A Study On The Metabolomics Profiling Alterations Associated With The High-concentrate Diet In Beef Cattle And The Effects Of Nutritional Modulations

In intensively beef cattle production,the high-concentrate diet that meets the nutritional requirements of fattening beef cattle is associated with a high risk of metabolic disorders,such as rumen acidosis.Nutritional metabolic disorders are derived from the metabolic imbalance,therefore,studies that mainly focused on a sigle class of metabolites cann't really reveal the mechanisms of occurance.It is necessary to elucidate this mechanism of occurance from the metabolic profiles,the internal relationships among different metabolites,the relationship between animal phenotypes and metabolites and so forth.As the end products of genome,metabolomics can effectively revealed the molecular mechanisms of animal pathology through dectecting the whole set of small molecular metabolites.Therefore,by ultra-high-performance liquid tandem chromatography quadrupole time of flight mass spectrometry?UHPLC-QTOF/MS?,the rumen and plasma metabolomics profiling associated with a high-concentrate diet in beef steers were analyzed in this study,which provided preferences for further elucidating the pathogenesis of nutritional metabolic diseases.On the basis,the effects of montmorillonite supplement and corn steeped by dilute acid on beef steers fed a high-concentrate diet were evaluated by metabolomics,which provided a new effective and feasible solution to decrease the negative effects of the high-concentrate diet on beef cattle production.This study is divided into the following three experiments:1.Study on the metabolomics profiling alterations associated with the high-concentrate diet in beef cattleIn order to clarify the mechanisms of the metabolic disorders occurrence in beef cattle fed a high-concentrate diet,the production performance,rumen pH value,lipopolysaccharide?LPS?content,immune response,and rumen and plasma metabolic profiling of beef cattle fed diets with different concentrate-to-roughage ratios were compared in this experiment.Twelve beef cattle?Charolais×Luxi?with the similar baseline metabolomics were paired according the similar body weight and body condition,and randomly allocated to one of two diets:the low concentrate diet?LCD?with a 40:60 ratio of concentrate to roughage and the high concentrate diet?HCD?with a 60:40 ratio of concentrate to roughage.The animal trial was conducted for 32 days with the first 7 days serving as an adaptation period.Based on UHPLC-QTOF/MS metabolomics and multivariate analyses,including principal component analysis?PCA?and orthogonal partial least-squares discriminant analysis?OPLS-DA?,rumen and plasma metabolic profiling of two diets were recognized.According VIP values>1 of OPLS-DA model and P<0.05 of the student's test,significantly differential metabolites between two groups were screened.Then,MetaboAnalyst was performed to pathway analysis,including enrichment analysis and topology analysis.The results showed that:?1?Compared with the low-concentrate diet,the high-concentrate diet significantly increased final body weight,average daily gain?ADG?and average dry matter intake?ADMI?of beef steers?P<0.05?,and significantly decreased feed conversion ratio?ADMI/ADG?of beef steers?P<0.05?.?2?Compared with the low-concentrate diet,the high-concentrate diet significantly increased the apparent digestibility of dry matter,organic matter,crude protein,ether extract,neutral detergent fiber,crude ash and calcium in beef cattle?P<0.05?.?3?Compared with the low-concentrate diet,the high-concentrate diet significantly reduced the ruminal pH values of beef cattle?P<0.01?.?4?Compared with the low-concentrate diet,the high-concentrate diet significantly increased the LPS concentrations in rumen fluid,plasma and feces of beef cattle?P<0.05?.?5?Compared with the low-concentrate diet,the high-concentrate diet significantly increased the concentrations of plasma C-reactive protein?CRP?,serum amyloid A?SAA?,LPS-binding protein?LBP?and haptoglobin?Hp?in beef cattle?P<0.05?.In addition,the high-concentrate diet also significantly increased the concentrations of plasma interleukin?IL?