| Subacute ruminal acidosis(SARA)is a metabolic disease in dairy cows that resulted in long-term health and economic consequences,including feed intake depression,reduced milk yield,diarrhea,rumenitis,liver abscesses,and laminitis,and finally caused significant economic losses to the industries of dairy cows.This research aimed to explore the effects of SARA on the rumen microbiome and body metabolism,as well as the effects on the health of mammary gland and milk quality in dairy cow.1.Effects of SARA on rumen microbiome and metabolome of dairy cowsThis study aims to explore the response of ruminal fermentation parameters,rumen microbiome and metabolites to SARA in diary cows.Eight ruminally cannulated Holstein cows in mid-lactation were randomly assigned to either a low-concentrate(40%)diet(CON)or a high-concentrate(60%)diet(HC)for 3 weeks.The feed intake was measured on the last 2 days of each week of the experimental period,and the rumen contents were collected from the abdominal sac of the rumen at 0,2,4,6,8 and 12 hours after the morning feeding on the last day of each week,after being filtered by 4layers of gauze,rumen fluid was used to measure p H and VFA concentrations.The rumen contents collected at 0h were used for microbial DNA extraction and further for sequencing analysis,rumen fluid collected at 0h was used for metabonomics analysis.The results showed that HC feeding significantly reduced rumen p H(P = 0.002),acetate concentration(P = 0.026),acetate ratio(P = 0.006)and the ratio of the concentrations of acetate to propionate(P < 0.001),meanwhile increased total VFA concentration(P= 0.001),propionate concentration(P = 0.001),valerate concentration(P = 0.016)and propionate ratio(P = 0.003)compared with the CON group.16 S r RNA gene sequencing analysis showed bacterial communities were significantly separated in the two groups,and HC feeding significantly reduced the alpha diversity indexes(P <0.05).As compared with the CON group,at the phylum level,HC feeding significantly decreased the relative abundance of Bacteroidetes(P = 0.008),Spirochaetae(P = 0.011),Fibrobacteres(P = 0.003),Tenericutes(P = 0.021),Candidate＿division＿SR1(P =0.028),Elusimicrobia(P = 0.016),Lentisphaerae(P = 0.028),SHA-109(P = 0.028)and Verrucomicrobia(P = 0.028).At the genus level,HC feeding significantly increased the relative abundance of Roseburia(P < 0.001)and Dialister(P = 0.015),and decreased the relative abundance of RC9＿gut＿group(P = 0.002),Treponema(P =0.011),unclassfied Christensenellaceae(P < 0.001)and Fibrobacter(P = 0.003).At the OTU level,HC feeding also caused the missing of 845 OTUs and the emergence of 500 OTUs.PLS-DA of Metabolomics results showed the clear separation between two groups.When compared with the CON group,the levels of PGE1,MG(12:0/0:0/0:0),Lyso PA(8:0/ 0:0),propionate,valerate,palmitoleic acid,adenine,5-methylthioadenosine,hippuric acid,benzoic acid,2-hydroxyvaleric acid,3-hydroxysebacic acid,L-ascorbic acid,deoxyribose and phosphorylcholine were significantly increased in the HC group(P < 0.05),while the content of acetate,3-hydroxytetradecanedioic acid,propionylcarnitine,2-octenoic acid,sebacic acid,suberic acid,undecadienoic acid,Lyso PE(0:0/16:0),L-Acetylcarnitine,1,11-undecanedicarboxylic acid,leukotriene E4,3-hexanoic acid,Lyso PC(16:0),Lyso PE(0:0/18:0),2’-deoxyuridine,3’-AMP,Cyclic AMP,4-hydroxyhippuric acid,3-hydroxyanthranilic acid,L-isoleucine,hexanoylglycine,N-acetyl-L-tyrosine,α-tocotrienol,spermidine,γ-tocotrienol,choline and homogentisic acid were significantly down-regulated in the HC group(P < 0.05).In conclusion,SARA can alter rumen fermentation of dairy cows,affect the structure and composition of rumen bacterial communities,and and further disrupt the rumen metabolism.2.Effects of SARA on plasma biochemical indexes and metabolomics profiling changes of dairy cowsThis chapter explored the changes in blood metabolic profiles in dairy cattle with SARA,the experimental design was mentioned in the earlier chapter.On the last day of each week of the