Zebrafish have become one of the most widely used vertebrate research animals, with their welfare requiring increasing attention as evidence of pain capacity and stress sensitivity accumulates.
Zebrafish welfare has evolved from an assumption of minimal sentience to recognition that the species shows responses to noxious stimuli and stressors that are consistent with welfare significance. The increasing use of larval stages to circumvent vertebrate regulations has been challenged as larvae show pain-like responses and the regulatory exemption does not reflect welfare reality. The 3Rs framework applied to zebrafish requires genuine consideration of Replacement (cell and organoid models where possible), Reduction (appropriate statistical power calculation), and Refinement (anesthesia protocols, housing quality, and experimental procedure refinement) rather than treating zebrafish as a regulatory loophole.