Biological Validation of Cortisol in Zebrafish Trunk, Skin Mucus, and Water as a Biomarker of Acute or Chronic Stress

The most used technique to assess cortisol in zebrafish is trunk sampling, a terminal procedure. Extracting cortisol non-terminally in adult zebrafish remains challenging, limiting longitudinal studies, and the reduction of the number of zebrafish used in research. This study explored non-terminal methods for cortisol measurement in adult zebrafish under acute and chronic stress, focusing on housing water and skin mucus as alternatives to terminal trunk sampling. Oxidative stress markers (cerebral and hepatic) were also assessed to confirm stress responses. In experiment A, zebrafish were exposed to no stress, acute stress (AS), or chronic stress for 14 days (CS14) to evaluate skin mucus and trunk cortisol as biomarkers. In experiment B, in addition to CS14, a 7-day unpredictable chronic stress protocol (CS7) was tested to discard stress habituation. Results showed significant effects on cerebral oxidative stress: AS increased ROS and AChE activity, CS7 reduced GPx and AChE, and CS14 raised GPx in experiment A, while it increased protein carbonyls and decreased ATPase levels in experiment B. Trunk and skin mucus cortisol increased following AS. Under chronic stress, trunk and skin mucus cortisol levels were not significantly altered, but water cortisol increased at CS7. In conclusion, skin mucus and trunk cortisol levels are reliable biomarkers for acute stress, while water cortisol holds promise for chronic stress.