,which contributed to the suppression of the C.perfringens infection.These results demonstrated that there are changes in the intestinal gene expression in response to dietary AGP and dietary probiotics supplementation,despite the C.perfringens challenge which efficiently improved the intestinal immune system and increased the nutrients absorption efficiency of the broiler chickens.
Eexperiment3.effect of dietary probiotics and antibiotics on short chain fatty acids,cecal microbiome and noxious gas emission of broilers challenged with C.perfringens induced NE
The3rd experiment was conducted to investigate the effect of treatments on cecal short chain fatty acids(SCFAs),relative abundance of cecal microbiota and fecal noxious gas emission of broiler chickens challenged with C.perfringens.The GC system was used to determine the concentration of SCFAs at7and21day PC,while the16S rRNA cecal microbiota relative abundance was determined using the qRT-PCR while the16S rRNA gene copies/g of the cecal content for the total bacteria,Bifidobacterium bifidum,Escherichia.coli,Clostridium perfringens,Enterobacter,Lactobacillus salivarius and Pediococcus at14,21and35days of age,while the fecal gaseous emission of ammonia(NH3)and hydrogen(H2S sulphide were determined at42days of age.The result of this study indicated that,at day7PC the concentration of butyric acid was significantly(P=0.023)higher in AGP and BL while concentration of propionic acid was significant(P=0.019)higher in BL than in the rest of the treatment groups,however,at21day PC,the total SCFA was significantly(P=0.005)higher in BL than in AGP,acetic acid was significantly(P=0.015)higher in BS and BL than in AGP,the propionic acid was significantly(P=0.017)higher in BL than in the rest of the group,while the butyric acid was significant(P=0.035)higher in BL than in AGP.The results for the mean log1016S rRNA gene copies/g of the total bacteria shows a significant(P=0.0249)higher population in the supplemented group than in NC group,similarly,the gene copies of B.bifidum and L.salivarius were significantly(P<0.05)higher in supplemented group than in the NC,whereas,the abundance of C.perfringens and E.coli tends to be higher in NC group compared to supplemented groups,while the abundance of Enterobacter was highly influenced by BS and BL at both14and35days of age,while the counts of Pediococcus was significantly(P<0.05)higher in BL at14days of age and in BS and BL at35days of age.However,results of this study indicated significant reduction of ammonia emission in the supplemented groups compared to NC group,while no significant effect(P>0.05)in treatments groups on the fecal hydrogen sulphide emission of broiler at42days of age was observed.Based on this trial results,these strains of Bacillus based probiotics showed a promising effect and can effectively enhanced the production of SCFA,and not only prevent the effects of necrotic enteritis but also indirectly encouraged the growth of other useful bacteria while discouraged the survival of harmful pathogens and improved the overall intestinal health status of broiler birds.In view of this,B.subtilis and B.licheniformis could be used as alternative strategy to improve gut health of broiler chicken under subclinical necrotic enteritis condition.
Experiment4.Effects of dietary treatments on cecal microbial composition and metabolic pathways of broilers challenged with C.perfringens induced NE
In this study,in addition to the16S rRNA qRT-PCR gene sequencing,the Illumina HiSeq analysis was also carried out,the indicators for species richness of Chao1and Ace indices shows highest species richness in AB and PB than in CON at both21and35days of age,while no noticeable difference of the percentage cecal microbial abundance of Actinobacteria,Firmicutes and Proteobacteria were observed between the supplemented and CON group at21days of age,whereas,the percentage Firmicutes which are known as beneficial group were higher in supplemented group of AB and PB than in CON fed-group,while at35days of age,the percentage composition of proteobacteria which are pathogenic bacterial group are higher in CON group than in supplemented fed-group,with only noticeable percentage of Bacteroidetes(an important bacteria group)in PB fed-group,important metabolism like carbohydrates and vitamin metabolism were highly enriched in PB group,while amino acid metabolism,membrane transport,replication and repair were enriched in AB group,in the same vein,CON group was enriched in cell translation,replication and repair。
Experiment1.Effect of dietary treatment on growth performance and growth-related parameters of broiler Challenged with C.perfringens induced NE
The first part of the experiment was to assess the effects of growth performance of broilers challenged with C.perfringens induced NE,broiler performance was measured weekly in terms of average daily weight gain(ADWG),Average daily feed intake(ADFI)and feed conversion ratio(FCR)from day1to42days of age while histomorphology and intestinal lesion scores were measured at21and35days of age,the growth performance,intestinal morphology and intestinal lesion scores,were assessed.In the present experiment,result of the study shown that, the NC and BL fed-groups showed significantly higher(P=0.005)average daily feed intake(ADFI)from d1to d21than in AGP and BS fed-groups,whereas,BS and AGP fed-group showed higher average daily weight gain(ADWG)from d22to d42and from d1to d42of age.FCR was significantly(P=0.047)lower in BS fed-group than in BL and NC at d1to21day of age.Higher mortality rate of(12.5%)and lower(5.5%)was recorded in AGP and NC groups respectively,lesion score was higher in BS and BL than in AGP,and the lesion scores recorded at21days of age were relatively higher than those recorded at day35of age,while no lesion was observed in NC group,results of this study also revealed higher duodenum and jejunum villus height to crypt depth(VH:CD)by NC,AGP and BL in duodenum and by AGP and BL in jejunum compared with other fed group.Based on this result,it indicated that both BS and BL had virtually similar effect with AGP in enhancing growth,improving feed efficiency,intestinal integrity and prevention or partially averting the negative effect of NE infection in broilers.
