#METABOLOMICS WORKBENCH lnvoker_20220702_082829 DATATRACK_ID:3327 STUDY_ID:ST002210 ANALYSIS_ID:AN003613 PROJECT_ID:PR001411 VERSION 1 CREATED_ON July 8, 2022, 11:35 am #PROJECT PR:PROJECT_TITLE Revealing the Social Biomarkers of Residual Feed Intake by Using 16s rRNA and PR:PROJECT_TITLE LC-MS/MS in Duroc Pig PR:PROJECT_SUMMARY Feed efficiency (FE) is a typical social affected trait. However, the mechanisms PR:PROJECT_SUMMARY involved are not fully elucidated. According to the rank of residual feed intake PR:PROJECT_SUMMARY (RFI)’s the social genetic effect (SGE), ten high and low pigs were selected, PR:PROJECT_SUMMARY named LRI and HRI groups. The sampling of jejunal chyme after slaughter. 16S PR:PROJECT_SUMMARY rRNA and LC-MS/MS were conducted to investigate the relationship between the gut PR:PROJECT_SUMMARY microbiome or metabolites and the SGE of RFI. The results showed significant PR:PROJECT_SUMMARY differences between HRI and LRI groups. Compared with the HRI group, PR:PROJECT_SUMMARY Escherichia, Eubacterium, and Gemmiger were enriched in the LRI group (P < PR:PROJECT_SUMMARY 0.01), whereas the abundance of Fusobacterium, Eubacterium, and Desulfovibrio in PR:PROJECT_SUMMARY the HRI group were significantly higher than that in the LRI group (P < 0.01). PR:PROJECT_SUMMARY In the metabolome, we found that Glycine, L-lysine, and L-tryptophan were PR:PROJECT_SUMMARY positively correlated with RFI’s SGE. KEGG pathway analysis revealed that most PR:PROJECT_SUMMARY differential metabolites were involved in amino acid metabolism. The Pearson PR:PROJECT_SUMMARY correlation analysis of the candidate social biomarkers was carried out. Amino PR:PROJECT_SUMMARY acid metabolites were discovered to have significant correlations with PR:PROJECT_SUMMARY Escherichia and Fusobacterium. Therefore, Escherichia and Fusobacterium may PR:PROJECT_SUMMARY influence the SGE of RFI through amino acid metabolism, thereby affecting feed PR:PROJECT_SUMMARY efficiency. PR:INSTITUTE Sichuan Agricultural University PR:LAST_NAME Wang PR:FIRST_NAME Shujie PR:ADDRESS Huimin Road, Chengdu, Sichuan, China PR:EMAIL 670186296@qq.com PR:PHONE 15680993607 #STUDY ST:STUDY_TITLE Revealing the Social Biomarkers of Residual Feed Intake by Using 16s rRNA and ST:STUDY_TITLE LC-MS/MS in Duroc Pig ST:STUDY_SUMMARY Feed efficiency (FE) is a typical social affected trait. However, the mechanisms ST:STUDY_SUMMARY involved are not fully elucidated. According to the rank of residual feed intake ST:STUDY_SUMMARY (RFI)’s the social genetic effect (SGE), ten high and low pigs were selected, ST:STUDY_SUMMARY named LRI and HRI groups. The sampling of jejunal chyme after slaughter. 16S ST:STUDY_SUMMARY rRNA and LC-MS/MS were conducted to investigate the relationship between the gut ST:STUDY_SUMMARY microbiome or metabolites and the SGE of RFI. The results showed significant ST:STUDY_SUMMARY differences between HRI and LRI groups. Compared with the HRI group, ST:STUDY_SUMMARY Escherichia, Eubacterium, and Gemmiger were enriched in the LRI group (P < ST:STUDY_SUMMARY 0.01), whereas the abundance of Fusobacterium, Eubacterium, and Desulfovibrio in ST:STUDY_SUMMARY the HRI group were significantly higher than that in the LRI group (P < 0.01). ST:STUDY_SUMMARY In the metabolome, we found that Glycine, L-lysine, and L-tryptophan were ST:STUDY_SUMMARY positively correlated with RFI’s SGE. KEGG pathway analysis revealed that most ST:STUDY_SUMMARY differential metabolites were involved in amino acid metabolism. The Pearson ST:STUDY_SUMMARY correlation analysis of the candidate social biomarkers was carried out. Amino ST:STUDY_SUMMARY acid metabolites were discovered to have significant correlations with ST:STUDY_SUMMARY Escherichia and Fusobacterium. Therefore, Escherichia and Fusobacterium may ST:STUDY_SUMMARY influence the SGE of RFI through amino acid metabolism, thereby affecting feed ST:STUDY_SUMMARY efficiency. ST:INSTITUTE Sichuan Agricultural University ST:DEPARTMENT animal science and technology ST:LABORATORY Guoqing Tang Group ST:LAST_NAME Wang ST:FIRST_NAME Shujie ST:ADDRESS Huimin Road, Chengdu, Sichuan, China ST:EMAIL 670186296@qq.com ST:PHONE 15680993607 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Sus scrofa SU:TAXONOMY_ID 9823 SU:AGE_OR_AGE_RANGE 174-196day SU:WEIGHT_OR_WEIGHT_RANGE 110-120kg SU:GENDER Female #FACTORS #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 LRI LRI1 Factor:high SGE of RFI RAW_FILE_NAME=LRI1_POS.raw LRI1_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI2 Factor:high SGE of RFI RAW_FILE_NAME=LRI2_POS.raw LRI2_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI3 Factor:high SGE of RFI RAW_FILE_NAME=LRI3_POS.raw LRI3_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI4 Factor:high SGE of RFI RAW_FILE_NAME=LRI4_POS.raw LRI4_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI5 Factor:high SGE of RFI