TY - JOUR

T1 - A Multi-Site Assessment of Anesthetic Overdose, Hypothermic Shock, and Electrical Stunning as Methods of Euthanasia for Zebrafish (Danio rerio) Embryos and Larvae

AU - Mocho, Jean Philippe

AU - Lang, Florian

AU - Valentin, Guillaume

AU - Bedu, Sébastien

AU - McKimm, Robin

AU - Ramos, Juan

AU - Torres, Yolanda Saavedra

AU - Wheatley, Sarah E.

AU - Higgins, Joseph

AU - Millington, Mollie E.

AU - Lundegaard, Pia Rengtved

AU - Valverde, Rubén Chamorro

AU - Jenčič, Vlasta

AU - von Krogh, Kristine

N1 - Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/4

Y1 - 2022/4

N2 - 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.

AB - 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.

KW - anesthesia

KW - benzocaine

KW - clove oil

KW - Danio rerio

KW - electrical stunning

KW - euthanasia

KW - hypothermic shock

KW - lidocaine

KW - tricaine

KW - zebrafish

UR - http://www.scopus.com/inward/record.url?scp=85128323955&partnerID=8YFLogxK

U2 - 10.3390/biology11040546

DO - 10.3390/biology11040546

M3 - Journal article

C2 - 35453745

AN - SCOPUS:85128323955

VL - 11

SP - 1

EP - 17

JO - Biology

JF - Biology

SN - 2079-7737

IS - 4

M1 - 546

ER -