experimental period,blood samples were obtained from the jugular vein at 6h after morning feeding,which were used to the biochemical indexes and metabolomics analysis.No difference was observed in dry matter intake(DMI)between the two groups(P = 0.524).The plasma biochemical indexes results showed that cows fed the HC diet had a lower plasma concentration of β-hydroxybutyrate(P =0.007),and a higher concentration of triglycerides(P = 0.014).Plasma metabolomics profiling were analyzed by high performance liquid chromatography/mass spectrometry(HPLC-MS).Both principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)revealed the clear separation between two groups.By multivariate analysis,26 differential plasma metabolites were identified(VIP > 1 and P < 0.05).Compared with CON group,the levels of 7-ketodeoxycholic acid,deoxycholic acid,cholic acid,12-ketodeoxycholic acid,12(13)Ep-9-KODE,12,13-DHOME and L-asparagine metabolites were significantly increased(P < 0.05),while the content of undecanedioic acid,hexadecanedioic acid,9-HODE,thromboxane B3,PGE2,L-arginine,L-tryptophan,L-threonine,isobutyrylglycine,isovalerylglycine,hippuric acid,4-hydroxyhippuric acid and 6-phospho-2-dehydro-D-gluconate were significantly reduced in HC group(P < 0.05).Overall,as compared with CON group,HC feeding affected the lipid metabolism,amino acid metabolism and glucose metabolism during SARA in dairy cows.3.Effects of SARA on milk performance and milk quality of dairy cowsThe objective of this chapter was to explore the impact of SARA on milk performance and milk quality in dairy cows.The experimental design was mentioned in chapter 1.The cows were milked twice daily,and the milk samples were collected on the last two days of each week.Milk composition was determined within 24 hours after sample collection,16 S r DNA sequencing and HPLC-MS methods were used to investigate the differences of the microbial diversity and the metabolites profiles of the milk,respectively.Results showed that there were no significant differences in DMI,milk yield,SCC,milk protein,and lactose between the two groups(P > 0.05),while the milk fat ratio was observed decreased in the HC group(P = 0.034).The milk 16 S r DNA gene sequence results showed that the alpha diversity indexes(Chao 1,Shannon index,and Simpson index)were not affected by the HC feeding.At the phylum level,the milk bacteria was dominated by Proteobacteria,Actinobacteria,Bacteroidetes,and Firmicutes,regardless of diet.At the genus level,most abundant bacteria were from the external environment and the HC feeding increased the proportion of Labrys(P =0.019).Both the principal components analysis and the partial least squares of discriminant analysis of the milk metabolomics indicated that the metabolites clustered separately between the two groups.The metabolic profile demonstrated that 20 metabolites were up-regulated including cholic acid,L-Palmitoylcarnitine,tetradecanoylcarnitine,stearoylcarnitine,decanoylcarnitine,lyso PA(8:0/0:0),glycocholic acid,L-octanoylcarnitine,lyso PE(0:0/18:0),3-phospho-D-glycerate,betaD-Fructose 6-phosphate,deoxyribose 1-phosphate,D-ribose 5-phosphate,N-acetylalpha-D-galactosamine 1-phosphate,phosphoenolpyruvic acid,flavine mononucleotide,7-methylguanosine,niacinamide,acetylcholine,and sphingosine(P< 0.05 and VIP > 1).On the contrary,11 metabolites were down-regulated in the HC group,including α-linolenic acid,PGE2,L-lactic acid,L-malic acid,3-hydroxysebacic acid,succinyladenosine,guanosine,pyridoxal,L-glutamic acid,hippuric acid,and trigonelline(P < 0.05 and VIP > 1).Pathway analysis results showed that three metabolic pathways were enriched in the HC group including pyruvate metabolism,pentose phosphate pathway,and citrate cycle.These results elucidated that the changes in the milk bacteria were negligible during SARA.However,the metabolite profiles were altered obviously by the HC feeding,which may finally result in the deterioration of the milk performance and milk quality. |
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