Behavioral and neurophysiological effects of electrical stunning on zebrafish larvae

Two methods dominate the way that zebrafish larvae are euthanized after experimental procedures: anesthetic overdose and rapid cooling. Although MS-222 is easy to apply, this anesthetic takes about a minute to act and fish show aversive reactions and interindividual differences, limiting its reliability. Rapid cooling kills larvae after several hours and is not listed as an approved method in the relevant European Union directive. Electrical stunning is a promising alternative euthanasia method for zebrafish but has not yet been fully established. Here we characterize both behavioral and neurophysiological effects of electrical stunning in 4-day-old zebrafish larvae. We identified the electric field characteristics and stimulus duration (50 V/cm alternating current for 32 s) that reliably euthanizes free-swimming larvae and agarose-embedded larvae with an easy-to-implement protocol. Behavioral analysis and calcium neurophysiology show that larvae lose consciousness and stop responding to touch and visual stimuli very quickly (<1 s). Electrically stunned larvae no longer show coordinated brain activity. Their brains instead undergo a series of concerted whole-brain calcium waves over the course of many minutes before the cessation of all brain signals. Consistent with the need to implement the 3R at all stages of animal experimentation, the rapid and reliable euthanasia achieved by electrical stunning has potential for refinement of the welfare of more than 5 million zebrafish used annually in biomedical research worldwide.