A Multi-Site Assessment of Anesthetic Overdose, Hypothermic Shock, and Electrical Stunning as Methods of Euthanasia for Zebrafish (Danio rerio) Embryos and Larvae

Simple Summary Zebrafish (Danio rerio) younger than five days post fertilization are not protected by legislation, and protocols for their euthanasia are poorly explored. In the present paper, we assess the euthanasic efficacy of anesthetic overdose, hypothermic shock, and electrical stunning on zebrafish at <12 h post fertilization, and 1 and 4 days post fertilization utilizing laboratories in different countries. Based on the survival/recovery rates 24 h after treatment, the most effective methods were an overdose with either clove oil or lidocaine with ethanol, and electrical stunning. For the oldest larvae, signs of aversion during treatment demonstrated that all the tested anesthetics, except lidocaine, induced aversive behavior. Therefore, the most suited anesthetic treatment was lidocaine hydrochloride, 1 g/L, buffered with 2 g/L of sodium bicarbonate and mixed with 50 mL/L of ethanol. Electrical stunning also euthanized embryos and larvae efficiently and without signs of aversion; this method is new and needs further assessment in other laboratories to draw firm conclusions. Euthanasia in zebrafish (Danio rerio) younger than 5 days post fertilization (dpf) is poorly described in the literature, and standardized protocols are lacking, most likely because larvae not capable of independent feeding are often not protected under national legislations. We assessed the euthanasia efficacy in laboratories in different countries of a one hour anesthetic overdose immersion with buffered lidocaine hydrochloride (1 g/L, with or without 50 mL/L of ethanol), buffered tricaine (1 g/L), clove oil (0.1%), benzocaine (1 g/L), or 2-phenoxyethanol (3 mL/L), as well as the efficacy of hypothermic shock (one hour immersion) and electrical stunning (for one minute), on zebrafish at <12 h post fertilization (hpf), 24 hpf, and 4 dpf. Based on the survival/recovery rates 24 h after treatment, the most effective methods were clove oil, lidocaine with ethanol, and electrical stunning. For 4 dpf larvae, signs of aversion during treatment demonstrated that all anesthetics, except lidocaine, induced aversive behavior. Therefore, the most suited euthanasic treatment was lidocaine hydrochloride 1 g/L, buffered with 2 g/L of sodium bicarbonate and mixed with 50 mL/L of ethanol, which euthanized both embryos and larvae in an efficient and stress-free manner. Electrical stunning also euthanized embryos and larvae efficiently and without signs of aversion; this method needs further assessment in other laboratories to draw firm conclusions.