| The small intestine is an important biorgan in the gastrointestinal tract,which is mainly responsible for the digestion and absorption of nutrients.And the small intestine epithelium is inhabited by mucosa-associated bacteria and represents an epithelial barrier function.High concentrate diet resulted in ruminal and hindgut acidosis.When excessive undigested concentrate flows into the small intestine from the rumen,it may accelerate the bacterial fermentation and change the bacterial composition and structure,finally,it can affect the normal physiological function in the small intestine.We study the fermentative capability of the different percentages of concentrate with the small intestinal microbiota in vitro.Based on the result,we investigate the effects of high concentrate diets on the small intestinal fermentation,the composition of bacterial community and mucosal morphology on goats.The thesis is composed of three parts.1.The effect of different percentages of concentrate in fermentable substrates on the jejunal fermentation in vitroThe objective of the study was to investigate the effect of different percentages of concentrate in the substrates on jejunal fermentation in vitro.In this study,we collected the terminal jejunal digesta as the inoculum,and added three different percentages of concentrate in substrate(A0:0%concentrate + 100%forage,A0.3:30%concentrate + 70%forage and A0.7:70%concentrate + 30%forage)in each fermentative bottle,and each group had five replications,then incubated for 24 h at 39?.The results showed that,compared with the A0,the additive of the concentrate in substrate(A0.3 and A0.7)significantly decreased the pH(P<0.001),and with the increase of the concentrate,the pH was linearly decreased(P<0.001).Compared with the A0,the additive of the concentrate in substrate(A0.3 and A0.7)significantly increased the total gas production(P<0.001),and with the increase on the percentage of concentrate in substrate,the total gas production was linearly increased(P<0.001).Compared with the A0,the additive of concentrate in substrate(A0.3 and A0.7)significantly increased the total volatile fatty acids,acetate,propionate,isobutyrate,butyrate,isovalerate,valerate and the percentage of propionate and isovalerate(P<0.05),while decreased the percentage of acetate,isobutyrate and butytate and the ratio of the acetate and propionate(P<0.05).Besides the isobutyrate,the other parameters were linearly changed with the increase on the percentage of concentrate in substrate(P<0.05).But,there was no significant difference on the percentage of valerate and the content of the lactate between the A0 and A0.3 or A0.7 group(P>0.05).The results indicated that there was difference on the small intestinal bacterial fermentation pattern after the different percentage daily ration,and the high concentrate diet accelerated the small intestinal bacterial fermentation.2.The effect of high concentrate diet feeding on small intestinal fermentation,morphological structure,microbial flora in goatsThe objective of the study was to investigate the effect of high concentrate(HC)diet feeding on microbial fermentation,epithelial tissue morphology and the quantity of microbial flora of small intestine in goats.Twelve male goats were randomly allocated to two groups,fed a Hay diet(0%concentrate,n=6)or a HC diet(56.5%concentrate + 43.5%hay,n = 6)for 6 weeks.After 6 weeks of feeding,six goats in each group were slaughtered,and then the small intestinal digesta and mucosal and epithelial tissue were collected to analysis.The result of bacterial metabolites showed that,as compared with the Hay group,HC diet feeding significantly increased the concentrations of total volatile fatty acid(P ?0.015),propionate(P = 0.008),butyrate(P = 0.004),isobutyrate(P = 0.035),while decreased the concentration of lactic acid(P = 0.008).However,HC diet feeding did not influence the pH,as well as the concentrations of acetate,valerate and isovalerate and LPS in jejunal digesta(P>0.05).HC diet feeding increased the concentrations of total volatile fatty acid(P = 0.007),propionate(P=0.013),butyrate(P = 0.008),valerate(P<0.001),lactic acid(P = 0.008)and lipopolysaccharide level(P<0.001),while decreased the pH value(P = 0.005)in ileal