The intrafollicular milieu influences mammalian fertility by providing the microenvironment for oocyte growth and maturation. A number of studies have linked abundance of follicular fluid metabolites to oocyte developmental competence and pregnancy outcome. Few studies have interrogated the preovulatory follicular fluid metabolome in cattle. This dataset includes preovulatory follicular fluid metabolome profiles from non-lactating Jersey cows. Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry was performed on preovulatory follicular fluid samples and Xcalibur (RAW) files were converted to an open-source mzML format (msconvert software; ProteoWizard package). The converted files were processed using the Metabolomic Analysis and Visualization Engine (MAVEN; mzroll software, Princeton University) to complete an untargeted analysis of the liquid chromatography mass spectrometry data. The pre-processed peak data tables generated by MAVEN are provided in this dataset. 

Ultra-High-Performance Liquid Chromatography - High Resolution Mass Spectrometry (UHPLC-HRMS) was performed on follicular fluid samples at the Biological and Small Molecule Mass Spectrometry Core (RRID: SCR_021368) at the University of Tennessee, Knoxville. Sixty μL aliquots of follicular fluid samples (n=29) were thawed at room temperature, and metabolites within each sample were extracted using a 40:40:20 methanol/acetonitrile/water solution with 0.1 M formic acid following a previously reported procedure. An extraction solvent was added to the samples which underwent agitation and vortexing before samples were then chilled at -20°C for 20 min. Once samples were properly chilled, the tubes were centrifuged to form pellets and remove debris from the sample. A second set of 2 mL microcentrifuge tubes were used to collect the supernatant from the first set of tubes. The extraction solvent was again dispensed into the first set of tubes, which contained the pellet, and the mixture was pipetted to resuspend the pellet. The tubes were then re-submitted to agitation and vortexing, chilling, and centrifugation as described above. The supernatant from the tube was added to the second set of tubes that contained the previous supernatant. The first tube set with the final pellet was then discarded. The second set of tubes containing the supernatant underwent drying using nitrogen gas. Once dried, tubes were filled with MilliQ water to resuspend samples and moved to new autosampler vials. Metabolomic samples were separated using columns (Synergi Hydro RP, 2.5 μm, 100 mm x 2.0 mm column; Phenomenex, Torrance, CA, USA) which were maintained at 25°C. Solvents to elute metabolites during the mobile phase were (i) 97:3 methanol/water with 15 mM acetic acid and 11 mM tributylamine and (ii) 100% methanol. At a flow rate of 0.2 μL/min, the solvent gradient was (i) 100% and (ii) 0% from 0 to 5 min, (i) 80% and (ii) 20% from 5 to 13 min, (i) 45% and (ii) 55% from 13 to 15.5 min, (i) 5% and (ii) 95% from 15.5 to 19 min, and (i) 100% and (ii) 0% from 19 to 25 min. An Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with an electrospray ionization probe attached was used, operating in negative mode with a scan range between 72–1000 m/z, a resolution of 140,000, and an acquisition gain control of 3x10⁶.

The mass spectrometry generated Xcalibur (RAW) files were converted to an open-source mzML format (msconvert software; ProteoWizard package). The converted files were processed using the Metabolomic Analysis and Visualization Engine (MAVEN; mzroll software, Princeton University) to complete an untargeted analysis of the liquid chromatography mass spectrometry data. MAVEN identifies metabolites using a variety of factors including peak shape, retention time, and signal-to-noise-ratio. The program then generated pre-processed peak data tables which were used for statistical analysis.

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