Mechanism Of Dietary Rumen Degradable Starch On The Gastrointestinal Function And Health Of Young Dairy Goats

Starch is an important energy source for ruminants.The difference of starch in digestion and metabolism in the rumen,small intestine and hindgut significantly affects the health and function of the gastrointestine,thereby influcing the energy efficiency and production performance.Rumen degradable starch（RDS）indicates the extent of ruminal starch degradation and digestion.RDS is a more accurate nutritional index to predict starch digestion and energy efficiency.Previous researches found that the low RDS level diet（LRDS）significantly reduced the feed conversion rate（FCR）,improved the daily gain（ADG）and slaughter performance of young dairy goats,and via the AMPK-m TOR-S6K pathway and GCN2-e IF2αpathway to promote muscle protein deposition.Therefore,basing on these results,we changed corn processing methods to prepare high and low RDS diets and explored the effects and mechanisms of dietary RDS on gastrointestinal health and function of growing goats with multi-omics study in the present study.Experiment 1 The effects of dietary RDS on the ruminal metabolism and epithelial function of young dairy goatsForty healthy,weaned 3-month-old male goats（BW=13.6±0.23 kg）were randomly enrolled to receive either a whole corn grain diet（low rumen degradable starch content group,LRDS,n=20）or crushed corn grain diet（high rumen degradable starch content group,HRDS,n=20）.All goats were housed individually in tie-stall barns and fed twice a day（0800and 1600）with free access to water.Feed samples and feed intake data were collected continuously for 3 days every week during the trial period.The feeding trial lasted for 90days,and the fecal score were evaluated for continuously 7 days（84-90 days）using a5-grade fecal scores evaluation system.After the goats had been fed for 90 days,basing on growth performance and the mean values of fecal scores,8 goats of each group were randomly selected and slaughtered at 3 h after morning feeding.The results showed that,HRDS significantly decreased the fecal score of dairy goats（P<0.01）.Fecal score was significantly positively correlated with ADG of dairy goats（r>0.5,P<0.01）,and significantly negatively correlated with feed conversion ratio（FCR）（r<-0.5,P<0.01）.HRDS significantly decreased rumen papilla length（P<0.01）,rumen papilla area（P<0.05）.HRDS reduced starch content in rumen starch content（P<0.05）.The proportion of rumen butyrate in HRDS group showed a trend of decreasing during 2-8 h after morning feeding.The 16S r RNA results showed that HRDS significantly decreased the relative abundances of Clostridiales＿unclassified,Candidatus＿Saccharimonas,and Fretibacterium in the rumen（P<0.05）.Untargeted metabolomic results showed that HRDS decreased aboudance of 17metabolites（VIP>1,P<0.05）.Transcriptome sequencing revealed that 147 differential genes（DEGs）were found in the rumen and enriched to 31 pathways.Dietary RDS affected rumen epithelial galactose metabolism,PPAR signaling pathway,arginine biosynthesis,arginine and proline metabolism,pancreatic secretion,and proximal tubule bicarbonate regeneration.Compared with LRDS,HRDS significantly decreased the expression of SLC26A3（DRA）,PRKCB,and UBC（FDR<0.05）;significantly decreased the expression of SLC4A4（P<0.01）;significantly increased the expression of FABP1（FDR<0.05）.Correlation analysis showed that Fretibacterium were significantly positively correlated with the proportion of propionate（r>0.50,P<0.05）and significantly negatively correlated with the proportion of acetate and the ratio of acetate to propionate（r>0.50,P<0.05）.There were 45 significant correlations（|r|>0.50,P<0.05）between the differential metabolites and differential genera in the rumen.This experiment showed that corn processing methods changed dietary RDS levels,thereby influencing rumen fermentation.Dietary RDS affected rumen epithelial development and gene expression,while HRDS upregulated rumen epithelial immune responses and transport of VFA and