digesta,and there were no significantly difference of the concentrations of acetate,isobutyrate,isovalerate between Hay and HC group(P>0.05).Morphology result showed that,compared with the Hay diet,HC diet feeding increased the villi height and crypt depth significantly in the duodenum,jejunum and ileum tissue(P<0.001).The ratio of villus height to crypt depth(V/C)was increased in the jejunum(P ?0.024).Transmission electron micrographs of jejunum and ileum tissue during the HC diet displayed a deterioration of the tight junction.The real-time PCR of gene expression of tight function protein result found that,compared with the Hay group,HC decreased the mRNA expression of claudin-1(P = 0.001),claudin-4(P = 0.033),occludin(P<0.001)and ZO-1(P = 0.010)in jejunal mucosa and had a reduction on the claudin-4(P = 0.034),occludin(P = 0.033)and ZO-1(P = 0.004)in ileal mucosa.Compared with the Hay diet,HC diet feeding increased the alkaline phosphatase activity of ileal mucosa significantly(P<0.05),but there was no influence on the alkaline phosphatase activity of jejunal mucosa(P>0.05);Real-time PCR analysis showed that the 16S rRNA gene copies of Bacteroidetes of the HC group were significantly lower than that in Hay group in ileal digesta(P = 0.037),HC group caused an increase of the ratio of the Firmicutes and Bacteroidetes(P<0.001),while there was no significant difference of 16S rRNA gene copies of Firmicutes(P>0.05).Additionally,no significant(P>0.05)difference was observed in the 16S rRNA gene copies of Bacteroidetes,Firmicutes,and the ratio of Firmicutes to Bacteroidetes between Hay and HC group in the jejunal digesta.Collectively,these results indicates that feeding goats high proportions of concentrate significantly affect the morphological characteristics of ileal epithelium and microbial fermentation in ileal digesta,and may have a negative effect on health of the goats.3.The effect of high concentrate diet on jejunal and ileal mucosal bacterial community and mucosal inflammatory cytokines of goatsIn this study,we set out to investigate the effect of the high concentrate(HC)diet on the jejunal and ileal mucosal bacterial composition and expressions of mucosal inflammatory cytokines in goats.Twelve,male goats were divided into two different groups(n = 6 for each group)in a completely randomized design.One group was the Hay group(a complete hay diet)and the other was the HC group(a high concentrate diet,56.5%concentrate + 43.5%hay).The experimental period was six weeks,and then the small intestinal mucosal tissues were collected to analysis.One part of the jejunal and ileal mucosal samples was extracted the mucosal DNA and determined the composition of bacterial community using Miseq sequencing method.The other part was extracted the mucosal RNA and determined the mRNA expressions of mucosal inflammatory cytokines.In our study,we chose five samples each group for data analysis.The microbial taxonomic analysis data showed that 19 phyla,245 genera and 498 operational taxonomic units(OTUs)were detected in jejunal mucosa,and 20 phyla,253 genera and 447 OTUs were found in ileal mucosa.A higher percentage of Stenotrophomonas(P = 0.028),Moraxella(P = 0.009),Lactobacillus(P = 0.009)and Pre.votella(P = 0.009)was observed in HC group in jejunal mucosal microbiota compared with the Hay group,and there was a lower abundance of Christensenellaceae R-7 group(P = 0.010)in the HC group in ileal mucosal samples.Real-time PCR found that HC increased the mRNA expressions of IL-1?(P = 0.015),IL-12(P = 0.013),IL-10(P = 0.025)and IFN-y(P = 0.033),while decreased the expressions of IL-2(P = 0.021)and IL-6(P = 0.009)in jejunal mucosa.In ileal mucosa,HC increased IL-2(P = 0.027)and IFN-?(P = 0.038)expressions,while decreasing the expressions of IL-1?(P = 0.017)and TNF-?(P = 0.045).In general,these results clearly demonstrated that HC feeding increased the proportion of mucosal pathogenic bacterial and the bacteria associated with the degradation of starch,and increased the expressions of inflammatory cytokines,suggesting that HC diet may affect the composition and structure of small intestinal mucosal bacteria,and induce the local inflammation in goats. |
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