Lab Animal ( IF 5.9 ) Pub Date : 2025-01-27 , DOI: 10.1038/s41684-024-01505-0
David-Samuel Burkhardt, Claire Leyden, Carina Thomas, Christian Brysch, Florian Alexander Dehmelt, Aristides B. Arrenberg
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.
中文翻译:
电击晕对斑马鱼幼虫的行为和神经生理学影响
两种方法主导了斑马鱼幼虫在实验程序后被安乐死的方式:麻醉剂过量和快速冷却。虽然 MS-222 很容易应用,但这种麻醉剂大约需要一分钟才能起作用,而且鱼表现出厌恶反应和个体间差异,限制了其可靠性。快速冷却可在几个小时后杀死幼虫,因此未在相关欧盟指令中被列为批准的方法。电击晕是一种很有前途的斑马鱼替代安乐死方法,但尚未完全建立。在这里,我们表征了电击对 4 日龄斑马鱼幼虫的行为和神经生理学影响。我们确定了电场特性和刺激持续时间(50 V/cm 交流电,持续 32 秒),它们通过易于实施的方案可靠地对自由游泳的幼虫和琼脂糖包埋的幼虫实施安乐死。行为分析和钙神经生理学表明,幼虫会很快失去意识并停止对触觉和视觉刺激做出反应 (<1 s)。电击晕的幼虫不再表现出协调的大脑活动。相反,在所有大脑信号停止之前的几分钟内,他们的大脑会经历一系列协同的全脑钙电波。与在动物实验的所有阶段实施 3R 的需要一致,通过电击晕实现的快速可靠的安乐死有可能改善全球每年用于生物医学研究的 500 多万只斑马鱼的福利。