| In recent years,the research on intestinal microbes in poultry has made great progress,including the microbial composition,the influence of dietary nutrition level,environmental factors and host genetics on microbial structure.However,how to achieve the accurate elaboration of the law of spatiotemporal succession of intestinal microflora,the staged expression of the function of the successional microorganisms with the age of the host is still more prominent.Therefore,when discussing the relationship between the cecum health of broilers and microbes,this study first used 16S r RNA gene sequencing technology and full-period coverage to ensure the accuracy and timeliness of bacterial structure analysis,and secondly,it is located in the common genus and dominant genus of various ages,focusing on the process of cecal flora involved in the digestion,absorption,metabolism and excretion of nutrients,and the local specific immune response that may participate.Through integrated analysis,we hope to have a comprehensive understanding of the structural characteristics of cecum flora and the potential impact on the nutritional function of the host.The test consists of three parts:Exp.1 Colonization and succession of cecum flora in broilersIn this test,280 1-day-old Avian chickens were randomly divided into 7 groups,each group of 40 chickens,and the test period was 42 days.The basic diet is fed throughout the production period.The cecal chyme was collected at 3、7、14、21、28、35 and 42-day-old respectively,and the structure of the cecum flora was analyzed using 16S r RNA gene sequencing technology.The results showed that:1)PCA analysis clarified that the development and succession of cecal flora in 3～42-day-old broilers were carried out in stages,and the similarity was high between the 3-day-old and 7-day-old groups and the structure of the 3-day-old flora is significantly different from other days,but with the increase of age,the structure of cecal flora is more stable,and the gap between groups and the gap within groups are getting smaller and smaller.2)Dynamic changes of phylum level at different days.The dominant phylums from 3-day-old to 42-day-old are Firmicutes,Bacteroidetes,Proteobacteria and Tenericutes.The rapid development period is 3～7-day-old,and Firmicutes and Proteobacteria jointly regulate the structure of 3-day-old bacteria;at7-day-old,the relative abundance of Proteobacteria was significantly reduced(P<0.01),and the microbial structure changed greatly,and Firmicutes dominates,followed by Tenericutes;7～28-day-old is a relatively stable stage of bacterial structure;at 35-day-old,because the relative abundance of Bacteroidetes increased significantly(P<0.01),the microflora structure changed greatly,and Bacteroidetes remained at the secondary position of the dominant phylum since then;35～42 day-old is the second relatively stable period.3)Dynamic changes of genus level at different days.The genus that significantly changed from 3-day-old to7-day-old include Ruminococcus,Coprobacillus,etc.(P<0.01).The genus that changed significantly from 3 to 7-day-old include Faecalibacterium and Bacteroides,etc.(P<0.01).The trend of the microflora structure at 7～21-day-old and28～42-day-old is not obvious.4)The trends of the dynamic change of the dominant flora at different taxonomic levels at different ages are consistent.Taking Firmicutes as an example,its relative abundance at different ages is consistent with the dynamic changes of Clostridia,Clostridiales,and Ruminococcaceae.5)Screening of common genus at each age.The main dominant genus are Unclassified Clostridiales,Unclassified Ruminococcaceae,Unclassified Lachnospiraceae,and the relative abundances of Unclassified Erysipelotrichacea,Coprobacillus and Blautia at 3-day-old are significantly higher than those at other days(P<0.01).6)Screening of dominant genus at each age.The relative abundance of most dominant genus at 3-day-old was significantly higher than that at other ages;the obvious feature of the dominant genus at 7-day-old was that the relative abundance of the flora was relatively low at 3-day-old,and that at 7-day-old significantly increased;the species of dominant genus of 14～21-day-old were few and the structure had no significant change;the large changes in the structure of 21～28-day-old flora resulted in an increase in the28-day-old dominant genus compared to the 14-day-old and 21-day-old;35-day-old dominant genus are abundant,and their main characteristics are between 3～21-day-old,the relative abundance of genus is almost zero;42-day-old dominant genus are rich in species,and the relative abundance of genus is relatively low.The main dominant genus are only colonized at 42-day-old.The results of this experiment showed that the developmental characteristics of cecal flora in broilers are obvious,and the succession is accelerated at3～7-day-old and 28～35-day-old;the structural changes at other days are relatively flat.The trend of the dynamic changes of the dominant microbial groups with subordinate relationship with different ages at different ages is similar.The characteristics of the staged colonization of the common genus and dominant genus at different ages are obvious.Exp.2 Prediction of nutritional function of common genus of broiler caecal in different agesIn this experiment,samples of cecal chyme,cecum mucosa,and cecal tonsil tissue were collected at 3、7、14、21、28、35 and 42-day-old.The cecal chyme short-chain fatty acid content was monitored in real time by the aid of gas chromatograph.The m RNA expression levels of G protein-coupled receptors were measured by RT-q PCR technique in cecum mucosa samples.The number of plasma cells and Ig A in cecum tonsil was detected by immunohistochemistry.The results showed:1)Acetic acid,butyric acid and propionic acid were higher in broiler chyme short-chain fatty acids.The relative abundance of the common genus of broiler caecal bacteria was correlated with the content of short-chain fatty acids.The content of acetic acid is extremely significantly positively correlated with the relative abundance of Coprobacillus(P<0.01);propionic acid content is