Effects Of Dietary Rumen Degradable Starch On Intestinal Microbiota In Dairy Goats

The composition and metabolites of gut microbiota affect nutrient metabolism and body health of ruminants.Although,the fermentation characteristics and microbial composition in the hindgut of dairy goats are affected by high concentrate diets,there still exist little systematic research on the metabolic function of gut microbiota metabolism and the effect of intestinal barrier.As an effective index,dietary rumen degradable starch(RDS)is to measure the intestinal health and nutrient utilization of ruminants in terms of dietary carbohydrate degradation sites.In this research,high concentrate diets can cause fecal microflora and dysfunction is first verified,and then dietary with different RDS levels that wheat partially replaced by corn is set,so as to study the effect of RDS level on intestinal fermentation parameters of dairy goats.In addition,microbial diversity analysis on the small intestine and hindgut via 16 S r RNA sequencing and microbial function analysis on cecum via metagenomic sequencing are employed to study carbohydrate metabolism,and immune and morphological indicators are employed to study the barrier function of intestinal epithelial,so as to clarify the molecular mechanism of dietary RDS levels regulates the intestinal flora and epithelial barrier.Experiment 1 The effect of diet concentrate on the structure of the fecal flora of dairy goatsIn order to study the effect of high concentrate diet on the fecal microbial structure and function of dairy goat,under the premise of consistent DMI control,diet was fed in the experiment,and two groups were divided: high-concentration group(HC 70: 30)and low concentration group(LC 30: 70).14 fistula dairy goats(30.1 ± 5kg)were selected for the experiment,and permanent rumen fistulas were installed.The experiment period was 21 days.The results showed that in the HC group,the concentration of LPS in feces was increased on the 14 th and 21 th day in the experiment period(P < 0.05),and the pH of feces was decreased on the 14 th day in the experiment period(P < 0.05).The Alpha diversity analysis and Beta diversity analysis on the flora showed that there existed differences in the structure of the fecal flora between the HC group and LC group(P < 0.05).In the HC group,the relative abundance of the bacterial phyla: Patescibacteria and Campilobacterota(P <0.05)and the relative abundance of the bacterial genus: unclassified c Clostridia,Candidatus Saccharimonas and Campylobacter(P < 0.05)were reduced,and the relative abundance of bacterial genus: Prevotellaceae UCG-001,Turicibacter and Fibrobacter(P < 0.05)was increased.The correlation analysis results showed that the bacterial genus: unclassified c Clostridia,Candidatus Saccharimonas and Campylobacter were significantly positively correlated with pH(P < 0.05).The PICRUSt2 function prediction results showed that the relative abundance of fecal flora metabolism(Metabolism)function was the highest.As for the LC group,the relative abundance of the circulatory system(0.05 ? P <0.10)was increased,and the relative abundance of the Xylene degradation function was increased(P <0.05).It is shown in this research that subacute rumen acidosis(SARA)induced by HC dietary in dairy goats,affects the fecal microflora and function,lowers the pH of the feces,and causes the accumulation of toxic substances free LPS.Experiment 2 The effect of diet rumen degradable starch on the intestinal microflora of dairy goatsBased on the results drawn from Experiment 1,in order to further study whether the specific components of the dietary affect the intestinal metabolism by changing the intestinal microflora of dairy goats,16 s r RNA sequencing technology was adopted in the experiment to evaluate the microbial composition of jejunum,cecum and rectum of dairy goats.18 dairy goats(45.8 ± 1.54 kg)were selected and randomly divided into 3 groups,with diet of different RDS levels,of which wheat were partially replaced by corn: the low RDS group(LRDS = 20.52%),the medium RDS group(MRDS = 22.15%),and the high RDS group(HRDS = 24.88%).The experiment period was 35 days.Adonis test indicated that the it is possible to clearly distinguish the hindgut flora of the HRDS group from that of the LRDS and MRDS groups(P < 0.05).Compared with the LRDS group,in the HRDS group,the relative abundance of Ruminococcaceae,Ruminococcaceae UCG-010 and Eubacterium coprostanoligenes was increased,and the relative abundance of Lachnospiraceae and Bacteroidales?S24-7(P < 0.05)was decreased.The structure of the rectum and cecal flora was similar: compared with the LRDS group,in the HRDS group,the relative abundance of bacterial family: Succinivibrionaceae was increased;the abundance of bacterial genus:Bacteroidales S24-7 group and Succinivibrio was decreased(P < 0.05).The results of gut fermentation products showed that compared with the LRDS group,in the HRDS group,the mole ratio of hindgut acetate(P < 0.05)was increased,the mole ratio of butyrate to acetate(B: A),however,in the rectum(P < 0.05)and cecum(0.05 ? P <0.10)was decreased.The correlation analysis indicated that Ruminococcaceae and Ruminococcaceae UCG-010 were positively correlated with the ratio of acetate(P < 0.05).The ratio of Lachnospiraceae,Succinivibrionaceae and Succinivibrio to butyrate was positively correlated with the