Behavioral and Neurophysiological Effects of Electrical Stunning on Zebrafish Larvae

In today’s scientific research, two methods dominate the way zebrafish larvae are euthanized after experimental procedures: anesthetic overdose and rapid cooling. Although easy to apply, the anesthetic MS-222 takes about a minute to act, fish show aversive reactions, and inter-individual differences limit its reliability. Rapid cooling only kills larvae after many minutes and is not listed as approved method in the relevant EU directive. Electrical stunning is a promising alternative euthanasia method but hasn’t been established yet for zebrafish. In this study, we characterize both behavioral and neurophysiological effects of electrical stunning in 4 dpf zebrafish larvae. We identified the electric field magnitude and stimulus duration (32 s at 50 V/cm) that reliably euthanize free-swimming larvae and agarose-embedded larvae and provide an easy-to-implement protocol. Behavioral analysis and calcium neurophysiology show that larvae lose consciousness very fast and stop responding to touch and visual stimuli (< 1 second). Electrically stunned larvae do not show coordinated brain activity anymore and their brains will instead undergo a series of concerted whole-brain calcium waves over the course of many minutes before the ultimate cessation of all brain signals. Consistent with the imperative of implementing the 3R at all stages of animal experiments, the rapid and reliable euthanasia achieved by electrical stunning has the potential to bring about a real refinement of the welfare of more than 5 million zebrafish used annually in biomedical research worldwide.