#METABOLOMICS WORKBENCH lizhipeng_20251117_204357 DATATRACK_ID:6712 STUDY_ID:ST004384 ANALYSIS_ID:AN007324 PROJECT_ID:PR002777 VERSION 1 CREATED_ON November 24, 2025, 5:14 am #PROJECT PR:PROJECT_TITLE Microbiome biogeography and ventral epithelial differences of rumen across PR:PROJECT_TITLE different regions in three different ruminant species PR:PROJECT_SUMMARY Ruminants thrive in diverse ecosystems by leveraging their rumen microbiome to PR:PROJECT_SUMMARY ferment fibrous plants. However, the spatial biogeography of the rumen PR:PROJECT_SUMMARY microbiome and genetic diversity of ventral rumen among the ruminants remain PR:PROJECT_SUMMARY unknown. Here, we present a multi-omics integrating region-resolved microbiome PR:PROJECT_SUMMARY across eleven ruminal sacs, metabolomics, and single-cell RNA sequencing, PR:PROJECT_SUMMARY ATAC-seq, and bulk RNA-seq of ventral epithelium of roe deer, sika deer and PR:PROJECT_SUMMARY sheep. Our results reveal species-specific rumen microbial compositions and PR:PROJECT_SUMMARY metabolic capacities that contribute to differences in short-chain fatty acids PR:PROJECT_SUMMARY and vitamin B production. We uncover functional divergence, genomic PR:PROJECT_SUMMARY specialization and metabolic changes across distinct ruminal sacs microbiome. PR:PROJECT_SUMMARY Single-cell profiling reveals changes of immune responses and structural PR:PROJECT_SUMMARY remodeling in ventral rumen. We demonstrate that vitamin B12 promotes epithelial PR:PROJECT_SUMMARY growth, and four novel genes that enhance stem cell differentiation. Our results PR:PROJECT_SUMMARY highlight variation in microbial ecology and epithelial architecture among three PR:PROJECT_SUMMARY ruminant species, offering insights to improve livestock productivity. PR:INSTITUTE Jilin Agricultural University PR:DEPARTMENT College of Animal Science and Technology PR:LABORATORY Ruminant Nutrition Laboratory PR:LAST_NAME Li PR:FIRST_NAME Zhi Peng PR:ADDRESS No. 2888 Xincheng Street PR:EMAIL zhplicaas@163.com PR:PHONE 18043213681 #STUDY ST:STUDY_TITLE Microbiome biogeography and ventral epithelial differences of rumen - vitamin ST:STUDY_TITLE profiling of rumen fluid from three ruminant species ST:STUDY_TYPE Targeted LC–MS/MS ST:STUDY_SUMMARY This study aims to quantify and compare vitamin concentrations in the rumen ST:STUDY_SUMMARY liquid of three ruminant species—roe deer, sika deer, and sheep—using a ST:STUDY_SUMMARY targeted LC–MS/MS metabolomics approach. Rumen liquid samples were collected ST:STUDY_SUMMARY immediately after euthanasia and processed for vitamin extraction and analysis ST:STUDY_SUMMARY on a Triple Quad 4500MD mass spectrometer. The resulting dataset provides ST:STUDY_SUMMARY species-specific vitamin profiles that can support studies on rumen physiology, ST:STUDY_SUMMARY nutritional adaptation, and metabolic differences among ruminants. ST:INSTITUTE Jilin Agricultural University ST:DEPARTMENT College of Animal Science and Technology ST:LABORATORY Ruminant Nutrition Laboratory ST:LAST_NAME Li ST:FIRST_NAME Zhi Peng ST:ADDRESS No. 2888 Xincheng Street ST:EMAIL zhplicaas@163.com ST:PHONE 18043213681 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Ovis aries, Capreolus pygargus, Cervus nippon SU:TAXONOMY_ID 9940, 48560, 9863 SU:AGE_OR_AGE_RANGE 4 years SU:GENDER Male SU:ANIMAL_HOUSING