Dissociation between plasma and oral mucus cortisol in bottlenose dolphins (Tursiops truncatus) under human care: implications for non-invasive assessment of physiological state

Cortisol is a fundamental biomarker of hypothalamic–pituitary–adrenal (HPA) axis activity, yet the relationship between plasma and oral mucus cortisol dynamics remains poorly understood in cetaceans. We investigated this relationship in eight bottlenose dolphins ( Tursiops truncatus ) under managed care, sampled on six days at two time points: a morning baseline (09:00–09:30 h) and immediately before a mid-day educational encounter with visitors (~ 12:00 h). All replicate-level observations were analysed with linear mixed-effects models, with dolphin as a random intercept. Plasma cortisol increased markedly between the two time points (fold-change 2.08×; permutation p = 0.008), whereas oral mucus cortisol rose only modestly (fold-change 1.09×; permutation p = 0.023). Both matrices increased in the same direction, but the magnitude of change differed significantly (time point × matrix interaction, parametric-bootstrap p < 0.001), indicating a dissociation in magnitude rather than direction. Between-individual differences accounted for a substantial share of variance (ICC = 0.56 and 0.64). Because plasma and oral mucus were quantified on different analytical platforms (ELISA and LC-MS/MS), the analysis was confined to relative within-matrix change rather than absolute equivalence. These results indicate matrix-specific HPA dynamics in which plasma tracks acute change while oral mucus changes more gradually, and position oral mucus cortisol as a complementary, minimally invasive indicator of physiological state rather than a substitute for plasma. Because sampling time was confounded with the educational encounter, a dedicated control day would be needed to fully isolate the event effect.