B: A ratio(P < 0.05).From Bug Base phenotypic prediction,it could be known that compared with the MRDS and HRDS groups,in the LRDS group,the oxidative stress tolerant phenotypes of the cecum and rectum(P < 0.05)was increased,the potential pathogenic phenotypes of the jejunal flora(P < 0.05)was decreased,and the cecal flora also shares the same trend(0.05 ? P <0.10).KEGG functional analysis showed that compared with the HRDS group,in the LRDS group and MRDS group,the gene abundance of the jejunal circulatory system(P < 0.05)was increased,the cardiac muscle contraction(P < 0.05)and gene abundance of the Renin-angiotensin system in the jejunum(0.05 ? P <0.10)were decreased.From this research,it can be said that the microbial diversity from the small intestine to the hindgut of dairy goats gradually increases.High RDS dietary affects hindgut fermentation,inhibits the proliferation of butyrogenic bacteria,and increases the abundance of acetogenic bacteria,the proportion of acetic acid,the instability of intestinal microbiota,and the risk of developing enteritis.Experiment 3 The effect of dietary rumen degradable starch on the microbial function in the cecum of dairy goatsExperiment 2 found that dietary RDS affected the microbial phenotype in the hindgut and the structure of SCFAs producing bacteria.For the purpose of exploring the effect of dietary RDS on the fermentation characteristics of the microbial carbohydrate function in the cecum,metagenomic sequencing analysis was adopted in this research to study the changes in gene expression related to carbohydrate degradation in the cecal microbiome,and the dominant species Succinatimonas?sp.?CAG:777 in the hindgut of ruminants was localized for the first time.Compared with the LRDS group,in the HRDS group,the relative abundance of isoamylase(EC 3.2.1.68)and the GH13?20 family for starch degradation(P <0.05)were decreased,cellulase encoding genes(EC 3.2.1.4)and the relative abundance of GH95(P < 0.05),GH10(P < 0.05)and GH44(0.05 ? P <0.10)for cellulose degradation were increased.Also,the cecal cellulose content(P < 0.05),and the cecal amylose content(P < 0.05)were decreased in the HRDS group.Results of Pro Bio probiotic database analysis indicated that compared with LRDS,the gene abundance of 7 probiotics was significantly decreased in the HRDS group.In addition,compared with the LRDS group(P < 0.05),the mRNA expression level of the cecal SCFAs transporter NHE3(P < 0.05)was increased in the HRDS group.This experiment shows that dietary RDS level affects cecal fermentation and carbohydrate degradation in dairy goats.In the HRDS group,by reducing the cecal amylose content,the abundance of cecal isoamylase genes is reduced;by increasing the cecal cellulose content,the abundance of cecal acetogens and endoglucanase genes and the output of acetate are increased,and the absorption capacity of intestinal epithelial acetate is enhanced.In the MRDS group,the gene abundance of propionic acetate bacteria Succinatimonas sp.CAG:777 is increased,and the ratio of propionate is increased through the Succinate pathway.Experiment 4 The effect of rumen degradable starch on the intestinal epithelial barrier of dairy goatsPrevious research reveals that high RDS dietary will change the amylose and fiber levels in the cecum of dairy goats,causing the disorder of the hindgut flora,and increasing the concentration of free LPS in the blood(see Chapter 3 & Chapter 4),but it is still left unknown what effect it imposed on intestinal epithelial barrier.On the basis of past research,the impact of bacterial flora disorder caused by high RDS content on the health of the gut barrier was analyzed in this research.The results showed that compared with the LRDS group,in the HRDS group,the height of duodenal villi/crypt depth(V/C)(0.05 ? P <0.10)and the mRNA expression of tight junction protein Claudin-7(P < 0.05)were decreased,the cecum Claudin-2(P < 0.05),ZO-1(P < 0.05)and Claudin-7 mRNA expression(0.05 ? P<0.10)were increased.The electrophysiological results indicated that compared with the LRDS and MRDS groups,in the HRDS group,the short-circuit current(Isc)in duodenum(0.05 ? P <0.10),jejunum(0.05 ? P <0.10)and cecal epithelial(P < 0.05)were decreased.Meanwhile,in the HRDS group,the total number of hemoglobin(HB)in the blood of dairy goats(P < 0.05),the total number of white blood cells(WBC)(0.05 ? P <0.10)and the mRNA expression level of cecal pro-inflammatory cytokine IL-1?(P < 0.05)were increased.In addition,compared with the LRDS group,in the HRDS group and MRDS groups,the protein expression of the cecal secretory immunoglobulin SIgA(P < 0.05)was increased,and the structure of the cecal mucin Muc2(P < 0.05)was damaged.This research indicates that HRDS dietary will cause multiple damages to the cecal gut barrier of dairy goats: high RDS dietary is able to destroy the chemical barrier of the cecal mucin,weaken the physical barrier of the cecum(increase the permeability of the intestinal epithelium,change the tight junction structure of the cecal epithelium,and increase the risk of developing inflammation in cecum.The results above indicate that high RDS dietary will cause changes in the structure of the gut flora of dairy goats,affect the expression of carbohydrate degradation-related enzymes,increase the pressure of hindgut cellulose degradation,and cause damage to mucus layer and inflammation.