System-biology Based Approaches To Explore The Physiological And Metabolic Mechanisms For Regulation Of Lactation Performance In Dairy Cows Receiving Different Quality Of Forages

Milk is an important protein food source for human beings,and utilization of human-inedible crop coproducts to produce high quality milk is an emerging global task.In this study,multi-omics system biology were employed to investigate the physiological and metabolic mechanisms for lactation performance in dairy cows receiving different quality of forages.Based on the results of lactation performance and metabolic phenotypes in dairy cows fed corn stover diet in comparison with those of alfalfa hay-fed cows,metabolomics was applied to analyze the metabolites of four biofluids including rumen fluids,blood,milk and urine,metagenomics method was used to approach the molecular mechanism in the metabolism of the rumen microbiome,and multi-omics methodologies were applied to compare and systematically analyse the key different metabolic function in the liver and mammary gland.Finally,dietary manipulation strategies were proposed by integrating the omics results with nutritional knowledge so that the lactation performance of the cows fed corn stover diet was successfully reached to that in alfalfa hay-fed cows,resulting in significant economic and environmental benefits.1.Lactation performance and metabolic phenotypes in dairy cows fed corn stover diet in comparison with those of alfalfa hay-fed cowsIn this part,the effects of feeding corn stover diet were studied on the dairy cow’s milk production,milk composition,rumen fermentation parameters and blood biochemistry traits in comparison with those of alfalfa hay-fed cows.Sixteen healthy multiparous Holstein dairy cows(milk yield = 29.4 ± 2.16 kg/d;day in milk=164 ±27.5 d)were divided into 2 groups.The diets were formulated to be isonitrogenous with a forage-to-concentrate ratio of 45:55(DM basis).The diets contained similar concentrate mixtures and 15%corn silage,with the following different forage sources(on a DM basis):(1)alfalfa-hay(AH),a diet containing 23%alfalfa hay and 7%Chinese wild rye hay;and(2)CS,inclusion of 30%corn stover.The DM intake of diet did not differ(P = 0.43)between the CS and AH diets.The milk yield,and milk efficiency in CS-fed cows were lower(P<0.01)than in AH-fed animals.The milk protein content(P = 0.04)and lactose content(P ＝ 0.02)were also lower in cows fed CS compared with those fed AH.The ruminal molar proportions of acetate and propionate were higher and lower(P<0.01),respectively,in CS-fed cows than in AH-fed animals,resulting in a significantly higher ratio of acetate to propionate in CS group(P<0.01).Blood glucose concentration in CS group was significantly lower(P＝0.03)than AH group.It is indicated that the reduce milk performance,lowered rumen propionate concentration,enhanced rumen acetate concentration,and lowered blood glucose concentration are associated with feeding of CS diet in comparison with AH diet.2.The physiological and metabolic mechanisms in which feeding of corn stover diet exerted the effect on cow’s milk performanceThe metabolomics,metagenomics,transcriptomics and the integrated association analysis were employed to study the key functions and metabolic pathways cross multi-biofluids and organs,in order to revealed the molecular mechanisms in which lactation performance was adversely affected in the cows receiving CS diet.2.1 The systematic metabolic mechanism based on four biofluid metabolomicsRumen fluid,serum,milk and urine samples were collected from 16 dairy cows before morning feeding on the 65-day of the experimental period.The gas chromatography-time of flight/mass spectrometry(GC-TOF/MS)were applied to identify the metabolites in the four biofluids.Totally,165,218,195 and 156 metabolites were identified in the rumen fluid,serum,milk and urine,respectively,with 29 mutual metabolites identified in all four biofluids.The mutual metabolites related pathways were glycine,serine and threonine metabolism,glycerolipid metabolism,phenylalanine metabolism and tyrosine metabolism pathways(P<0.05&Impact value>0.1).A total of 55,28,8 and 31 significantly different metabolites(VIP>1&P<0.05)were identified in the rumen