RAW_FILE_NAME=LRI5_POS.raw LRI5_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI6 Factor:high SGE of RFI RAW_FILE_NAME=LRI6_POS.raw LRI6_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI7 Factor:high SGE of RFI RAW_FILE_NAME=LRI7_POS.raw LRI7_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI8 Factor:high SGE of RFI RAW_FILE_NAME=LRI8_POS.raw LRI8_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI9 Factor:high SGE of RFI RAW_FILE_NAME=LRI9_POS.raw LRI9_NEG.raw SUBJECT_SAMPLE_FACTORS LRI LRI10 Factor:high SGE of RFI RAW_FILE_NAME=LRI10_POS.raw LRI10_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI1 Factor:low SGE of RFI RAW_FILE_NAME=HRI1_POS.raw HRI1_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI2 Factor:low SGE of RFI RAW_FILE_NAME=HRI2_POS.raw HRI2_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI3 Factor:low SGE of RFI RAW_FILE_NAME=HRI3_POS.raw HRI3_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI4 Factor:low SGE of RFI RAW_FILE_NAME=HRI4_POS.raw HRI4_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI5 Factor:low SGE of RFI RAW_FILE_NAME=HRI5_POS.raw HRI5_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI6 Factor:low SGE of RFI RAW_FILE_NAME=HRI6_POS.raw HRI6_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI7 Factor:low SGE of RFI RAW_FILE_NAME=HRI7_POS.raw HRI7_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI8 Factor:low SGE of RFI RAW_FILE_NAME=HRI8_POS.raw HRI8_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI9 Factor:low SGE of RFI RAW_FILE_NAME=HRI9_POS.raw HRI9_NEG.raw SUBJECT_SAMPLE_FACTORS HRI HRI10 Factor:low SGE of RFI RAW_FILE_NAME=HRI10_POS.raw HRI10_NEG.raw #COLLECTION CO:COLLECTION_SUMMARY After ranking the RFI values of 294 pigs, the model equation was used to CO:COLLECTION_SUMMARY calculate RFI’s SGE. The top 10 highest RFI’s SGEs and 10 lowest RFI’s CO:COLLECTION_SUMMARY SGEs were selected as the LRI and HRI groups, respectively. The jejunal contents CO:COLLECTION_SUMMARY of 20 pigs were collected after being humanely slaughtered. Stores samples in CO:COLLECTION_SUMMARY liquid nitrogen immediately. Then, the samples were stored at − 80 °C CO:COLLECTION_SUMMARY refrigerators. CO:SAMPLE_TYPE Jejunum #TREATMENT TR:TREATMENT_SUMMARY After ranking the RFI values of 294 pigs, the model equation was used to TR:TREATMENT_SUMMARY calculate RFI’s SGE. The top 10 highest RFI’s SGEs and 10 lowest RFI’s TR:TREATMENT_SUMMARY SGEs were selected as the LRI and HRI groups, respectively. The jejunal contents TR:TREATMENT_SUMMARY of 20 pigs were collected after being humanely slaughtered. Stores samples in TR:TREATMENT_SUMMARY liquid nitrogen immediately. Then, the samples were stored at − 80 °C TR:TREATMENT_SUMMARY refrigerators. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Weigh a 25 mg sample of jejunal content and add 400μl of extract (methanol: SP:SAMPLEPREP_SUMMARY water = 4:1). Then, using the high-throughput tissue grinder, pulverize at -20 SP:SAMPLEPREP_SUMMARY °C (60 Hz). After 30 minutes of vertexing at 40 kHz for 30 minutes at 5 °C for SP:SAMPLEPREP_SUMMARY mixing and sonication, put the extracted samples at -20 °C for 30 minutes. SP:SAMPLEPREP_SUMMARY After centrifuging the solution at 13,000 g for 15 minutes (4 °C), the SP:SAMPLEPREP_SUMMARY supernatant was collected and fed into the LC-MS/MS apparatus for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Waters 2D UPLC CH:COLUMN_NAME Waters Acquity BEH C8 (100 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Ion trap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) in the MS:MS_COMMENTS positive mode, and in the negative mode, the mobile phase consisted of 10 mM MS:MS_COMMENTS ammonium formate (A) and acetonitrile (B). The gradient conditions were as MS:MS_COMMENTS follows: 0-1 min, 2% B; 1-9 min, 2%-98% B; 9-12 min, 98% B; 12-12.1 min, 98% B MS:MS_COMMENTS to 2% B; and 12.1-15min, 2% B. The flow rate was 0.35 mL/min and the injection MS:MS_COMMENTS volume was 5 μL. The mass spectrometric settings for positive/negative MS:MS_COMMENTS ionization modes were as follows: spray voltage, 3.8/−3.2 kV; sheath gas flow MS:MS_COMMENTS rate, 40 arbitrary units (arb); aux gas flow rate, 10 arb; aux gas heater MS:MS_COMMENTS temperature, 350 °C; capillary temperature, 320 °C. The full scan range was MS:MS_COMMENTS 70–1050 m/z with a resolution of 70000, and the automatic gain control (AGC) MS:MS_COMMENTS target for MS acquisitions was set to 3e6 with a maximum ion injection time of MS:MS_COMMENTS 100 ms. Top 3 precursors were selected for subsequent MSMS fragmentation with a MS:MS_COMMENTS maximum ion injection time of 50 ms and resolution of 17500, the AGC was 1e5. MS:MS_COMMENTS The stepped normalized collision energy was set to 20, 40 and 60 eV. MS:MS_RESULTS_FILE ST002210_AN003613_Results.txt UNITS:peak area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END