Each animal was housed in an individual pen (3 m × 3 m). SU:ANIMAL_FEED Roe deer, sika deer and sheep fed a total mixed ration consisting of alfalfa and SU:ANIMAL_FEED concentrate (45:55, dry matter basis) for two months were used in this study. SU:ANIMAL_WATER Free water #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS MD MLY01 Sample source:rumen liquid | Host:Cervus nippon RAW_FILE_NAME(Raw file name)=MLY01.mzML SUBJECT_SAMPLE_FACTORS MD MLY02 Sample source:rumen liquid | Host:Cervus nippon RAW_FILE_NAME(Raw file name)=MLY02.mzML SUBJECT_SAMPLE_FACTORS MD MLY03 Sample source:rumen liquid | Host:Cervus nippon RAW_FILE_NAME(Raw file name)=MLY03.mzML SUBJECT_SAMPLE_FACTORS MD MLY04 Sample source:rumen liquid | Host:Cervus nippon RAW_FILE_NAME(Raw file name)=MLY04.mzML SUBJECT_SAMPLE_FACTORS MD MLY05 Sample source:rumen liquid | Host:Cervus nippon RAW_FILE_NAME(Raw file name)=MLY05.mzML SUBJECT_SAMPLE_FACTORS PD PLY01 Sample source:rumen liquid | Host:Capreolus pygargus RAW_FILE_NAME(Raw file name)=PLY01.mzML SUBJECT_SAMPLE_FACTORS PD PLY02 Sample source:rumen liquid | Host:Capreolus pygargus RAW_FILE_NAME(Raw file name)=PLY02.mzML SUBJECT_SAMPLE_FACTORS PD PLY03 Sample source:rumen liquid | Host:Capreolus pygargus RAW_FILE_NAME(Raw file name)=PLY03.mzML SUBJECT_SAMPLE_FACTORS PD PLY04 Sample source:rumen liquid | Host:Capreolus pygargus RAW_FILE_NAME(Raw file name)=PLY04.mzML SUBJECT_SAMPLE_FACTORS PD PLY05 Sample source:rumen liquid | Host:Capreolus pygargus RAW_FILE_NAME(Raw file name)=PLY05.mzML SUBJECT_SAMPLE_FACTORS SD SLY01 Sample source:rumen liquid | Host:Ovis aries RAW_FILE_NAME(Raw file name)=SLY01.mzML SUBJECT_SAMPLE_FACTORS SD SLY02 Sample source:rumen liquid | Host:Ovis aries RAW_FILE_NAME(Raw file name)=SLY02.mzML SUBJECT_SAMPLE_FACTORS SD SLY03 Sample source:rumen liquid | Host:Ovis aries RAW_FILE_NAME(Raw file name)=SLY03.mzML SUBJECT_SAMPLE_FACTORS SD SLY04 Sample source:rumen liquid | Host:Ovis aries RAW_FILE_NAME(Raw file name)=SLY04.mzML SUBJECT_SAMPLE_FACTORS SD SLY05 Sample source:rumen liquid | Host:Ovis aries RAW_FILE_NAME(Raw file name)=SLY05.mzML #COLLECTION CO:COLLECTION_SUMMARY Rumen liquid samples were collected from three ruminant species—roe deer, sika CO:COLLECTION_SUMMARY deer, and sheep—immediately after euthanasia. Each animal was humanely CO:COLLECTION_SUMMARY euthanized 3 hours after morning feeding, followed by postmortem dissection. The CO:COLLECTION_SUMMARY rumen was opened through a midline abdominal incision, and rumen fluid was CO:COLLECTION_SUMMARY obtained by gently squeezing fresh digesta through four layers of cheesecloth to CO:COLLECTION_SUMMARY remove coarse particulate matter. The filtered rumen fluid was transferred into CO:COLLECTION_SUMMARY sterile tubes, kept on ice, and transported to the laboratory for subsequent CO:COLLECTION_SUMMARY vitamin extraction and LC–MS/MS analysis. CO:SAMPLE_TYPE rumen liquid CO:COLLECTION_METHOD Rumen liquid was obtained by filtering rumen digesta through four layers of CO:COLLECTION_METHOD cheesecloth. CO:COLLECTION_LOCATION Liquid:Rumen liquid CO:COLLECTION_FREQUENCY 15 CO:COLLECTION_DURATION 1 year CO:VOLUMEORAMOUNT_COLLECTED 50ml CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Animals received no experimental treatment and were maintained under identical TR:TREATMENT_SUMMARY feeding and housing conditions. