Effect And Mechanism Of Lactobacillus Plantarum PA01 On Intestinal Health Of Broilers At Different Stages

Most poultry diseases are related to altered gastrointestinal health,and this is a serious obstacle to the healthy development of China’s poultry industry.The intestine is the main site of digestion and nutrient absorption in broiler chickens.It has powerful endocrine,immunomodulatory and barrier functions.The balance of intestinal flora,which is affected by factors such as age,diet,environment and disease,is essential for the health of the broiler.By altering the structure of the broiler gut microbiota,probiotics may regulate host mucosal immunity,nutrient absorption and intestinal health.As one of the most mature probiotics in the broiler industry,Lactobacillus have a good regulatory effect on the growth performance and intestinal health in broilers.Previously our team obtained the strain Lactobacillus plantarum PA01 with good probiotic characteristics.Therefore,in this study,the effect and preliminary mechanism of Lactobacillus plantarum PA01 on intestinal health,and growth performance of broilers were investigated.Experiment 1 Effect of Lactobacillus plantarum PA01 on growth performance and intestinal health of broiler chickens.The aim of this experiment was to investigate the effects of drinking water supplemented with Lactobacillus plantarum PA01 on growth performance and jejunal health of 14d broilers.A total of 480 1d AA chicks were selected and randomly divided into2 groups with 6 replicates each.The treatment group was fed daily with Lactobacillus plantarum PA01(PA-01)at 3×10~8 CFU/each chicken in the drinking water.The results showed that PA01 increased BW,ADG,ADFI and FCR(P<0.05)in broilers at 7d and 14d.16S sequencing of the 7d jejunum morsels revealed that the enriched microorganisms in the PA-01 group were Lactococcus and Veillonella.The Con group were Devosia,Shinella,Streptococcus,Oscillospira,Ruminococcus and Rhodobacter;similar results were obtained at 14d.The PA01 increased jejunal villus height and crypt depth at 7d and 14d(P<0.05),decreased serum levels of DAO(P<0.05),and enhanced intestinal barrier function.14d jejunal transcriptome analysis yielded 12 differential pathways,of which the Adherens Junction pathway was associated with the mucosal barrier.PA01 increased jejunal mucosal7d ZO-1,Occludin and 14d ZO-1 Occludin gene mRNA expression(P<0.05).In conclusion,PA01 altered the broiler jejunal microflora,regulated the expression of intestinal mucosal tight junction protein genes,and promoted broiler intestinal health.Experiment 2:Effect of Lactobacillus plantarum PA01 on productive performance and intestinal health in broiler chickens with necrotizing enteritisThe aim of this experiment was to investigate the effect of Lactobacillus plantarum PA01 on the production performance,jejunum microbiota,jejunal celiac metabolites and mucosal transcription of broilers with necrotizing enteritis.A total of 180 1d AA chicks were selected and randomly divided into three groups with six replicates each and 10 chicks per replicate.They were control group(Con),Enteritis group(NE)and Lactobacillus+enteritis group(PA-NE)respectively.The results showed that PA-NE increased BW at 21d,28d and 42d(P<0.05).The jejunenteritis score was found to be significantly higher in NE and PA-NE groups than in Con groups at 28d(P<0.05).NE decreased the villus height and crypt depth in the 21d jejunum compared with the Con groups(P<0.05).At 28 days of age,LPS was significantly lower in the PA-NE group compared to the Con and NE groups(P<0.05).It is evident that enteritis damages the mucosal barrier of broiler jejunum and Lactobacillus plantarum PA01 has a restorative resistance effect.16S sequencing of jejunal surimi at 21d and 28d revealed no significant changes in microbial diversity index and composition(P>0.05).At 21d,Con enriched microorganism was Bacilli,PA-NE were Enterococcus,Lactococcus,Ruminococcus,Oscillospira,and Subdoligranulum.NE were Clostridium,Ruminococcus,Catenibacterium,and Faecalibacterium.At 28d,PA-NE enhanced the enrichment of Lactobacillus and reduced the harmful marker microorganisms associated with enteritis.The metabolome and transcriptome results showed that KEGG enrichment analysis,Con vs NE were:Phenylalanine metabolism and Tyrosine metabolism,NE and PA-NE were:Arachidonic acid metabolism,Sphingolipid metabolism,Serotonergic synapse and Inflammatory mediator regulation of TRP channels.The transcriptomic GO enrichment analysis focused on 18 enriched pathways,of which cell junction is closely related to the intestinal barrier.Multi-omics analysis showed that Lactobacillus,Clostridium,Veillonella,and Peptostreptococcus are responsible for the differential metabolites.In conclusion,Lactobacillus plantarum PA01 altered substance metabolism and mucosal transcription in the jejunum,associated with genera such as Lactobacillus.Experiment 3 Effect of embryonic injection of Lactobacillus plantarum PA01 on microflora of broiler chickens at 20 embryonic age and 1 day oldThe total number of microorganisms in the embryonic stage is low,and the intestinal tract is rapidly and imperfectly developed.This particular physiological stage of the embryonic stage is an important time window to