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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14063/m_MTBLS14063_LC-MS_alternating_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14063/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14063/a_MTBLS14063_LC-MS_alternating_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14063/s_MTBLS14063.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14063Targeted Bile Acid Analysis A volume of 100 μL of sample was accurately transferred and mixed with 50 μL of internal standard working solution 2 (200 ng/mL), followed by the addition of 350 μL of extraction solvent (methanol). The mixture was vortexed for 30 s, subjected to low-temperature ultrasonication for 30 min (5 °C, 40 kHz), and then left at −20℃ for 30 min. After centrifugation at 13,000 rpm for 15 min at 4℃, the supernatant was collected and evaporated to dryness under nitroge  arrow_drop_downmoreMetaboLightsPublicLiquid Chromatography MS - alternating - reverse-phaseThis experiment employed LC-ESI-MS/MS (UHPLC-Qtrap) for the qualitative and quantitative detection of target compounds in the samples. The specific parameters are as follows:Chromatographic conditions: ExionLC AD system, Waters BEH C18 (150*2.1 mm, 1.7 μm) liquid chromatography column, column temperature 50°C, injection volume 5 μL. Mobile phase A (0.1% formic acid in water), mobile phase B (0.1% formic acid in acetonitrile).Multi-omics and intervention analyses reveal a hindgut microbiota–secondary bile acid–mammary axis linked to milk yield in dairy cows.jilin universityjiaxin wangSerummass spectrometry assayAccurately transfer 100 μL of the sample, add 50 μL of internal standard working solution 2 (200 ng/mL), then add 350 μL of extraction solvent (methanol). Vortex for 30 seconds, perform low-temperature ultrasonication for 30 minutes (5°C, 40 KHz), and let stand at -20°C for 30 minutes. Centrifuge at 4°C and 13,000 rcf for 15 minutes, collect the supernatant, and evaporate it to dryness under nitrogen. Reconstitute with 100 μL of 50% acetonitrile in water, vortex for 30 seconds, perform low-temperature ultrasonication for 10 minutes (5°C, 40 KHz), and centrifuge at 4°C and 13,000 rcf for 15 minutes. Collect the supernatant for instrument analysis.Mus musculushttps://www.ebi.ac.uk/metabolights/MTBLS14063jiaxin wang. Jilin university. 1252978953@qq.com.shoupeng fu. jilin university. fushoupeng@jlu.edu.cn.Each ion fragment was automatically identified and integrated using default parameters in the AB Sciex quantification software OS, assisted by manual inspection. The ratio of the peak area of the analyte to the peak area of the internal standard was plotted as the ordinate, and the concentration of the analyte was plotted as the abscissa. sample This concentration calculation: the ratio of the peak area of the sample analyte to the peak area of the internal standard was substitute  arrow_drop_downmoreTreatment1252978953@qq.comBriefly, fresh feces from each group were pooled and homogenized in sterile saline (50 mg feces/mL).    In addition, a total of 12 fecal samples (0.5 g/sample) were mixed and aliquoted according to the experimental design.    These samples were centrifuged at 100 × g for 2 min at 4℃, and the supernatants were collected.    Glycerol was added to a final concentration of 10%, and the samples were frozen at −80℃.    During the transplantation, pregnant mice were treated with antibiotics by oral gavage for 5 days to eliminate the commensal microbiota.    The antibiotic cocktail included 200 mg/kg ampicillin, metronidazole (Sigma-Aldrich, USA), and neomycin (Sigma-Aldrich, USA), and 100 mg/kg vancomycin (Sigma-Aldrich, USA).    