Short-term feed withdrawal does not alter morpho-physiological stress response in adult zebrafish

Fish welfare is increasingly gaining attention in both aquaculture and research, aligning with the "One Welfare" principles that connect animal welfare, human wellbeing, and environment. Here we evaluate whether shortterm feed withdrawal, a common practice used in research and aquaculture prior to handling, transportation, and sedation determines a stress response in the adult zebrafish (Danio rerio), through the assessment of key biomarkers across different biological matrices. Adult zebrafish were assigned either to a control group, fed with standard diet twice daily, or to a group subjected to 96 h of complete food deprivation. Cortisol levels were measured in fins, while advanced oxidation protein products (AOPP) and malondialdehyde (MDA) levels were analyzed in muscle tissue. Cellular and oxidative stress markers were assessed through the immunohistochemical localization of Heat Shock Protein-70 (HSP70) and 8-hydroxy-2 '-deoxyguanosine (8-OHdG). The levels of cortisol did not have significant differences by treatment, sex, or their interaction. AOPP levels exhibited a significant interaction between treatment and sex, with a 10 % increase in fasted males compared to control females, while MDA levels were unchanged. Consistently, immunohistochemical analyses revealed no significant differences between groups in HSP70 and 8-OHdG positivity. These findings suggest that short-term feed withdrawal does not significantly affect key physiological stress markers in zebrafish. Finally, we validated the use of the caudal fin as a matrix for measuring individual cortisol levels in zebrafish, providing an alternative to terminal whole-body analyses as well as to group-level water-borne cortisol.