| In ruminants,the rate and extent of ruminal starch degradation are important nutritional factors affecting rumen metabolism and health.Rumen degradable starch（RDS）play vital roles on gut health and nutrient utilization.Dietary RDS affects rumen fermentation and production parameters,and their effect on microbial metabolic functions and liver metabolism warrants deep studies.In the present study,we increased RDS through replacing corn with wheat and intended to investigate the effect of RDS on rumen fermentations,microbial metabolic functions and liver metabolism.Metagenomics method was applied to analyze the molecular mechanism in the carbohydrate metabolism of the rumen microbiome,transcriptome was used to profile the rumen gene expression,metabolomics was used to approach the metabolites of liver.So that the mechanism of dietary RDS regulates rumen and liver metabolism were explored.Experiment 1 The effect of dietary RDS on rumen dynamic fermentation of dairy goatsSix ruminally cannulated dairy goats were assigned to 3 treatments in a repeated 3×3Latin square design.Three diets consisted of different RDS:low RDS（LRDS=20.52%）,medium RDS（MRDS=22.15%）and high RDS（HRDS=24.88%）,which were achieved by replacing dietary corn with wheat.After morning feeding,the rumen p H,acetate proportion,butyrate proportion and the ratio of acetate to propionate in each group decreased first and then increased;the redox potential,total VFA concentration,propionate proportion and increased first and then decreased.On day 21,the HRDS group significantly decreased the acetate proportion and the ratio of acetate to propionate,and significantly increased the propionate proportion in the HRDS group,compared with the MRDS group and LRDS group at 08:00,12:00 and 16:00（P<0.05）.Ruminal redox potential was negatively correlated with rumen p H（P<0.05）and the ratio of acetate to propionate（P<0.05）.HRDS group significantly increased the concentration of free LPS in rumen fluid（P<0.05）and plasma（P<0.05）,compared with LRDS and MRDS groups.MRDS and HRDS groups significantly decreased 24 h DM,NDF,and ADF degradability,and 48 h CP degradability of alfalfa,compared with LRDS group.These results indicated that dietary RDS can significantly affect the rumen fermentation of dairy goats:higher RDS decreased ruminal p H,alfalfa NDF and ADF degradability;increased ruminal redox potential,total VFA concentration,propionate proportion;increased free LPS concentration in rumen fluid and plasma.Experiment 2 The effect of dietary RDS on the carbohydrate metabolism of the rumen microbiome of dairy goatsThis study analyzed CAZymes and Kyoto Encyclopedia of Genes and Genomes Orthology functional categories to explore carbohydrate degradation under different RDS in dairy goats by metagenomics analyses.Eighteen dairy goats（2nd lactation,45.8±1.54 kg）were divided in three groups fed low RDS（LRDS）,medium RDS（MRDS）,and high RDS（HRDS）diets.The results showed that,HRDS treatment group significantly decreased the ruminal p H（P<0.05）,NH3-N concentration（P<0.05）,NAD+concentration（P<0.05）,and the ratio of NAD+and NADH（P<0.05）;and increased the propionate proportion（P<0.05）,fumarate（P<0.05）and succinate concentrations（P<0.05）,compared with LRDS group.The most abundance phylum were:Bacteroidetes,Firmicutes,Proteobacteria,and Spirochaetes.The relative abundance of acetogens,such as family Clostridiaceae and Ruminococcaceae,genera Clostridium and Blautia were higher in HRDS than LRDS feeding goats.The GH9 family（responsible for cellulose degradation）genes were lower in HRDS than MRDS diet samples.The abundance of GH13＿9 and CBM48（responsible for starch degradation）were reduced in HRDS group indicating the decreased binding activity from catalytic modules to starch.The abundance of GH43＿4 and GH43＿5 was increased in HRDS group.Amylose（EC3.2.1.3）genes under HRDS treatment were more abundant than under LRDS treatment.These results indicated that dietary RDS can significantly affect the rumen microbial metabolic functions of dairy goats.Experiment 3 The effect of dietary RDS on ruminal epithelial barrier function of dairy goatsBased on the results of experiment 2,16S r RNA sequence and transcriptome were used to profile rumen epimural bacterial communities and rumen gene expression changes,respectively.HRDS significantly increased the relative abundance of Ruminiclostridium＿9,Ruminiclostridium＿5,Stenotrophomonas,Nocardioides,and Paracoccus（P<0.05,LDA>2）,and decreased the proportion of Rikenellaceae＿RC9,Pseudoramibacter,Alloprevotella,Bradyrhizobium,succinivibrio,and Howardella（P<0.05,LDA>2）.The ruminal p H had positive correlations with Spirochaeta＿2 and Desulfovibrio,and negative correlations with Streptocoocus and Lachnospiraceae＿NK3A20.Genus Campylobacter was negatively correlated with the propionate proportion.Dietary RDS influence vascular smooth muscle contraction,regulation of actin cytoskeleton,c GMP-PKG signaling pathway,calcium signaling pathway,and Focal adhesion et al.In the HRDS group,the expressions of KRT3,KRT10,KRT79,and DSG1 related to barrier function were significantly reduced（P<0.05）,and the expressions of MLCK,KLK9,KLK10,and KLK13 related to inflammation were significantly increased（P<0.05）,the expression of PDK4 related to energy supply for tissue cells were significantly increased（P<0.05）,compared with LRDS group.These results indicated that dietary RDS can affect rumen epimural bacterial communities,impair ruminal epithelial barrier and proliferative function,activating the local immune response in rumen epithelium.Experiment 4 The effect of dietary RDS on liver metabolism of dairy goatsOur previous studies showed that the HRDS increased free LPS concentration in both rumen liquid and plasma,impaired ruminal epithelial barrier function,suggesting the ruminal free LPS translation into the blood.Thus,this study was further investigated the effect of HRDS-increased LPS on liver metabolism in dairy goats.Compared with LRDS group,HRDS diet significantly enhanced the activity of alkaline phosphatase（P<0.05）and aspartate aminotransferase（P<0.05）in plasma,trended to increase the TNF-αm RNA expression in liver（P=0.098）,significantly increased PPARαm RNA expression（P<0.05）and CPT1 protein expression（P<0.05）.The HRDS treatment significantly decreased the concentrations of certain fatty acids（included linolenic acid,oleic acid,etc.）,and phospholipids（included PC（18:0/18:1）,LPC（16:0）,LPC（18:0）,etc.）,and increased the concentrations of L-carnitine,L-palmitoylcarnitine,taurochenodeoxycholate and taurodeoxycholic acid in the liver compared to the LRDS treatment（VIP>1,P<0.10）.Compared with LRDS group,HRDS diet significantly increased CYP7A1 m RNA expression in liver（P<0.05）,and decreased FXR（P<0.05）and ASBT（P<0.05）m RNA expression in the ileal mucosa.These results indicated that dietary RDS can affect lipid metabolism in the liver and enterohepatic circulation of bile acids.To sum up,this study showed that HRDS diet affect rumen fermentation characteristics by inhibiting rumen microbial fiber degradation,damage rumen epithelial barrier function;damage liver,promote fatty acidβ-oxidation and disrupt enterohepatic circulation of bile acids in dairy goats by free-LPS which was translocated from fluid to peripheral blood circulation. |
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