86c5dda23eaf1a52aff528f0f3da1a11 animals-15-02409.pdf c15152e32b23997e6d84a77c2154ca57dcc74e7a animals-15-02409.pdf 3aeb3f52371c57391f1e4513cd25674f18241cb4c1af6ff9bc7785450d5ca554 animals-15-02409.pdf Title: Development of a Methodology for Measuring Oxytocin in Feces: Insights from a Preliminary Study in Captive Lions (Panthera leo) Subject: Oxytocin (OT) has emerged as a central focus in stress regulation and animal welfare research. While commonly measured in blood, saliva, and urine, its presence in feces remains unexplored. Fecal OT (fOT) could be highly valuable for wildlife studies, where direct handling is impractical. This study explored OT detection in feces using lions (Panthera leo) as a model species. The objectives included developing a reliable fOT extraction protocol, describing baseline fOT patterns in captive lions, and evaluating its relationship with fecal glucocorticoid metabolites (fGM) under non-stressful conditions. Fecal samples from 16 lions in stable social groups across five Spanish zoos were collected over two to six weeks. A methanol-based extraction and commercial enzyme immunoassay were successfully validated for fOT quantification. Concentrations ranged from 3.00 to 296.64 ng OT/g of feces, with notable intra- and inter-individual variability. Age, sex, and contraceptive status had no significant influence on fOT (p > 0.05), but concentrations differed significantly between zoos (p < 0.001). No consistent relationship was found between fOT and fGM levels under baseline conditions. This study is the first to demonstrate OT detection in feces. The validated methodology provides a foundation for investigating fOT as a biomarker of stress and welfare in wild and domestic species. Keywords: fecal oxytocin; peptide hormone; non-invasive; extraction; enzyme immunoassay; animal welfare; wildlife Author: Paula Serres-Corral, Vanessa Almagro, Loles Carbonell, Santiago Borragán, Eva Martínez-Nevado, Miguel Angel Quevedo, Hugo Fernández-Bellon, Annaïs Carbajal and Manel López-Béjar Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Sun Aug 17 09:07:31 2025 CEST ModDate: Sun Aug 17 09:15:16 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 957605 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JRSRGE+URWPalladioL-Bold Type 1 Custom yes yes yes 166 0 YKZBCB+URWPalladioL-Roma Type 1 Custom yes yes yes 167 0 QKZKVH+VnURWPalladioL Type 1 Custom yes yes yes 168 0 FIUUUW+URWPalladioL-BoldItal Type 1 Custom yes yes yes 169 0 XAZVUP+URWPalladioL-Ital Type 1 Custom yes yes yes 170 0 QMDDXF+CMSY10 Type 1 Builtin yes yes yes 281 0 JHYTSG+CMR10 Type 1 Builtin yes yes yes 320 0 RFECCF+EURM10 Type 1 Builtin yes yes yes 330 0 MDIKPC+CMBSY10 Type 1 Builtin yes yes yes 346 0 EIHNPN+PalatinoLinotype TrueType WinAnsi yes yes no 341 0 EIHNPN+PalatinoLinotype TrueType WinAnsi yes yes no 384 0 EIHNPN+PalatinoLinotype TrueType WinAnsi yes yes no 371 0 MJJBJB+PalatinoLinotype TrueType WinAnsi yes yes no 394 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-11 03:37:03 CEST RepresentationInformation: animals-15-02409.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-10 14:25:23 CEST Size: 957605 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 654 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Material and Methods Children: Item: Title: Study Animals and Sample Collection Item: Title: Selection of Solvents and Extraction Procedure Item: Title: Extraction Efficiency of MeOH and ACN Item: Title: Oxytocin Assay Validation Children: Item: Title: Precision Item: Title: Specificity Item: Title: Lower Limit of Quantification Item: Title: Accuracy Item: Title: Quantification of fOT in Lions and Relationship with fGM Levels Item: Title: Statistical Analysis Item: Title: Results Children: Item: Title: Extraction Efficiency for the Two Solvents Item: Title: Assay Validation Children: Item: Title: Precision Item: Title: Specificity Item: Title: Lower Limit of Quantification Item: Title: Accuracy Item: Title: Selection of the Extraction Solvent Item: Title: fOT Concentrations in Captive Lions Item: Title: Relationship Between fOT and fGM Concentrations in Lions Item: Title: Discussion Children: Item: Title: Development of a Reliable Methodology for the Measurement of OT in Feces Item: Title: Exploring the fOT Levels in Captive Lions Item: Title: Relationship Between fOT and fGM Concentrations in Lions Item: Title: Limitations and Future Directions Item: Title: Conclusions Item: Title: References Info: Title: Development of a Methodology for Measuring Oxytocin in Feces: Insights from a Preliminary Study in Captive Lions (Panthera leo) Author: Paula Serres-Corral, Vanessa Almagro, Loles Carbonell, Santiago Borragán, Eva Martínez-Nevado, Miguel Angel Quevedo, Hugo Fernández-Bellon, Annaïs Carbajal and Manel López-Béjar Subject: Oxytocin (OT) has emerged as a central focus in stress regulation and animal welfare research. While commonly measured in blood, saliva, and urine, its presence in feces remains unexplored. Fecal OT (fOT) could be highly valuable for wildlife studies, where direct handling is impractical. This study explored OT detection in feces using lions (Panthera leo) as a model species. The objectives included developing a reliable fOT extraction protocol, describing baseline fOT patterns in captive lions, and evaluating its relationship with fecal glucocorticoid metabolites (fGM) under non-stressful conditions. Fecal samples from 16 lions in stable social groups across five Spanish zoos were collected over two to six weeks. A methanol-based extraction and commercial enzyme immunoassay were successfully validated for fOT quantification. Concentrations ranged from 3.00 to 296.64 ng OT/g of feces, with notable intra- and inter-individual variability. Age, sex, and contraceptive status had no significant influence on fOT (p > 0.05), but concentrations differed significantly between zoos (p < 0.001). No consistent relationship was found between fOT and fGM levels under baseline conditions. This study is the first to demonstrate OT detection in feces. The validated methodology provides a foundation for investigating fOT as a biomarker of stress and welfare in wild and domestic species. 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