extremely significantly negatively correlated with the relative abundance of Oscillospira,Ruminococcus and Unclassified Erysipelotrichaceae(P<0.01);the isobutyric acid content is significantly positively correlated with the relative abundance of Unclassified Clostridiales and Unclassified Ruminococcaceae(P<0.05);the relative genus of butyric acid content and isobutyric acid content were more similar;isovaleric acid content is negatively correlated with the relative abundance of[Ruminococcus]and Coprococcus;the related genus of valeric acid content is similar to butyric acid.2)Relatively expressed m RNA levels of short-chain fatty acid G protein coupled receptors in cecal chyme of broilers.The relative expression levels of GPR41 and GPR43 m RNA at 3-day-old were significantly higher than those at other days(P<0.01).3)The dynamic changes of the number of plasma cells in the caecum tonsils of broilers.The number of plasma cells at 3-day-old was significantly higher than that of other days of age(P<0.01);4)The dynamic change of the quantity of Ig A in the caecum tonsils of broilers.The number of Ig A at 3-day-old was significantly higher than that at other days(P<0.01).5)Correlation analysis of 3-day-old significant indicators.The relative abundance of Coprobacillus、the content of Butyric acid、the relative expression of m RNA levels of GPR41 and GPR43 and the number of plasma cells and Ig A in the cecum tonsil showed a positive correlation,the relative abundance of Coprobacillus showed a significant positive correlation with the amount of Ig A and the relative expression of GPR41(P<0.05).The results of this experiment show that based on correlation analysis,it is reasonably inferred that the dominant commensal genus(Coprobacillus)in the cecum is involved in carbohydrate metabolism to produce short-chain fatty acids(acetic acid),which are recognized and bound by the G protein-coupled receptor(GPR43)on the cecum mucosa,thereby recruiting the secretion of Ig A by plasma cells triggers a local specific immune response.Exp.3 Prediction of nutritional function of dominant genus of broiler caecal in different agesIn this experiment,broiler cecum chyme,mucosa,intestinal segment and serum samples were collected at 3、7、14、21、28、35 and 42 day-old.The cecum morphology was observed through paraffin sections.The cecal chyme was dried to determine the moisture content.The cecum mucosa was determined by RT-q PCR technique to determine the m RNA expression level of the basic amino acid and sodium glucose co-transporter.Detect the concentration of serum urea nitrogen,glucose,total cholesterol,triglyceride,low density lipoprotein and high density lipoprotein.The results showed:1)Correlation analysis of cecum intestinal morphology and relative abundance of dominant genus.Villous height is extremely significantly positively correlated with Unclassified [Mogibacteriaceae](P<0.01);crypt depth is significantly positively correlated with the relative abundance of Unclassified YS2(P<0.05);mucosal thickness is significantly positively correlated with Unclassified Coriobacteriaceae(P<0.05).2)Correlation analysis of cecal mucosal transporter expression and relative abundance of dominant genus.The relative expression of CAT1 is significantly positively correlated with the relative abundance of Ruminococcus,Butyricicoccus and Erwinia(P<0.05);The relative expression of CAT4 is extremely significantly positively correlated with the relative abundance of Odoribacter,Unclassified Dehalobacteriaceae,Anaerofustis,etc.(P<0.01);The relative expression of r BAT is negatively correlated with the relative abundance of[Ruminococcus],Unclassified Clostridiaceae and Unclassified Betaproteobacteria;the relative expression level of y~+LAT2is positively correlated with the relative abundance of Prevotella;the relative expression level of SGLT1 is significantly positively correlated with the relative abundance of Parabacteroides,Butyricimonas and Sutterella(P<0.01).3)Correlation analysis of broiler serum metabolite concentration and relative abundance of dominant genus.The four blood lipid concentrations are positively correlated with the relative abundance of[Ruminococcus],Unclassified Erysipelotrichaceae,Coprobacillus and Dorea(P<0.05);the concentration of urea nitrogen is extremely significantly positively correlated with the relative abundance of Unclassified Lachnospiraceae and Unclassified Desulfovibrionaceae(P<0.01);the correlation coefficient between blood glucose concentration and Oscillospira relative abundance is the highest.4)Correlation analysis between the water content of cecal chyme in broilers and the relative abundance of dominant genus.The highest correlation coefficient with chyme moisture content is Bacteroides.The results of this test showed that the main dominant genus at various ages such as Unclassified[Mogibacteriaceae]、Unclassified YS2and Unclassified Coriobacteriaceae are related to the development of cecum morphology;Ruminococcus,Anaerofustis,Preputolacis and Butyricimonas are related to basic amino acid and glucose transport;[Ruminococcus],Coprobacillus,Unclassified Lachnospiraceae and Oscillospira are related to the metabolism of the three major nutrients;Bacteroides are related to chyme moisture content.In summary,this study integrated the colonization of broiler cecum flora and the nutritional function of its host.First,the basic rules of the staged colonization and succession of broiler caecal flora were clarified.Secondly,it is located in the common genus of various ages,and through correlation analysis,it is reasonably inferred that the potential mechanism of the common genus in the cecum of broilers affecting the local specific immune response mediated by B lymphocytes in the cecum tonsils.Finally,the predominant genus of bacteria of various ages that could potentially affect the nutritional function of the host were screened,which provides a viable target for microflora for precise nutrition.It provides a research direction to further clarify the staged regulation mechanism of host function presented by broiler cecum flora with age. |
PDF Full Text Request