explore the regulation of organismal health by lactobacilli.Therefore,this experiment was conducted to investigate the effect of embryonic injection of Lactobacillus plantarum PA01 on the microorganisms of the digestive tract by measuring the microorganisms of the contents of the 20-embryonic-age myogastric,cecum and 1-day-old cecum by 16S sequencing.PA01 altered the Shannon index andβ-diversity(P<0.05),increased the abundance of Firmicutes,and decreased Proteobacteria,Bacteroidetes and Actinobacteria(P<0.05)in the 20-embryonic age myogastric.At the genus level,PA01 significantly increased the relative abundance of Lactobacillus(P<0.05).P01 altered theαandβdiversity index of the 20 embryonic age cecum(P<0.05)and decreased the relative abundance of Salmonella(P<0.05).For LefSe analysis,the marker microorganisms in the Con group were Salmonella,PA-01 group were Lactobacillales,Blautia,Lachnospiraceae,and Asinibacterium.PA01 altered theβ-diversity index(P<0.05)and had no significant effect on microbial phylum and genus level composition of the 1d cecum(P>0.05).LefSe analysis revealed Lactobacillaceae,Lactiplantibacillus,Moraxellaceae and Acinetobacter in the PA-01 group;Devosia,Bacillus,Nordella,Mesorhizobium and Pseudolabrys in the Con group of the 1d cecum.Embryonic injection of Lactobacillus plantarum PA01 can colonize the gastrointestinal tract of chicken embryos and chicks and change the microflora structure,especially promoting the colonization of Lactobacillus.Experiment 4 Effect of embryonic injection of Lactobacillus plantarum PA01 on performance and intestinal health of broiler chickens at 14 days of ageThis experiment aimed to investigate the effects of embryonic injection of PA01 on the production performance and intestinal health of 14d broilers.The experiment was designed as a single-factor test,and 720 embryonated AA broiler eggs of uniform weight and normal development at E14 were selected and randomly divided into four groups:control group(no treatment,C),saline group(0.1 mL 0.9%saline,S),medium supernatant group(0.1 mL lactobacillus medium supernatant,Q),and lactobacillus group(0.1 mL 9×108 CFU/mL of physiological saline resuspension,J).There was no difference in hatching rate among the four groups(P>0.05).So,the C,Q and J group were subsequently selected for breeding,and the blood and liver biochemical indices,cecum microorganisms and their metabolites,and mRNA of jejunal mucosa-related genes in each group were detected at 14d.The results showed that J reduced FCR from 1-7 and 1-14 compared to C(P<0.05)and did not affect broiler body weight(P>0.05).In order to analyze the relationship between microflora and growth phenotypes,correlation analysis was performed for differential microbial and production phenotypic indicators.Roseburia and coprobacillus were positively correlated with body weight(P<0.05),Roseburia and Lachnospira were significantly correlated with FCR(P<0.05).The J group increased the abundance of the g＿Anaerostipes(P<0.05).Determination of intestinal short-chain fatty acids revealed that propionic,butyric and total acid contents were higher in the cecum of groups Q and J than in group C(P<0.05).Group J increased acetic,propionic,butyric and total acid contents in the jejunum compared to groups C and Q(P<0.05).Group J increased total serum cholesterol at 1 day of age(P<0.05),total triglycerides in serum at 7 and 14 days of age,and TC,TG,and HDL in liver at14 days of age(P<0.05);and decreased LDL in liver at 14 days of age(P<0.05).The expression of target genes related to jejunal development was examined,and group J promoted the expression of mRNAs for the 1-day-old chick jejunal mucosal stem cell renewal genes Wnt3,Notch1,stem cell marker genes Lgr5 and Lrp5 genes compared with group C(P<0.05).groups Q and J increased the relative expression of the 14-day-old jejunal lipid transport carrier L-FABP,stem cell renewal-related genes Wnt3,Notch1 and Lrp5,cupula cell differentiation pathways Dll1 and Hes1 and the relative expression of the physical barrier Ocludin gene(P<0.05).In conclusion,embryonic injection of Lactobacillus plantarum PA01 promoted intestinal physical barrier development by altering the microbiota,metabolically producing butyric acid to regulate lipid metabolism,and activating the Wnt pathway of intestinal stem cell renewal.In conclusion,this study showed that feeding Lactobacillus plantarum PA01 to broilers during early growth altered the jejunal microflora,especially Lactococcus and Veillonella,modulated gene transcription in the jejunal mucosa,especially the Adherens Junction pathway,and improved the physical barrier of the mucosa.In the case of mucosal damage by enteritis,PA01 improves the enrichment of Lactobacillus in the jejunum,reduces the harmful microorganisms associated with enteritis,alters the cell junction of metabolism and mucosal transcription,promotes villi development and reduces intestinal permeability,and improves mucosal barrier function.Lactobacillus plantarum PA01injection at embryonic stage alters intestinal microorganisms,especially butyric acid producing bacteria,at embryonic stage and after fledging,activates intestinal stem cell renewal,and promotes intestinal physical barrier function and affects organism growth and metabolism.