b24feba1365e44c0d086b3d39c1b5673 animals-15-02628-v4.pdf 7258fec6914c8d58e9c3c7d0e14fd84de6ac91be animals-15-02628-v4.pdf 646b0e949dbe152d396a619a0d530e5a2eafd3c2863c4c321e0ed7319a8804a8 animals-15-02628-v4.pdf Title: Tracking Epidermal Cortisol and Oxytocin in Managed Bottlenose Dolphins as Potential Non-Invasive Physiological Welfare Indicators Subject: Growing concern over cetacean welfare has highlighted the need for rigorous, science-based assessment methods. Within this context, epidermal cortisol (ECC) and oxytocin (EOC) concentrations have emerged as potentially valuable physiological indicators. In this study, we first validated the analytical measurement of ECC and EOC in bottlenose dolphins (Tursiops truncatus) using AlphaLISA assays. Subsequently, weekly ECC and EOC levels were measured over an extended period in five managed dolphins and analyzed alongside aggregated environmental and welfare-related variables, using various time lags to account for delays between physiological activity and hormone deposition in the epidermis. ECC was negatively associated with mild weight loss and diazepam administration, exhibiting seasonal variability. In contrast, EOC was negatively associated with negative welfare indicators and COVID-19 park closures but positively associated with diazepam administration and peak visitor seasons, also showing seasonal variability. However, the interpretation of EOC remains complex due to a limited understanding of the cetacean oxytocin system and its dual role in positive and negative affective states. Overall, ECC and EOC show promise as non-invasive biomarkers for monitoring long-term welfare changes in cetaceans, although further research is necessary to validate these biomarkers across broader populations and contexts and to clarify their temporal dynamics in the epidermis. Keywords: cetaceans; welfare assessment; welfare indicators; non-invasive methods; wellbeing; odontocetes; bottlenose dolphin; cortisol; oxytocin; epidermis Author: Clara Agustí, Oriol Talló-Parra, Enrique Tejero-Caballo, Daniel Garcia-Parraga, Marina López-Arjona, Teresa Álvaro-Álvarez, José Joaquín-Cerón and Xavier Manteca Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Thu Sep 11 11:10:05 2025 CEST ModDate: Thu Sep 11 11:26:58 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 31 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1245449 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- FGZOTS+URWPalladioL-Roma Type 1 Custom yes yes yes 150 0 AIRHKL+VnURWPalladioL Type 1 Custom yes yes yes 151 0 UPTXOD+URWPalladioL-Ital Type 1 Custom yes yes yes 152 0 YYGPST+URWPalladioL-Bold Type 1 Custom yes yes yes 153 0 EQPGOB+CMSY10 Type 1 Builtin yes yes yes 287 0 CEZMQP+EURM10 Type 1 Builtin yes yes yes 308 0 ANCFIM+PalatinoLinotype TrueType WinAnsi yes yes no 411 0 ANCHII+TimesNewRoman TrueType WinAnsi yes yes no 412 0 ANCHJK+TimesNewRoman,Italic TrueType WinAnsi yes yes no 413 0 ANCFIM+PalatinoLinotype TrueType WinAnsi yes yes no 425 0 ANCFIM+PalatinoLinotype TrueType WinAnsi yes yes no 433 0 ANCFIK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 434 0 ANCFIM+PalatinoLinotype TrueType WinAnsi yes yes no 461 0 ANCFIK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 462 0 ANCFIM+PalatinoLinotype TrueType WinAnsi yes yes no 491 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 507 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-26 03:08:09 CEST RepresentationInformation: animals-15-02628-v4.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-25 14:50:03 CEST Size: 1245449 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 994 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: Materials and Methods Children: Item: Title: Ethics Statement Item: Title: Study Area and Individuals Item: Title: Sample Collection and Preparation Item: Title: Sample Storage, Preparation, and Hormone Extraction Item: Title: Hormone Detection and Assay Validation Item: Title: Data Collection and Individual Welfare Profiling Based on Aquarium Reports Item: Title: Data Management Item: Title: Statistical Analyses Item: Title: Results Children: Item: Title: Assay Validation Item: Title: Descriptive Overview, Individual Variation, and Correlation of Epidermal Cortisol and Oxytocin Concentrations Item: Title: Descriptive Analysis of Environmental and Welfare-Related Predictors Item: Title: Effects of Environmental and Welfare-Related Predictors on Epidermal Cortisol Concentrations at Different Time Lags Item: Title: Effects of Environmental and Welfare-Related Predictors on Epidermal Oxytocin Concentrations at Different Time Lags Item: Title: Discussion Children: Item: Title: Assay and Method Validation Item: Title: Descriptive Overview and Correlation Between Epidermal Cortisol and Oxytocin Concentrations Item: Title: Effects of Environmental and Welfare-Related Predictors on Epidermal Cortisol Concentrations Item: Title: Effects of Environmental and Welfare-Related Predictors on Epidermal Oxytocin Concentrations Item: Title: Effects of Temporal Dynamics of Cortisol and Oxytocin Incorporation into the Epidermis Item: Title: Study Limitations and Future Directions Item: Title: Conclusions Item: Title: References Info: Title: Tracking Epidermal Cortisol and Oxytocin in Managed Bottlenose Dolphins as Potential Non-Invasive Physiological Welfare Indicators Author: Clara Agustí, Oriol Talló-Parra, Enrique Tejero-Caballo, Daniel Garcia-Parraga, Marina López-Arjona, Teresa Álvaro-Álvarez, José Joaquín-Cerón and Xavier Manteca Subject: Growing concern over cetacean welfare has highlighted the need for rigorous, science-based assessment methods. Within this context, epidermal cortisol (ECC) and oxytocin (EOC) concentrations have emerged as potentially valuable physiological indicators. In this study, we first validated the analytical measurement of ECC and EOC in bottlenose dolphins (Tursiops truncatus) using AlphaLISA assays. Subsequently, weekly ECC and EOC levels were measured over an extended period in five managed dolphins and analyzed alongside aggregated environmental and welfare-related variables, using various time lags to account for delays between physiological activity and hormone deposition in the epidermis. ECC was negatively associated with mild weight loss and diazepam administration, exhibiting seasonal variability. In contrast, EOC was negatively associated with negative welfare indicators and COVID-19 park closures but positively associated with diazepam administration and peak visitor seasons, also showing seasonal variability. However, the interpretation of EOC remains complex due to a limited understanding of the cetacean oxytocin system and its dual role in positive and negative affective states. Overall, ECC and EOC show promise as non-invasive biomarkers for monitoring long-term welfare changes in cetaceans, although further research is necessary to validate these biomarkers across broader populations and contexts and to clarify their temporal dynamics in the epidermis. 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