| The shortage of dietary protein source is one of the bottleneck problems that restrict the healthy development of livestock system.Urea is a low-cost,non-protein nitrogen source,which has been widely used in ruminant production because urea can replace a portion of the normal protein source in diet of ruminant productivity.However,how urea affect rumen microbial composition and function,and rumen epithelial barrier is still unclear.Thus,the toxicity is sometimes observed because of the improper use of urea.Therefore,the present study used Hu sheep as a model,examined the effects of urea on growth performance,host metabolism,rumen microbial community and function,rumen epithelial morphology and gene expression through the supplementation of urea in the diet,which can provide theoretical guidance for the rational use of urea in production.Results were listed as follows:1 Effects of dietary urea on growth performance,rumen fermentation and blood metabolites of Hu sheepThe present study aims to examine the effects of urea in the diet on growth performance,rumen fermentation parameters and blood metabolism of fattening Hu sheep.Forty-two male Hu lambs(24.3 ±1.7 kg,3 months old)were fed a total mixed ration based on concentrate and roughage(55:45,dry matter basis)and randomly assigned to one of three experimental diets:a basal diet with no urea(UC group,0 g/kg DM),a basal diet supplemented with low levels of urea(LU group,10 g/kg DM),and a basal diet supplemented with high levels of urea(HU group,30 g/kg DM).The experiment conducted 11 weeks,with 1 week of adaptation followed by 10 weeks of dietary treatment.At the end of the experiment,the body weight was measured,and six Hu lambs with similar body weight from each group were slaughtered,and then the plasma and rumen fluid were collected.The results showed that the average daily gain and dry matter intake of Hu sheep in the LU and HU groups increased quadratically in comparison with that in the UC group(P<0.05),with the highest value form Hu sheep in the LU group(10 g/kg DM).The concentrations of urea nitrogen in LU and HU groups significantly increased in comparison with the UC group(P<0.01).The concentration of blood ammonia in the HU group is significantly higher than that in the LU group(P<0.01).The amount of total VFA and pH are not significantly different among the three groups(P>0.05).The concentrations of ammonia(P<0.01),butyrate,and butyrate(P=0.03),valerate(P=0.02),and isobutyrate(P<0.01)linearly increased with urea level in the diet.The amount of propionate linearly decreased with urea level in the diet(P=0.01).Collectively,these results suggest that the urea supplementation in the diet affects the rumen fermentation,blood physiological and biochemical parameters,and growth performance of Hu sheep,with the best growth performance under the level of 10 g/kg DM urea.2 Effects of urea in the diet on rumen solid-,liquid-and epithelium-associated bacterial community composition of Hu sheepThe aim of this experiment was to investigate the effect of urea in the diet on bacterial community in rumen solid,liquid and epithelial fractions of Hu sheep.The design of the experiment is the same as that in first chapter.Samples of rumen solid,liquid and epithelial fractions in Hu sheep were collected,and microbial genomic DNA was extracted,and the bacterial 16S rRNA gene V3-V4 region was amplified and then was sequenced on Illumina Miseq.QIIME 1.9.1 was applied to analyze the microbial composition,and the microbial function was predicted based on PICRUSt.The results showed that the phyla Bacteroidetes,Firmicutes and Proteobacteria were prevalent bacteria in the rumen of Hu sheep.Prevotella 1 is the most abundant genus among the UC,LU and HU groups.The unclassified Muribaculaceae,bacteria within the families Lachnospiraceae and Ruminococcaceae,and Christensenellaceae R7 were abundant in the solid and liquid fractions.Butyrivibrio 2 and Treponema 2 were the abundant bacteria in the epithelial fraction.The principal coordinates analysis results showed the levels of urea in the diet has a significant effect on the solidassociated bacterial community,and only has limited effects on the liquid-associated bacterial community,but no significant effect on the epithelium associated bacterial community.In the solid and liquid fractions,Succinivibrionaceae UCG 001 and Prevotella 1 showed decreased abundance with dietary urea supplementation(P<0.05),whereas the abundance of Oscillospira spp.significantly increased(P<0.05).Howardella spp.and Desulfobulbus spp.were higher in the epithelial fraction of the UC and LU groups relative to HU group(P<0.05).Comparison of predictive function among the three groups revealed that the relative abundance of glycine,serine and threonine metabolism in rumen solid and liquid fractions from the LU and HU groups decreased in comparison with UC group(P<0.05),while the relative abundance of butyrate metabolism pathway increased(P<0.05).Collectively,these results reveal that the supplementation of urea in the diet affected rumen solid-,liquid-and epitheliumassociated microbial community membership and structure,and the influencing degree is different among the