fluid,serum,milk and urine,respectively,between the CS and AH diets.The key pathways were glycine,serine and threonine metabolism,tyrosine metabolism and phenylalanine metabolism.By the integrated analysis of the metabolites from 8 cows in AH group,creatine was identified as key metabolite to explain the biological variation amongst 4 biofluids(FDR<0.01,VIP =1.537).Five pathways,including gluconeogenesis,pyruvate metabolism,the tricarboxylic acid cycle,glycerolipid metabolism and aspartate metabolism,demonstrated most functional enrichment amongst 4 biofluids(Fold Enrichment>2,FDR<0.05).The above results indicated the main metabolic difference between 2 groups were amino acid metabolism,and the most enrichment pathways based on integrated analysis were correlated with carbohydrates metabolism.2.2 The mocular mechanism for the rumen metabolism based on combined analysis of metagenome-metabolomeTotal DNA was extracted from the rumen content sample in the above 16 dairy cows.Illumina Miseq was used to obtain 2 × 300 bp sequence data;and Metagenomics Rapid Annotation or Subsystems Technology(MG-RAST)analysis platform was applied to analyze the microbial diversity and function.The taxonomic assessment of rumen microbiota revealed 60 phyla,784 genera and 3560 microbial species including archaea,bactreia and eukaryota.Proteobacteria,Fibrobacteres and Lentisphaerae(P<0.05)bacteria phyla and the Bacteroides,Parabacteroides,Porphyromoas,Paludibacter,Fibrobacter,and Victivallis bacteria genera were significantly higher(P<0.05),whereas the relative abundances of Treponema,and Succinimonas genera were significantly lower in the rumen of CS fed cows(P<0.05)compared to the AF-fed cows.Among dominant bacteria species(relative abundance≥ 0.01%),T.maltophilum was only identified in the rumen of the AH group,while the relative abundance of T.saccharophilum and T.succinifaciens were significantly lower in CS group(P<0.05).For the function analysis,only carbohydrate metabolism showed significantly lower in the CS group(P = 0.032),including lactaldehyde reductase,glutamine synthetase type I,methylmalonyl-CoA decarboxylase,duccinate dehydrogenase,and alpha-xyloside ABC transporter(P<0.05).Integrated analysis between 10 key metagenomics elements and 17 significantly decreased microbial metabolites in CS group showed thatthe Fibrobacter had significantly negative correlation with 11 metabolites(R<-0.5,P<0.05).The relative abundance of the methylmalonyl-CoA decarboxylase gene had significant negative correlation with 3 metabolites(R<-0.5,P<0.05).Succinate dehydrogenase served as a core function and had a significantly positive correlation with 14 metabolites(R>0.5,P<0.05).The lower content of non fibrous carbohydrate(pectin content,fructose,fucose,etc.)in the CS caused the changed rumen microbe density and down-regluated carbohydrates-related functions,which reduced amount of 17 metabolites and succinic pathway activity,resulting in lower production of propionate.The higher acetate in CS group was correlated with the higher abundance of genera Fibrobacteres and up-regulated activity of methylmalonyl-CoA decarboxylase.2.3 The key metabolic functions in the liver based on metabolome-transcriptome analysisSix cows were selected from 8 cows in AH and CS groups for liver GC-TOF/MS metabolomics and Illumina Hiseq 2000 transcriptomics measurements.Totally,515 valid peaks including 270 metabolites were identified,with 28 metabolites that showed significant difference between two groups(VIP>1&P<0.05).Fifteen metabolites had lower relative abundance and 13 had significantly higher abundance in the CS.Ingenuity pathway analysis(IPA)revealed 11 pathways that were significantly different between two groups(P<0.01,|z-score|>2).The most two different function pathways were amino acid metabolism and gluconeogenesis(P<0.001,z-score<-2).The pathway topology view based on all the metabolites involved in the amino acid metabolism showed that the metabolism of glycine,serine,and threonine was the most difference and enrichment pathway(P = 0.029,Impact value = 0.597).Leucine,cystine and hippuric acid were 3 biomarkers in the liver that could explain 99.9%of the discriminations.Using RNA-seq,expression of 11,3 71 genes was detected in the