MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13639/m_MTBLS13639_GC-MS_positive__metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13639/s_MTBLS13639.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13639/a_MTBLS13639_GC-MS_positive__metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13639/i_Investigation.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13639<p>Compound identification was performed by matching the acquired mass spectra with the NIST 20 library and retention index data (https://webbook.nist.gov/chemistry/) using MS-Search software version 3.0 with the NIST 20 database (Wallace and Moorthy, 2023). Metabolites were identified based on their retention times and by comparing one target ion and two qualifier ions with those of authentic standards and spectra from the NIST library. The identified compounds were further categorized into chemical classes using the Human Metabolome Database (HMDB) version 5.0 (https://www.hmdb.ca).</p>MetaboLightsPublicGas Chromatography MS - positive<p>The metabolites of Bali bull spermatozoa were analyzed using a gas chromatography–mass spectrometry (GC–MS) system (GCMS-QP2010, Shimadzu Corporation, Japan) equipped with an Rtx-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness; Agilent Technologies, USA). A 1 μL aliquot of each liquid sample was injected into the GC–MS. The injector, interface, and ion source temperatures were set to 230, 250, and 200°C, respectively. Helium (99.9% purity) was used as the carrier gas at a constant flow rate of 3 mL/min. The oven temperature program was as follows: initially maintained at 60°C for 2 min, then increased to 300°C at a rate of 15°C/min, and held for 20 min. The solvent-cut time was set to 5 min. </p>Integrative analysis of blood biochemistry and sperm metabolome profiles reveals metabolic determinants of cryotolerance in Bali bulls under standardized nutritional conditions.National Research and Innovation AgencyMuhamad Aldi NurdiansyahSpermatozoamass spectrometry<p>Semen samples used for metabolomic analysis were derived from archived frozen semen straws and corresponded to the same ejaculates for which post-thaw sperm quality records were available in the RAIC archives, thereby enabling the correlation of sperm metabolite profiles with phenotypic parameters without re-evaluation of semen traits in the present study. Straws were thawed at 37 °C for 30 s, and the contents were transferred to sterile 15 mL conical tubes. The extender and cryoprotectant were removed by centrifugation at 3,000 × g for 30 min at 4 °C, followed by two washes with phosphate-buffered saline (PBS, pH 7.4) to minimize the carry-over of non-cellular components. The washed sperm pellets were aliquoted into cryogenic vials, snap-frozen in liquid nitrogen, and stored at −80 °C until metabolomic analysis. The handling times and temperatures were standardized across bulls to minimize preanalytical variation. All ten fertile Bali bulls available at the RAIC and meeting the semen quality criteria were included in this exploratory analysis; therefore, no a priori sample-size calculation was performed. Randomisation was not applicable because all animals were maintained under identical husbandry conditions and underwent the same sampling procedures. Laboratory personnel performing semen processing and GC-MS analyses were aware of sample identity; however, quantification relied on automated instruments to minimise observer bias. Semen collection followed standard operating procedures of the RAIC using minimal restraint to ensure animal welfare and to reduce handling-related stress</p>Bos javanicus<p>Raw data were transformed using Microsoft Excel 365.</p>Agemuha371@brin.go.idhttps://www.ebi.ac.uk/metabolights/MTBLS13639<p>This study was conducted using Bali bulls (B. javanicus) maintained at the Regional Artificial Insemination Center (RAIC) Pucak, Maros, South Sulawesi, Indonesia. Ten healthy, fertile Bali bulls aged 5–10 years were included in the study. The animals were maintained under standard husbandry conditions according to the institutional management guidelines of the RAIC, with routine health examinations and balanced feeding programs. All bulls exhibited a minimum fresh semen motility of 70% based on secondary evaluation data from the center’s reproductive laboratory. </p>MetabolomicsSperm MotilityBali (Cattle)chebiuntargeted metabolitesBos javanicusSperm MotilityBali (Cattle)chebiuntargeted metabolitesBos javanicus<p>The mass spectrometer was operated in electron ionization (EI) mode at 70 eV, with a mass scan range of m/z 30–600. </p>EthanolamineSerineCalcitriol1-MonopalmitinGlycinePalmitic acidHexanoic acidDodecanoic acidCitric acidCholesterolketo-D-fructose2-Pyridinecarboxylic acidMyristic acidL-Lactic acidNonadecanoic acidStearic acidGlycerol monostearatePentanoic acidfalseIntegrative analysis of blood biochemistry and sperm metabolome profiles reveals metabolic determinants of cryotolerance in Bali bulls under standardized nutritional conditions<p>Enhancing the nation's beef supply and guaranteeing long-term food security requires increasing the effectiveness of artificial insemination (AI) in Bali cattle (Bos javanicus). Cryotolerance, or post-thaw variability in semen quality, limits AI results and probably reflects systemic and cellular metabolic variations. To determine the metabolic determinants of cryotolerance under standardized nutrition and supervision, this study combined blood biochemistry with the sperm metabolome. Ten healthy breeding bulls at the regional artificial insemination center (RAIC) were maintained on a uniform forage–concentrate diet. Blood biochemical parameters were measured using ethylenediaminetetraacetic acid (EDTA) plasma. For metabolomics, washed sperm pellets were prepared from frozen–thawed semen straws and profiled using untargeted gas chromatography-mass–mass spectrometry (GC–MS). Multivariate analyses (hierarchical clustering, K-means, and partial least squares–discriminant analysis (PLS-DA) with variable importance in projection (VIP) scores) summarized the global patterns. Spearman correlations were used to integrate blood indices, sperm metabolites, and post-thaw traits (motility, viability, plasma membrane integrity (%PMI), and morphological abnormalities). Eighteen intracellular metabolites were identified, predominated by fatty acyls.​ Unsupervised clustering and PLS-DA revealed clear inter-individual separation, with palmitic and stearic acids being among the most discriminant features (VIP ≥ 1.0). Systemic markers of lipid carriage and ionic tone aligned with sperm lipid composition: albumin and potassium were associated with higher intracellular palmitate levels and related metabolites. Functionally, lipid and short-chain fatty acid features, 1-monopalmitin, nonadecanoic acid, caproic acid, and valeric acid, were positively associated with viability and/or PMI %, whereas dodecanoic acid and glycerol monostearate were inversely related to morphological abnormalities. However, no robust association was detected with motility. Under a controlled dietary baseline, a lipid-centric blood-to-sperm metabolic axis emerges as a key determinant of cryotolerance in&nbsp;B. javanicus. The prioritized metabolites constitute practical biomarker candidates for sire screening and provide a mechanistic basis for refining extenders and cryopreservation protocols at AI centers. Targeted tandem mass spectrometry, membrane-focused lipidomics, and mitochondrial functional assays offer immediate paths to translation, with the potential to improve reproductive efficiency and, ultimately, bolster sustainable beef production and food security.</p>2026-01-202026-01-08MTBLS13639MTBLC28747MTBLC174137MTBLC422MTBLC30776MTBLC16000MTBLC57305MTBLC17822MTBLC30805MTBLC30769MTBLC178059MTBLC48095MTBLC168544MTBLC17823MTBLC184306MTBLC39246MTBLC64757MTBLC75456MTBLC16113CHEBI:28747CHEBI:174137CHEBI:422CHEBI:30776CHEBI:16000CHEBI:57305CHEBI:17822CHEBI:30805CHEBI:30769CHEBI:178059CHEBI:48095CHEBI:168544CHEBI:17823CHEBI:184306CHEBI:39246CHEBI:64757CHEBI:75456CHEBI:16113