three fractions.3 Metagenomic analysis reveals the effects of urea in the diet on rumen microbial function of Hu sheepThe aim of this study was to examine the effect of urea in the diet on rumen microbial composition and metabolic function of Hu sheep.The experimental design is the same as the first chapter.After collecting Hu sheep rumen content,extracting microbial genomic DNA,Illumina HiSeq for sequencing,and software such as SOAP was used to analyze microbial composition and function.The results showed that the relative abundance of Succinivibrio spp.,Prevotella spp.,Lactobacillus spp.,Succinatimonas spp.,Selenomonas spp.,and Megasphaera spp.were significantly higher in the LU group than in the UC and HU groups(P<0.05).In contrast,the relative abundances of Butyricicoccus spp.,Streptococcus spp.,Clostridium spp.,Ruminococcus spp.,Butyrivibrio spp.,and Treponema spp.were greater in the UC and HU groups than in the LU group.The relative abundance of tyrosine metabolism,histidine metabolism,alanine,aspartate and glutamate metabolism,valine,leucine and isoleucine biosynthesis,arginine and proline metabolism,starch and sucrose metabolism were more abundant in the LU group than in the UC and HU groups(P<0.05).The relative abundance of cysteine and methionine metabolism,lysine degradation,and fructose and mannose metabolism pathways were less abundant in the LU group than those in the UC and HU groups(P<0.05).In comparison with the UC group,the significantly increased KOs in rumen content of Hu sheep from the LU group were related to the pathways of the metabolism of arginine,proline,glycine,serine,threonine,isoleucine,valine,leucine,and citric acid cycle(pyruvate,oxaloacetate,fumarate,and α-ketoglutarate).In comparison with the LU group,the significantly increased KOs in rumen content of Hu sheep from the HU group were related to the pathways of fructose and mannose metabolism,glycolysis,pentose phosphate pathway,cysteine metabolism and lysine metabolism.Collectively,these results show that dietary urea affect the rumen microbial function,resulting in changes of pathways of amino acid,and mannose and fructose metabolism.4 Effects of dietary urea on rumen epithelial morphology and gene expression of Hu sheepThe purpose of this experiment was to study the effect of urea in the diet on the epithelial morphology and gene expression of Hu sheep.The experimental design is the same as the first chapter.Rumen epithelium was collected,ruminal papillae was analyzed,RNA was extracted,Illumina HiSeq was used for de novo sequencing,and software such as SOAP and DEseq2 were used to analyze the differential genes and functions.The result showed that the length(P<0.01)and surface of the ruminal papillae(P<0.01)in the UC and LU groups were significantly higher than that of HU group.In addition,the surface of the ruminal papillae in the LU groups is higher than that of UC group(P<0.05).The principal component analysis results showed that the rumen epithelial function of HU group was different from that of UC and HU groups.In comparison with the UC group,102 genes in rumen epithelium of the LU group were significantly up-regulated,including CTGF,PROB1,CCL5 and NR4A1,and 231 genes were significantly down-regulated,including SLC30A1,CHORDC1,TWF1,PRSS35,STK26,HSPA4L,SLC35A3,CMTR2 and MRPS36.GO analysis showed that the differently expressed genes were enriched in functions of keratin fibers,intermediate fibers,intermediate fiber skeleton,immune response regulation,rough endoplasmic reticulum and lysozyme activity.In comparison with the LU group,60 genes in rumen epithelium of the HU group were significantly up-regulated,including KRT18,CTSL,CXCL8,ACTBL2,and ADORA3,and 79 genes were significantly down-regulated including CNN1,ACTG2,ACTA1,IFIT3 and CCL5.GO analysis showed that the differently expressed genes were enriched in immune response,MHC class Ib protein complex,MHC class I receptor activity,regulation of regulatory T cell differentiation,antigen presentation,defense response,and innate immune response.Correlation analysis showed that the concentration of ammonia positively correlated with the relative abundance of NR4A1(P<0.001,R=0.72)and CLDN23(P<0.001,R=0.85),and CLDN23 negatively correlated with the relative abundance of Desulfobulbus spp.(P<0.001,R=-0.73),and positively correlated with Treponema 2(P<0.001,R=0.82)and Ruminobacter spp.(P<0.001,R=0.93).The concentration of butyrate negatively correlated with the expression level of BST-2A(P<0.001,R=-0.79).The results show that dietary urea affects the rumen epithelial barrier function and induces an increase of immune response under high level of urea.In summary,the present study found that the supplementation of urea in the diet affected the growth performance of Hu sheep,with the best performance from the level of 10 g/kg DM urea.The supplementation of urea in diet affected the bacterial community in rumen solid-,liquid-,and epithelium-associated fractions,resulting in the significant change of the pathways of amino acid,and mannose and fructose metabolism,which further affected morphology and gene expression of rumen epithelium. |
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