Experiment2.Effect of dietary probiotics and antibiotic on mucosal barrier tight junction mRNA expression,nutrient transporters mRNA expression and cytokines mRNA genes expression of broilers challenged with C.perfringens
The2nd experiment was conducted to investigate the effects of treatments on the tight junctions gene expression,nutrients transporters/absorption and cytokines mRNA relative genes expression,from the intestinal tissue sample of the intestinal scrapings at7and21day post challenged(PC),In this experiment,supplementation of AGP,BS and BL in broiler challenged with C,perfringens,had resulted to the upregulation of mRNA expression of TJ protein of claudin-1in duodenum and jejunum at7and21d PC and markedly enhanced(P<0.05)the expression of ZO-1in duodenum and ileum,while supplementation of AGP,BS and BL upregulated the mRNA expression of occludens in jejunum and ileum.Similarly,mRNA expression of GLUT-2,and SGLT-1in duodenum and ileum were significantly(P<0.05)up-regulated by BS fed-group at7and21d PC,more so,the mRNA expression of rBAT was significantly(P<0.05)higher in BS fed-group in both duodenum,jejunum and ileum compared to other treatment groups.While the results revealed that the mRNA expression of LAT-2was significantly(P<0.05)upregulated in AGP and BS fed-groups,while CAT-2expression was significantly(P<0.05)upregulated by AGP than in other fed-groups.Nevertheless,the expression of PePT-1was significantly(P<0.05)higher in BS and BL fed-groups,while the expression of proinflammatory cytokines of IL-10and NF-kB were significantly(P<0.05) higher by AGP and BS fed-groups whereas,the expression of SigA was significantly(P<0.05)influenced by BL fed group than in other fed-groups,while low expression level of proinflammatory cytokine of TNF-αwas observed both at7and21d PC,even though the expression of IL-6and IFN-γcould be regulated by AGP and probiotics supplementation,which contributed to the suppression of the C.perfringens infection.These results demonstrated that there are changes in the intestinal gene expression in response to dietary AGP and dietary probiotics supplementation,despite the C.perfringens challenge which efficiently improved the intestinal immune system and increased the nutrients absorption efficiency of the broiler chickens.
Eexperiment3.effect of dietary probiotics and antibiotics on short chain fatty acids,cecal microbiome and noxious gas emission of broilers challenged with C.perfringens induced NE
The3rd experiment was conducted to investigate the effect of treatments on cecal short chain fatty acids(SCFAs),relative abundance of cecal microbiota and fecal noxious gas emission of broiler chickens challenged with C.perfringens.The GC system was used to determine the concentration of SCFAs at7and21day PC,while the16S rRNA cecal microbiota relative abundance was determined using the qRT-PCR while the16S rRNA gene copies/g of the cecal content for the total bacteria,Bifidobacterium bifidum,Escherichia.coli,Clostridium perfringens,Enterobacter,Lactobacillus salivarius and Pediococcus at14,21and35days of age,while the fecal gaseous emission of ammonia(NH3)and hydrogen(H2S sulphide were determined at42days of age.The result of this study indicated that,at day7PC the concentration of butyric acid was significantly(P=0.023)higher in AGP and BL while concentration of propionic acid was significant(P=0.019)higher in BL than in the rest of the treatment groups,however,at21day PC,the total SCFA was significantly(P=0.005)higher in BL than in AGP,acetic acid was significantly(P=0.015)higher in BS and BL than in AGP,the propionic acid was significantly(P=0.017)higher in BL than in the rest of the group,while the butyric acid was significant(P=0.035)higher in BL than in AGP.The results for the mean log1016S rRNA gene copies/g of the total bacteria shows a significant(P=0.0249)higher population in the supplemented group than in NC group,similarly,the gene copies of B.bifidum and L.salivarius were significantly(P<0.05)higher in supplemented group than in the NC,whereas,the abundance of C.perfringens and E.coli tends to be higher in NC group compared to supplemented groups,while the abundance of Enterobacter was highly influenced by BS and BL at both14and35days of age,while the counts of Pediococcus was significantly(P<0.05)higher in BL at14days of age and in BS and BL at35days of age.However,results of this study indicated significant reduction of ammonia emission in the supplemented groups compared to NC group,while no significant effect(P>0.05)in treatments groups on the fecal hydrogen sulphide emission of broiler at42days of age was observed.Based on this trial results,these strains of Bacillus based probiotics showed a promising effect and can effectively enhanced the production of SCFA,and not only prevent the effects of necrotic enteritis but also indirectly encouraged the growth of other useful bacteria while discouraged the survival of harmful pathogens and improved the overall intestinal health status of broiler birds.In view of this,B.subtilis and B.licheniformis could be used as alternative strategy to improve gut health of broiler chicken under subclinical necrotic enteritis condition.
Experiment4.Effects of dietary treatments on cecal microbial composition and metabolic pathways of broilers challenged with C.perfringens induced NE
In this study,in addition to the16S rRNA qRT-PCR gene sequencing,the Illumina HiSeq analysis was also carried out,the indicators for species richness of Chao1and Ace indices shows highest species richness in AB and PB than in CON at both21and35days of age,while no noticeable difference of the percentage cecal microbial abundance of Actinobacteria,Firmicutes and Proteobacteria were observed between the supplemented and CON group at21days of age,whereas,the percentage Firmicutes which are known as beneficial group were higher in supplemented group of AB and PB than in CON fed-group,while at35days of age,the percentage composition of proteobacteria which are pathogenic bacterial group are higher in CON group than in supplemented fed-group,with only noticeable percentage of Bacteroidetes(an important bacteria group)in PB fed-group,important metabolism like carbohydrates and vitamin metabolism were highly enriched in PB group,while amino acid metabolism,membrane transport,replication and repair were enriched in AB group,in the same vein,CON group was enriched in cell translation,replication and repair。