HCO₃^-,and reduces the ability of animals to maintain rumen homeostasis.Experiment 2 The effects of dietary RDS on the small intestinal immune function and nutrient absorption of young dairy goatsIn this experiment,the effect of dietary RDS on small intestinal function was analyzed by multi-omics anlysis.The results showed that HRDS significantly increased the content of starch in the ileum（P<0.05）.HRDS significantly increased the concentration of LPS in the jejunum（P<0.05）.16S r RNA sequencing results showed that the HRDS group significantly increased the relative abundances of inflammation-related Prevotella＿1,Erysipelotrichaceae＿unclassified,and Mollicutes＿RF39＿unclassified in the jejunum and ileum（P<0.05）,while significantly decreased the beneficial commensal bacteria such as Ruminococcus＿2,Lachnobacterium,Lactobacillus,and Romboutsia（P<0.05）.The metabolomic results showed that HRDS significantly increased the raletive contents of Lys-Ile,Pro-Ala,Pro-Phe,Pro-Arg,Lys-Thr,Ile-Asn,Gly-Arg,Ser-Lys,PC（16:0/16:0）,hesperetin and glycyl-L-leucine in the jejunum（VIP>1,P<0.05）.HRDS also decreased the raletive contents of stearic acid andα-linolenic acid in the ileum（VIP>1,P<0.05）.Transcriptome sequencing revealed that dietary RDS affected 5 and 9 immune-related pathways in the jejunal and ileal mucosa,respectively.HRDS significantly increased the gene expression of CD19,CD79A,CD79B,and CXCL13（FDR<0.05）;significantly increased the m RNA expression of C3 and IL1β（P<0.05）.HRDS significantly decreased the gene expression of CD86,MRC1,TLR2,IL1β,CD40,and CXCL9（FDR<0.05）;significantly decreased the m RNA expression of C3,TGFβ1,EAAT5,and ASBT（P<0.05）;tended to decrease m RNA expression of TLR3,TLR4,TNFα,IL10,SGLT1,FXR and TGR5（P<0.1）.Correlation analysis showed that there were 33 significant correlations between jejunal immune-related DEGs and the microbiome（|r|>0.50,P<0.05）.There were 88 and 19significant correlations between the immune-related DEGs and microbiome,immune-related DEGs and differential metabolites in the ileum,respectivly（|r|>0.50,P<0.05）.There were five significant correlations between the differential metabolites in the ileum and the microbiome（|r|>0.50,P<0.05）.This experiment showed that HRDS activated adaptive immunity in the small intestine by reducing the abundance of beneficial commensal bacteria in the small intestine and increasing the abundance of inflammation-related microbes,disrupting the balance between immune tolerance and inflammatory immune response in the small intestine,reducing the ability to maintain the microbial homeostasis,and affecting small intestinal gene expression of dairy goats.HRDS affects ileal bile acid reabsorption and inhibits FXR and TGR5 activation.Experiment 3 The effects of dietary RDS on colonic barrier and immune function of young dairy goatsCompared with LRDS,HRDS significantly decreased fecal scores of goats（P=0.001）and increased colonic pathology scores（P<0.05）.Using immunofluorescence and IHC,HRDS decreased colonic mucosal MUC2 protein expression（P<0.05）.HRDS downregulated the m RNA expression of colonic mucosal Claudin4（P<0.1）,Occludin（P<0.05）,and upregulated the m RNA expression of DUOX2（P<0.05）,IL-6（P<0.05）,and C3（P<0.01）in colonic mucosa.The serum level of DAO（P<0.05）in the HRDS goats were higher than those in LRDS goats.Transcriptomic analysis of colonic mucosa demonstrated the variation in host immune function.HRDS largely enhanced the immune pathway-related gene expression and m RNA expression of T_H2 cytokines secretion including GATA3,IL-5,and IL-13.Metagenomics analysis revealed that cellulolytic bacteria were significantly enriched in the colon of the goats under LRDS diet,conducing to maintain hindgut homeostasis and integrity of colonic mucosal barrier.The interactions between the microbiome and host transcriptome were detected and found that,the Prevotella species（Prevotella sp.P4-67 and Prevotella sp.PINT）and Bacteroides species（Bacteroides sp.CAG:927）showed the enhanced pathogenic ability,antigen processing and presentation,and