-1?,IL-6,IL-8 and tumor necrosis factor-??TNF-??in beef cattle?P<0.05?.?6?717 rumen metabolites and 386 plasma metabolites were identified in this study.Moreover,rumen and plasma metabolomics profiling of beef cattle fed a high-concentrate diet were obviously different from those of beef cattle fed a low-concentrate diet.The multivariate analysis identified 144 rumen and 56 plasma metabolites that were significantly different between the two groups.When steers were fed with the high-concentrate diet,rumen and plasma carbohydrate metabolites and amino acids were up-regulated.There were some noteworthy differential metabolites,such as DL-lactate,propionic acid,hypoxanthine,1-methylhistidine,indole-3-carboxylicacid,5-hydroxyindoleacetate,5-methyoxydimethyltryptamine,1,2,3-trihydroxybenzene,3-methylxanthine,picolinic acid and thymidine in the rumen,as well as tyramine,indole,5-hydroxyindoleacetate and phenyllactic acid in plasma,which were increased by feeding the high-concentrate diet.Galactose metabolism and vitamin B₆ metabolism were screened as the key rumen metabolic pathways associated with a high-concentrate diet,while phenylalanine metabolism and phenylalanine,tyrosine and tryptophan biosynthesis were also identified as the key plasma metabolic pathways associated with a high-concentrate diet.2.Effects of corn steeped with dilute acids on the rumen and plasma metabolic profiling of beef cattle fed a high-concentrate dietThe purpose of this experiment was to evaluate the effects of steeping corn by 1%lactic acid or 1%hydrochloric acid on the production performance,rumen pH value,LPS content and immune response of beef cattle fed a high-concentrate diet,which provided new ideas for improving the health value of high concentrate diet in production.In addition,based on metabolomics analysis,the effects of steeping corn by1%lactic acid or 1%hydrochloric acid on rumen and plasma metabolic profiling were also investigated,which provided theoretical basis for dilute acids processing to change the structure of corn starch and modulate abnormal rumen fermentation.Eighteen Charolais×Luxi hybrid steers with a similar initial body weight,body condition score and baseline metabolomics were selected for this experiment.These steers were randomly assigned to 1 of 3 diets.The 3 diets were identical,with the only difference being the treatment of corn:48.76%?dry matter basis?corn grain in the diet was steeped for 48 h in an equal quantity?i.e.,in a 1:1 ratio,wt/vol?of tap water?CON?,1%lactic acid?LA?or 1%hydrochloric acid?HA?before being mixed with other ingredients of the total mixed ration?TMR?.The steers were offered a TMR with a concentrate to roughage ratio of 60:40.The results showed that:?1?Compared with the CON group,there was no significant difference in final body weight,ADG,ADMI and ADMI/ADG in the LA group and the HA group?P>0.05?.?2?Compared with the CON group and the HA group,the apparent digestibility of neutral detergent fiber and ether extract of the LA group was significantly increased?P<0.05?,while the apparent digestibility of other feed nutrients was not different among the three groups?P>0.05?.?3?Compared with the CON group,the LA group and the HA group significantly increased the rumen pH values of beef cattle?P<0.01?.?4?Compared with the CON group,the feces LPS concentrations of the LA group and the HA group significantly decreased?P<0.05?,the plasma LPS concentrations of the LA group was also significantly decreased?P<0.05?,and the LA group and the HA group had a lower trend in rumen LPS concentrations?P=0.057?.?5?There were lower concentrations of plasma CRP,SAA,Hp,IL-1?and IL-8 in beef steers fed LA-or HA-treated corn?P<0.05?.The LA treatment decreased the concentrations of plasma LBP and TNF-??P<0.05?.?6?There were obviously differences in ruminal and plasma metabolic profiling among these three groups.85rumianl differential metabolites and 25 plasma differential metabolites were obtained in this study.Feeding beef steers corn steeped in 1%LA or 1%HA was associated with lower relative abundance of carbohydrate metabolites,amino acid,thymine,uracil,xanthine and DL-lactate in the rumen.Compared with the other two groups,the LA group decreased the relative abundance of L-phenylalanine,DL-phenyllactic acid and tyramine in plasma and upgrated the relative abundance of urea in plasma.The relative abundance of urea in plasma was the lowest in the HA group,and the HA treatment increased the relative abundance of all amino acids in the