Laboratory Animal Science Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.
Zebrafish. 2022 Aug;19(4):148-159. doi: 10.1089/zeb.2022.0001. Epub 2022 Jun 27.
The rapid increase in zebrafish use needs to be accompanied by research into the refinement of procedures. The European (EU) Directive lists three possible euthanasia methods for fish: anesthetic overdose, electrical stunning, and concussion. However, for small fish such as zebrafish, concussion and electrical stunning are difficult to perform, leaving anesthetic overdose as the most used method. Our aim was to test the efficacy and side effects of anesthesia overdose using different anesthetics and the rapid cooling method to euthanize adult zebrafish. Adult mixed-sex AB zebrafish were randomly assigned to: 250 mg/L MS222; 20 mg/L propofol +100 mg/L lidocaine; 6 mg/L etomidate; 50 mg/L clove oil; and rapid cooling (water at 2°C-4°C). Two minutes after opercular movement ceased, animals were transferred into clean water for 20 min and recovery assessed, or decapitated and used for biochemical analysis of the gills, muscle, liver, and brain; for the histological analysis of the gills and muscle; or for the assessment of cortisol levels. No animal recovered; rapid cooling was the quickest and etomidate overdose was the slowest method to cease the opercular movements. There were no major differences between euthanasia methods regarding the biochemical or histological data. Cortisol levels were higher in the rapid cooling group, but only when compared with the propofol/lidocaine group. The use of a physical method of euthanasia, such as rapid cooling, is essential when chemicals, such as anesthetics, may interfere with postmortem analyses. Although anesthetic overdose can be used without major effects on the analyses conducted in this work, rapid cooling can be another option with the advantage of being simple to administer, easily available, affordable, and very quick; this decreases the potential duration of suffering, being more humane. Therefore, a change in EU legislation should be considered to include additional humane options for euthanasia, such as rapid cooling, for zebrafish and other small tropical fish.
斑马鱼使用量的快速增加需要伴随对程序精细化的研究。欧盟指令列出了三种可能的鱼类安乐死方法：麻醉过量、电击和震荡。然而，对于像斑马鱼这样的小鱼来说，震荡和电击很难实施，因此麻醉过量成为最常用的方法。我们的目的是测试使用不同麻醉剂和快速冷却方法对成年斑马鱼进行麻醉过量安乐死的效果和副作用。成年混合性别 AB 斑马鱼被随机分配到以下组别：250mg/L 的 MS222；20mg/L 丙泊酚+100mg/L 利多卡因；6mg/L 依托咪酯；50mg/L 丁香油；以及快速冷却（2°C-4°C 的水）。在鳃盖运动停止两分钟后，将动物转移到干净的水中 20 分钟，然后评估恢复情况，或者将其斩首并用于鳃、肌肉、肝脏和大脑的生化分析；用于鳃和肌肉的组织学分析；或者用于评估皮质醇水平。没有动物恢复；快速冷却最快，依托咪酯过量最慢。在安乐死方法方面，没有关于生化或组织学数据的主要差异。与丙泊酚/利多卡因组相比，快速冷却组的皮质醇水平更高。使用物理安乐死方法，如快速冷却，对于化学物质（如麻醉剂）可能干扰死后分析的情况至关重要。尽管麻醉过量不会对本工作中的分析产生重大影响，但快速冷却可以是另一种选择，其优点是操作简单、易于获得、价格实惠且非常快速；这减少了潜在的痛苦持续时间，更加人道。因此，应该考虑修改欧盟法规，将包括快速冷却在内的其他人道安乐死选项纳入斑马鱼和其他小型热带鱼。