stimulated T_H2-mediated cytokine expression in the colon of HRDS goats.Metabolomics analysis showed that HRDS increased the relative content of CDCA and DCA,and decreased the relative content of hypoxanthine in colonic digesta,showing the negative correlations with the colonic mucosal barrier.This experiment showed that the malabsorption of ileal bile acids caused the accumulation of bile acids in the colon,thereby weakening the expression of MUC2 in the host mucosa and epithelial tight junctions,leading to macromolecular substances in the colon to break through the physical barrier.Colonic microbes and their metabolites stimulate colonic inflammation and tissue damage by promoting antigen presentation and promoting T_H2-mediated inflammatory processes.In addition,the ability of the colon to absorb water is also inhibited.Experiment 4 Integration analysis of microbiome,metabolome and transcriptome in the gastrointestine of young dairy goatsThis study analyzed gastrointestinal nutrients distribution,microbiome,and metabolome to explore the changes in luminal nutrients,microbiome,and metabolome under different RDS in dairy goats.Ruminococcaceae＿NK4A214＿group,Unclassified＿genera,Ruminococcaceae＿UCG-013,Ruminococcaceae＿UCG-014,Firmicutes＿unclassified,Lachnospiraceae＿unclassified,Eubacterium＿coprostanoligenes＿group,and Christensenellaceae＿R-7＿group were identified as gastrointestinal core genera by calculating the niche width.Metabolomics analysis revealed that metabolites were incorporated into 3 modules（including increased in the current site,ICS;undifferentiated inflows in the current site,UICS;absorbed or degraded in the former site,AD）.Among them,the metabolites of UICS in jejunum,ileum,cecum,and colon accounted for 35%,67%,64%,and 99%of the total metabolites’types,respectively.Linear mixed effect model was used to evaluate the explainability to the variation in host phenotypes by gastrointestinal microbiome,metabolome and transcriptome.The explainability of jejunal microbiome,jejunal metabolome,and ileal metabolome to the variation in ADG were 48.15%,39.04%,and56.70%,respectively.The explainability of ruminal microbiome,ruminal metabolome,jejunal metabolome,and colonic metabolome to the variation in FCR were 98.21%,86.24%,11.88%,and 14.30%,respectively.The explainability of colonic microbiome and microbial function to the variation in MUC2 expression were 10.32%and 25.96%,respectively.The explainability of colonic microbiome and metabolome to the variation in pathological scores were 75.50%and 57.06%,respectively.The explainability of LPS to the variation in jejunal DEGs and ileal DEGs were 16.62%and 10.65%,respectively.Colonic UICS explained49.77%of the colonic DEGs variation.1-palmitoylglycerol,CDCA,and DCA explained72.03%,10.41 and 12.59%of the gene expression variation in colonic DEGs,respectively.These findings provide a framework of longitudinally effects in shaping the subsequent GS functions by the nutrients,metabolites,and microbes coming from the former GS in dairy goats.Taken together,this study showed that HRDS represses the ruminal transport of VFAs and epithelial homeostasis in the rumen of young dairy goats,decreasing the ileal transport of nutrients and bile acids,and inhibiting the reabsorption of water in the colon.HRDS induces dysbiosis of intestinal microbes,disrupting the balance of intestinal tolerance and inflammatory response,and causing intestinal inflammation.HRDS down-regulates the ileal reabsorption of bile acids,resulting in the accumulation of bile acids in the hindgut,thereby weakening the colonic barrier function and causing colonic inflammation.Nutrients,microbiota,and metabolites have impacts on the gastrointestinal gene expression of young dairy goats.Our study provids a framework for subsequent analyses of crosstalk in shaping the subsequent GS functions by the nutrients,metabolites,and microbes coming from the former GS.It will be instrumental in isolating the impact of former GS when discussing the host-microbe interaction in a particular GS.