After one day of antibiotic withdrawal, mice were gavaged with 300 μL of fecal supernatant daily for 5 consecutive days, and then once every two days with the prepared microbiota suspension until the end of the experiment, and collected serum.Metabolomicsmilk productionMus musculusdeoxycholic acidmammary glandtargeted metabolite profilingmilk productionMus musculusdeoxycholic acidmammary glandtargeted metabolite profilingAB SCIEX QTRAP 6500+, detected in negative ion mode, with Curtain Gas (CUR) at 35, Collision Gas (CAD) set to Medium, IonSpray Voltage (IS) at -4500, Temperature (TEM) at 550, Ion Source Gas 1 (GS1) at 50, and Ion Source Gas 2 (GS2) at 50.apocholic acid23-Nordeoxycholic acidChenodeoxycholic acid-3-¦Â-D-glucuronidedehydrolithocholic acidUrsocholic acidTaurohyodeoxycholic acidTauroursodeoxycholic acidCholic acid12-ketolithocholic acidhyocholic acidDehydrocholic acidGlycocholic acidLithocholic acidTauro-alpha-muricholic acidUrsodeoxycholic acidOmega-Murichoclic acid3¦Â-Cholic acidAllocholic Acid7,12-Diketolithocholic acidTaurocholic acidGlycoursodeoxycholic acidChenodeoxycholic Acid 24-Acyl-¦Â-D-glucuronide7-ketoLithocholic acidnorcholic acidGlycochenodeoxycholic acidIsodeoxycholic acidTaurodeoxycholate acid7-ketodeoxycholic acidAlpha-Muricholic acidTauro-omega-muricholic acidtaurolithocholic acidDeoxycholic acidHyodeoxycholic acid12-ketochenodeoxycholicacidTaurohyocholic acidGlycohyocholic acidisolithocholic acidmurideoxycholic acidGlycolithocholic acidGlycodeoxycholic acidBeta-Muricholic acidChenodeoxycholic acid3-Dehydrocholic acidTaurochenodeoxycholic acidTauro-beta-muricholic acidLithocholic acid 3-sulfate3¦Â-Ursodeoxycholic acidfalseMulti-omics and intervention analyses reveal a hindgut microbiota–secondary bile acid–mammary axis linked to milk yield in dairy cows_Mus musculus_SerumBackgroundIn large scale dairy farming systems, maintaining stable milk yield post-lactation peak is crucial for dairy farms’ economic benefits. While sufficient evidence shows the microbiota profoundly impacts host production efficiency, the specific mechanisms by which hindgut microbiota support mid lactation milk performance via host-microbe interactions remain unclear. This study focused on the hindgut microbial ecosystem. By analyzing hindgut microbial composition and metabolic differences between high and low yield cows during mid-lactation, and conducting metabolite intervention, we clarified the biological mechanism by which hindgut microbiota regulates milk production through host interactions.ResultsShotgun metagenomics, untargeted metabolomics, and targeted metabolomics of high versus normal-yield cows revealed that high-yield animals harbored distinct hindgut microbial communities enriched in bile salt hydrolase–positive taxa, and elevated levels of deoxycholic acid (DCA). Fecal microbiota transplantation from high-yield donors into antibiotic-treated recipient mice increased their milk production, accompanied by a shift in circulating bile acid profiles towards higher DCA. Oral administration of DCA to antibiotic-treated mice partially recapitulated these mammary and lactation phenotypes, supporting a causal contribution of bile acids downstream of the microbiota. In lactating cows, supplementation with a rumen-protected DCA formulation enhanced cumulative energy-corrected milk yield and tended to increase daily milk yield. DCA can directly upregulate the expression of genes related to the cell cycle progression in dairy cow mammary glands, thereby promoting and maintaining the activity of mammary epithelial cells in mid-lactation cows to support milk production.ConclusionsOur multi-layered evidence demonstrates that hindgut microbiota-derived secondary bile acids, particularly DCA, act along a gut–mammary axis to enhance mammary epithelial function and lactation performance in dairy cows. These findings broaden the conceptual framework of microbiota–epithelial crosstalk and suggest microbial bile acid metabolism as a tractable target to improve lactation efficiency in ruminants.2026-04-152026-03-17MTBLS14063C01921C05466C05122C05465C17737C07880C04483C16868C15375C05464C15557C17647C17726C15515C02592C05463C03990C00695C02528C17649C17658C17662C13154C17644C17661