plasma.In addition,compared with the CON group,the relative levels of His-Ser and L-histidine were significantly increased in the LA group and the HA group.3.Effects of corn steeped with dilute lactic acid and dietary supplementation of montmorillonite on the rumen and plasma metabolic profiling of beef cattle fed a high-concentrate dietSteeping corn with dilute lactic acid can reduce the release of LPS in the gastrointestinal tract,and supplementation of montmorillonite?MMT?in diet can absorb the gastrointestinal LPS.Therefore,the purpose of this experiment was to evaluate the effects of the combination of these two treatments on the production performance,rumen pH value,LPS content,immune response and rumen and plasma metabolic profiling in beef cattle fed a high-concentrate diet.The results of this study provide references for preventing abnormal metabolism associated with a high-concentrate diet in beef cattle production.According to the complete random design,twenty-four Charolais×Luxi hybrid steers with the similar initial body weight,body condition and baseline metabolomics were randomly divided into four groups.The treatments consisted of?1?the basal diet?CON?,?2?the basal diet+2 g/kg MMT?MMT?,?3?steeping 48.76%?dry matter basis?corn in the diet for 48 h in an equal quantity?i.e.,in a 1:1 ratio,wt/vol?with 1%lactic acid?LA?,and?4?after steeping corn with 1%lactic acid,the basal diet+2 g/kg MMT?MMT+LA?.All steers were fed the same basic ration with a 60:40 ratio of concentrate to roughage.The results showed that:?1?Compared with the CON group,the MMT+LA group significantly increased ADG and decreased ADMI/ADG?P<0.05?.?2?The rumen pH values of the LA group or the MMT group was significantly higher than that of the CON group and the MMT+LA group?P<0.05?.?3?The LPS concentrations of the rumen,plasma and feces in the MMT+LA group were significantly lower than that of the CON group?P<0.05?.?4?Compared with the CON group,the plasma concentrations of IL-8,SAA and LBP of the MMT+LA group were significantly reduced?P<0.05?,but compared with the LA group,the concentrations of SAA,Hp,IL-1?and IL-8 of the MMT+LA group was increased and compared with the MMT group,the concentrations of CRP,Hp and IL-8of the MMT+LA group was also increased?P<0.05?.?5?There were differences in the rumen metabolic profiling between the MMT+LA group and the CON group,the LA group and the MMT group.In the MMT+LA group,the relative contents of carbohydrate metabolites,vitamin B₆ and phenylpropionic acid?3-PP?in rumen were significantly increased,while the relative content of xanthine content was decreased.However,compared with the MMT group,the ruminal relative contents of tyramine and other biogenic amines in the MMT+LA group were increased and the ruminal contents of uracil and hypoxanthine were also decreased.?6?There were differences in the plasma metabolic profiling between the MMT+LA group and the CON group,the LA group and the MMT group.The MMT+LA group significantly reduced the lipid metabolites,such as glycerol,creatinine,lauric acid and so forth.However,compared with the LA group,the relative content of DL-lactate of plasma in the MMT+LA group was also increased.And compared with the MMT group,the relative contents of non-esterified fatty acids?NEFA?of plasma in the MMT+LA group were increased.In conclusion,the high-concentrate diet changed the rumen and plasma metabolomics profiling of beef cattle,for example,increased the relative contents of carbohydrate metabolites and protein metabolites in rumen and plasma,which was conductive to the growth and fattening of beef cattle.However,the high-concentrate diet also decreased ruminal pH,increased the release of LPS,caused immune responses and produced some harmful metabolites.Therefore,long-term feeding of high-concentrate diet will increase the incidence of metabolic diseases for beef cattle.1%LA or 1%HA treatment of corn improved the rumen and plasma metabolic profiles and reduced inflammatory responses in beef cattle fed a high-concentrate diet.1%LA or 1%HA treatment of corn can effectively prevent the occurrence of metabolic diseases associated with the high-concentrate diet.Corn steeped with 1%LA and dietary supplementation of MMT modulated the metabolic disorders caused by the high-concentrate diet,but it didn't show the combination effects of these two treatments.