| There has been accumulating evidence supporting the improvement of growth and enhancing performance of Bacillus subtilis.However,the mechanisms for these effects still remain obscure.To clarify the mechanism of benefical effects of Bacillus subtilis,the paper was conducted to research the effects of supplementation with Bacillus subtilis CGMCC 1.921 on the performance,egg quality,and blood index of laying hens.Study the fecal/intestinal microbiota and ileum mucosa gene expression of laying hens with 16S rRNA gene and transcriptomics sequence analysis to explore the mechanism of enhancing performance.A total of 360 27-week-old Hy-Line Brown laying hens were randomly allocated into 5 treatments.The experimental treatments included a basal diet without additions and the basal diet supplemented with 1.0×10~5,1.0×10~6,1.0×10~7,and 1.0×10~8 CFU/g B.subtilis CGMCC 1.921.Egg numbers and egg weight per pen were recorded daily.The weight of feed consumption per pen was recorded weekly.Egg production,feed intake,egg weight,feed egg ratio,egg weight per hen and mortality were calculated.On the last d of wk 0,1,2,3,4,8,12,16,20,and 24,all eggs from each replicate were collected for egg quality assessment.And 4 birds were randomly selected to collecet blood and measured.And one cage per replicate was chosen randomly for fecal sample collection to analyze E.coli counts.At the end of experiment,one hen per replicate was randomly selected for intestinal content to analyze E.coli,Lactobacillus,Bifidobacterium,Campylobacter,Enterococcus,and C.perfringens counts.And cecal content between T0(control)and T1(10~8 CFU/g B.subtilis CGMCC 1.921)were used to analyse intestinal flora with 16S rRNA gene sequence.And sequencing was carried out on the Illumina HiSeqTM 4000 platform to research ileal mucosa gene expression by transcriptomics.The results were as follows:1.Supplementation with B.subtilis CGMCC 1.921 improved feed egg ratio and eggshell strength.Dietary with various levels of B.subtilis CGMCC 1.921 decreased feed egg ratio during wk 8 to 16 and17 to 24(P<0.05).Groups 2,4 and 5 decreased feed egg tatio during wk 1 to 24(P<0.05).Eggshell strength significantly improved(P<0.05)in groups 2(wk 8,16,20,and 24),3(wk 20 and 24),and 4(wk 8,16,20,and 24).Supplementation with B.subtilis CGMCC 1.921 increased serum glucose content(wk 2,3,12,16 and 20),increased blood leucocytes(wk 1,3,4,8,12,16,20 and 24)and lymphocytes(wk 2,3,4,8,12,16,20 and 24)counts,and increased serum IgG(wk 1 and 4)and IgM(wk 2,4,8 and 16)content(P<0.05).2.Supplementation with B.subtilis CGMCC 1.921 decreased fecal E.coli counts and modulated cecal flora.Fecal E.coli counts decreased(P<0.05)from wk 12.In cecal digesta,Lactobacillus(groups 2,4,and 5)and Bifidobacterium counts(groups 2,3,4,and 5)increased(P<0.05).E.coli(groups 3 and 5)and C.perfringens counts(groups 4 and 5)decreased(P<0.05).E.coli Lactobacillus ratio decreased in group 2,3,4,and 5(P<0.05).Bifidobacterium counts(groups 4 and 5)of jejunal and ileal content increased(P<0.05).Lactobacillus counts(group 5)of jejunal content increased(P<0.05).Supplementation with B.subtilis CGMCC 1.921 increased Shannon index,enhancing the cecal community diversity and modulated cecal microbiota by increasing the relative proportion of Tenericutes at phylum level,Peptococcaceae,Erysipelotrichaceae,Family XIII,Bacillaceae,Firmicutes bacterium CAG822,Oxalobacteraceae at family level,and Alistipes,Subdoligranulum,Ruminococcaceae UCG-014,Anaerotruncus,Ruminiclostridium 5,Ruminococcaceae UCG-010,Erysipelatoclostridium,Ruminococcaceae UCG-009,Family XIII AD3011 group,Bacillus,Faecalicoccus,Firmicutes bacterium CAG822,Oxalobacter,Dielma at genus level.3.Supplementation with B.subtilis CGMCC 1.921 up regulated differentially expressed genes related to the carbohydrate metabolism,amino acid metabolism,lipid digestion,and intestinal absorption.Differentially expressed genes associated with starch and sucrose metabolism(SI,MGAM,ENPP3,PYGB,G6PC,HK1,GYS2),glycine,serine and threonine metabolism(SRRL,GCSH,GLYCTK,GLDC,MAOA,PSAT1,SDSL,AMT)and galactose metabolism(LCT,G6PC,SI,MGAM,AKR1B10,HK1)were significantly riched which may increase the nutrient absorption efficiency.Therefore,the probiotic B.subtilis CGMCC 1.921 effectively improved performance.Enhancing microbial diversity,beneficial modulation of cecal microbiota and the increaseing of gene expression associated to substance metabolism may play an important role in the improvement of production performance.This result reveals the undering molecular mechanism from intestinal flora and gene level. |
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