| The objective of this study was to evaluate how the metabolic pathway and rumen bacterial community structure of carbohydrates and milk composition are affected by ratios of dietary neutral detergent fiber (NDF): starch, examine the ruminal metabolic response of dairy cows to heat stress (HS) using a combination of LC-MS, GC-MS and 1H NMR and metabolic changes in the mammary tissue and liver associated with milk synthesis under HS.1. Eight primiparous dairy cows were assigned to 4 total mixed rations (TMR)with NDF to starch ratios of 0.86,1.18, 1.63 and 2.34 in a replicated 4×4 Latin square design. The duration of each period was 21 d including a 14 d for adaptation and 7 d for sampling. At the beginning of the study, milk production and days in milk were 35.1 ± 0.5 kg/day and 146 ± 21.6 days, respectively. The NDF to starch ratios were achieved by altering the proportions of forage (corn silage and oat hay) and concentrate (corn), with the same composition and inclusion proportion of other dietary ingredients in the TMR. Plasma, urine, milk, rumen fluid, and feces were collected from each cow once daily on the last three days of each period. The metabolic profiles were analyzed using HPLC Q-TOF MS, and the identified metabolites were confirmed by both retention time and mass spectra, and total DNA was extracted from each rumen fluid sample was detected by the V4 hypervariable region of 16S rRNA gene amplified and subjected to paired-end Illumina sequencing.The results showed that, (1)31 different metabolic profiles in feces, 5 in rumen fluid,3 in plasma, and 6 in urine were detected, including leukotriene increased (P< 0.05)in rumen fluid, fatty acids increased (P< 0.05) in plasma, vitamin D3 increased (P<0.05) in plasma, glucuronide decreased (P< 0.05) in urine, isovalerate decreased and then increased (P< 0.05) in urine and L-isoleucyl-L-proline increased (P< 0.05) in feces. All the different metabolic profiles were related to several metabolic pathways,including fatty acid biosynthesis, the metabolism of vitamin D3, amino acids, protein and alkaloids, bile secretion, and inflammatory mediator regulation. Taken together,different NDF: starch ratio in diets can affect metabolism inside and outside of the rumen of dairy cows, and some of these metabolic responses may be attributable to the observed effects of NDF: starch ratio on animal productivity and health. (2) As dietary NDF: starch ratio increased from 0.86 to 2.34, the relative abundance of the phylum Firmicutes linearly increased, while that of Bacteroidetes, Tenericutes,Lentisphaerae, and candidate division SR1 linearly declined in the rumen fluid samples (P<0.05). Among the classified bacterial genera detected, as the NDF: starch ratio increased, the relative abundance of the genera Ruminococcus, Coprococcus,Syntrophococcus increased linearly, while Anaeroplasma and the RC9 gut group declined in relative abundance. The richness of the rumen bacterial community, as indicated by Chao 1 and ACE estimates, declined linearly (P<0.05) with the increased dietary NDF: starch ratio. The beta diversity analyses indicated that the rumen bacteria communities were clustered into two distinct groups(P<0.05): corresponding to NDF: starch ratio of 0.86 and 1.18 vs. 1.63 and 2.34. Taken together, increase in NDF: starch ratio decreased rumen bacterial diversity and favored fibrolytic bacteria.2. Four multiparous Holstein dairy cows (101 ± 10 DIM; 574± 36 kg of BW,38±2 kg of milk/d) were randomly assigned to four environment chambers with a crossover design. Cows were either subjected to HS [HS: 36? with light and 32?without light; THI = 87.2 and 81.8] or kept under thermal neutral conditions [TN:20?; THI = 65.5] for 9 d for adaptation and then for another 9 d of pair-feeding to eliminate confounding effects of dissimilar feed intake. There was a 30 d washout period between periods. Rumen fluid was collected at 0500 (before feeding) and 1000(after feeding) on d 9. Mammary tissue and liver were collected on the day before the end of the trial. The ruminal metabolic response of dairy cows to HS was detected by a combination of LC-MS, GC-MS and 1H NMR and the changes of protein abundance in the mammary tissue and liver between heat-stressed cows with ad libitum intake and pair-fed thermal neutral cows were investigated using the iTRAQ proteomic approach. The results showed that, (1) The OPLS-DA results showed that all the metabolic profiles in the rumen fluid changed were separated into two groups in response to HS and TN. Based on LC-MS, GC-MS and 1H NMR results, the metabolites of glucose, galactose, glycerol, butyrate, glucosamine and heptacosane,hentriacontane were increased by HS (VIP > 1, P < 0.05, Fold change > 0), while fatty acids and amino acids were decreased (VIP > 1, P< 0.05, Fold change < 0).Most of the rumen metabolites affected by HS were related to several metabolic pathways, including urine cycle pathway, metabolism of amino acids, tryptophan metabolism and citrate cycle, which likely affect the precursor supply for milk component synthesis. These findings indicate that the use of multiple metabolomics platforms permits a far more detailed understanding of HS-induced metabolic changes in rumen digestion: potential mechanism by which HS decreases milk production and component synthesis. (2) Most of the differentially expressed proteins from mammary tissue and liver between heat-stressed and pair-fed cows were involved in GO category of protein metabolic process. Pathway analysis indicated that differentially expressed proteins in the mammary tissue were related to pyruvate,glyoxylate and dicarboxylate metabolism pathways, while those in the liver participated in oxidative phosphorylation and antigen processing and presentation pathways. Several heat shock proteins directly interact with each other and were considered as central "hubs" in the protein interaction network. These findings provide new insights into understanding the turnover of protein biosynthesis pathways within hepatic and mammary tissue that likely contribute to changes in milk composition from heat-stressed cows. |
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