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Rumen liquid samples (200 μL) were transferred into 1.5-mL microcentrifuge SP:SAMPLEPREP_SUMMARY tubes, and 20 μL of internal standard solution was added. The mixture was SP:SAMPLEPREP_SUMMARY vortexed at 2,000 rpm for 10 min and then incubated at room temperature for 5 SP:SAMPLEPREP_SUMMARY min. Protein precipitation was performed by adding 600 μL of precipitating SP:SAMPLEPREP_SUMMARY reagent, followed by mixing at 2,000 rpm for 10 min at room temperature. Samples SP:SAMPLEPREP_SUMMARY were centrifuged at 12,000 × g for 10 min, and 400 μL of the resulting SP:SAMPLEPREP_SUMMARY supernatant was transferred into a V-shaped 96-well plate. The plate was dried SP:SAMPLEPREP_SUMMARY under a gentle stream of nitrogen at 37°C. Dried residues were reconstituted in SP:SAMPLEPREP_SUMMARY 60 μL of reconstitution solvent, sealed with a plate film, vortexed at 2,000 SP:SAMPLEPREP_SUMMARY rpm for 5 min, and centrifuged at 2,811 × g for 5 min. Finally, 55 μL of the SP:SAMPLEPREP_SUMMARY clarified extract from each well was transferred into a fresh V-shaped 96-well SP:SAMPLEPREP_SUMMARY plate for LC–MS/MS analysis. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE Room temperature #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation of vitamins was performed using reversed-phase liquid CH:CHROMATOGRAPHY_SUMMARY chromatography on an LC system coupled to a SCIEX Triple Quad 4500MD mass CH:CHROMATOGRAPHY_SUMMARY spectrometer. Samples were injected onto the analytical column and eluted with a CH:CHROMATOGRAPHY_SUMMARY binary mobile phase under gradient conditions at a constant flow rate. The LC CH:CHROMATOGRAPHY_SUMMARY method was optimized to ensure efficient separation of water-soluble vitamins CH:CHROMATOGRAPHY_SUMMARY prior to MS/MS detection. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME AB Sciex Triple Quad 4500MD CH:COLUMN_NAME Waters XSelect HSS T3 XP (100 x 2.1mm, 2.5um) CH:SOLVENT_A 100% Water; 0.1% formic acid CH:SOLVENT_B 100% Methanol; 0.1% formic acid CH:FLOW_GRADIENT 0.00–1.00 min, 99% A / 1% B at 0.35 mL/min; 1.00–1.30 min, linear gradient CH:FLOW_GRADIENT from 99% A / 1% B to 60% A / 40% B at 0.35 mL/min; 1.30–2.80 min, 60% A / 40% CH:FLOW_GRADIENT B at 0.35 mL/min; 2.80–3.80 min, linear gradient from 60% A / 40% B to 1% A / CH:FLOW_GRADIENT 99% B at 0.35 mL/min; 3.80–4.30 min, 1% A / 99% B at 0.35 mL/min; 4.30–4.35 CH:FLOW_GRADIENT min, linear gradient from 1% A / 99% B back to 99% A / 1% B at 0.35 mL/min; CH:FLOW_GRADIENT 4.35–5.20 min, 99% A / 1% B at 0.35 mL/min (re-equilibration). CH:FLOW_RATE 0.35mL/min CH:COLUMN_TEMPERATURE 35℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex Triple Quad 4500MD MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS/MS data were acquired on a SCIEX Triple Quad™ 4500MD mass spectrometer in MS:MS_COMMENTS multiple reaction monitoring (MRM) mode. In MRM, the first quadrupole selects MS:MS_COMMENTS the precursor ion of each target analyte, which is then fragmented in the MS:MS_COMMENTS collision cell, and a characteristic product ion is selected by the third MS:MS_COMMENTS quadrupole for highly selective and reproducible quantification. Instrument MS:MS_COMMENTS parameters and MRM transitions were optimized based on authentic