liver,and 18 gene modules were obtained using gene co-expression network.The correlated analysis of biomarkers and gene modules showed that the lowered functions in CS group were gluconeogenesis,pyruvate metabolism,monosaccharide biosynthesis,glucose homeostasis and hexose biosynthesis.From the metabolome-transcriptome integrated analysis results,it is indicated that the key metabolic difference was the down-regulated enrichment and function of gluconeogenesis in CS group,with the decreased metabolites and key enzyme encoded genes,which may be attributed to the less propionate produced in rumen in the CS diet and hence less glucose supply in mammary gland.2.4 The key metabolic functions in the mammary gland based on metabolome-transcriptome analysisThe above cows were used to analyze the difference in the key metabolic function in the mammary gland.Totally,493 valid peaks consisting of 273 metabolites were identified in the mammary gland,with 9 metabolites showing significant difference between the AH and CS groups(VIP>1&P<0.05).Three metabolites had lower relative abundance and 6 had significantly higher abundance in CS group,compared to the AH group.Among the down-regulated function pathways in the CS group,the most different pathways included uptake of amino acids,uptake of L-amino acid,carbohydrate metabolism and oxidation of glucose-6-phosphate(P<0.001,z-score<-2).Combination of the biomarkers,maleic acid,pyrogallol,and succinic acid,represented 99.6%difference between 2 groups.A total of 10,174 genes left after filtering.The correlated analysis of biomarkers and gene modules showed that the down-regulated functions in CS group included calcium ion binding,acetylation,secretion,membranes and transmembranes.The insufficient supply of amino acid to mammary gland and down-regulated functions related to lactose synthesis resulted in the lowered lactose synthesie in mammary gland,and hence reduced the mik production.The decreased milk protein was mainly attributed to the lowered amino acid in blood stream and down-regulated functions of amino acid uptake,metabolism,secretion and membranes.3.The nutritionally manipulated strategies based on multi-omics resultsBased on omics findings and nutritiaonl knowledge as well as ecomomic and environmental considerations,a CS based manipulated diet(CSM)was formulated to opitimize the nutritional balance.Validated experiment was conducted in three groups using 39 healthy dairy cows with average mik production of 27 kg/d:CS(n=13),AH(n=13),and CSM(n=13).The CS and AH diets were same as in the previous trials.The CS diet was manipulated as below to formulate the CSM diet:corn stover reduced to 23%(DM basis),alfalfa hay(7%)and soluble carbohydrate(4.8%cane molasses)added,corn grain(3.9%)and wheat bran(5%)reduced,amino acids(41.4 g/d lysine hydrochloride +14.1 g/d methionine)added,and oilseed meals manipulated to make all the 3 diets isonitrogeneously.The net energy contents were 1.45,1.52 and 1.57 Mcal/kg DM for CS,AH and CSM diets,respectively.During the experimental period,milk production was measured continuously for a total of 1404 times(3 times/week/cow),with weekly measurement of milk compositions.Average milk production in CSM-fed cows was significantly higher(P<0.0001)than that in CS group,with no difference between AH and CSM groups(P = 0.435).Milk protein content was significantly higher(P = 0.008)in CSM group than CS group,with no difference between MSR and AH groups(P = 0.946).Similarly,lactose content in CSM group was significantly higher(P = 0.0006)in the MSR than in CS group,with no difference between MRS and AH(P = 0.308).The above results indicated the dietary manipulation strategies based on omics results and nutritional knowkedge can effectively upgrade the corn stover diet along with reduce feed cost,and environment benefits.In summary,the multi-omics across rumen,liver and mammary gland tissues revealed the comprehensive biological and metabolic mechanisms involved in dairy cow milk production when they were fed CS.The systematic comparison between cows fed high and low quality forage sources provided a fundamental understanding and practical experience for enhancing the bioavailability of human-inedible forages for milk production.