standards and MS:MS_COMMENTS the MWDB (Metware Database). Raw LC–MS/MS data were processed using the MWDB MS:MS_COMMENTS (Metware Database) and vendor software for peak detection and integration. MS:MS_COMMENTS Chromatographic peaks corresponding to target vitamins were integrated across MS:MS_COMMENTS all samples, and quantitative analysis was performed using internal and/or MS:MS_COMMENTS external standard calibration. Peak area (or peak area ratios of analyte to MS:MS_COMMENTS internal standard) was used to calculate the concentrations of each vitamin in MS:MS_COMMENTS rumen fluid. Qualitative assignment of vitamins was based on the MWDB (Metware MS:MS_COMMENTS Database) constructed from authentic standards, using matching of MRM MS:MS_COMMENTS transitions, retention time, and ion response patterns. Only compounds that MS:MS_COMMENTS matched the standard library criteria were retained as confidently identified MS:MS_COMMENTS targets. No untargeted feature discovery workflow was applied. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ml MS_METABOLITE_DATA_START Samples PLY01 PLY02 PLY03 PLY04 PLY05 MLY01 MLY02 MLY03 MLY04 MLY05 SLY01 SLY02 SLY03 SLY04 SLY05 Factors Sample source:rumen liquid | Host:Capreolus pygargus Sample source:rumen liquid | Host:Capreolus pygargus Sample source:rumen liquid | Host:Capreolus pygargus Sample source:rumen liquid | Host:Capreolus pygargus Sample source:rumen liquid | Host:Capreolus pygargus Sample source:rumen liquid | Host:Cervus nippon Sample source:rumen liquid | Host:Cervus nippon Sample source:rumen liquid | Host:Cervus nippon Sample source:rumen liquid | Host:Cervus nippon Sample source:rumen liquid | Host:Cervus nippon Sample source:rumen liquid | Host:Ovis aries Sample source:rumen liquid | Host:Ovis aries Sample source:rumen liquid | Host:Ovis aries Sample source:rumen liquid | Host:Ovis aries Sample source:rumen liquid | Host:Ovis aries Thiamine 115.854 105.02 107.019 105.985 106.283 167.046 136.916 142.336 120.725 77.441 203.769 209.817 202.068 200.254 196.317 Riboflavin 145.86 84.382 106.592 91.318 93.932 253.578 211.161 209.219 195.535 154.533 156.118 158.777 149.112 147.054 146.147 Nicotinamide 91.804 33.804 80.289 23.308 41.81 8.331 9.03 6.396 6.707 68.239 10.756 8.09 5.993 13.568 7.462 Pantothenic acid 362.45 278.509 289.64 297.254 270.926 751.633 549.493 592.514 575.273 424.723 1363.166 1253.961 1277.439 1202.957 1119.371 Pyridoxic acid 185.366 128.633 135.207 135.063 138.471 113.063 118.87 136.421 114.036 117.723 85.854 71.554 83.117 81.161 73.218 Biotin 5.891 4.598 5.151 4.904 4.696 7.605 2.291 2.934 2.064 1.631 18.636 17.158 16.915 17.097 15.713 5-Methyltetrahydrofolate (5-MTHF) 4.167 4.892 3.461 4.009 3.852 0.345 4.181 5.817 4.426 3.734 4.267 3.607 2.746 5.421 4.738 Methylmalonic acid 53.755 27.077 33.938 27.362 21.873 75.803 23.745 18.708 29.708 53.681 177.698 29.875 127.791 23.442 93.187 Ascorbic acid 0.213 0.351 0.238 0.239 0.19 0.397 0.549 0.474 0.539 0.471 0.932 0.856 0.828 0.576 0.923 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID Thiamine 1130 C00378 Riboflavin 493570 C00255 Nicotinamide 936 C00153 Pantothenic acid 6613 C00864 Pyridoxic acid 6723 C00847 Biotin 171548 C00120 5-Methyltetrahydrofolate (5-MTHF) 135398561 C00440 Methylmalonic acid 487 C02170 Ascorbic acid 54670067 C00072 METABOLITES_END #END