50d8e3caf8ad292266ffe3871bf60e48 pmc_9027676.pdf 2a868fc2a8a33966d35244d0778313871de178dd pmc_9027676.pdf e9372db80ee47883cfa7e3c3d22eeb6ded8506a178e83ca85d1adb89ab54e4db pmc_9027676.pdf Title: A Multi-Site Assessment of Anesthetic Overdose, Hypothermic Shock, and Electrical Stunning as Methods of Euthanasia for Zebrafish (Danio rerio) Embryos and Larvae Subject: 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. Keywords: zebrafish; Danio rerio; euthanasia; anesthesia; lidocaine; tricaine; benzocaine; clove oil; hypothermic shock; electrical stunning Author: Jean-Philippe Mocho, Florian Lang, Guillaume Valentin, Sébastien Bedu, Robin McKimm, Juan Ramos, Yolanda Saavedra Torres, Sarah E. Wheatley, Joseph Higgins, Mollie E. Millington, Pia Rengtved Lundegaard, Rubén Chamorro Valverde, Vlasta Jenčič and Kristine von Krogh Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Apr 1 10:50:52 2022 CEST ModDate: Fri Apr 1 10:53:14 2022 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2031341 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MXTWPO+VnURWPalladioL Type 1 Custom yes yes no 10 0 UBHZRH+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 WISCOY+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 AGKCLH+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 WFZCXP+URWPalladioL-BoldItal Type 1 Custom yes yes no 28 0 BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 40 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WinAnsi yes yes no 267 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 271 0 Times New Roman,Bold TrueType WinAnsi no no no 278 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 288 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-01-16 02:42:42 CET RepresentationInformation: pmc_9027676.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-11-28 03:36:17 CET Size: 2031341 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 491 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Animals and Husbandry Destination: subsection.2.1 Item: Title: Experimental Design Destination: subsection.2.2 Children: Item: Title: Anesthetic Overdose Destination: subsubsection.2.2.1 Item: Title: Electrical Stunning Destination: subsubsection.2.2.2 Item: Title: Hypothermic Shock Destination: subsubsection.2.2.3 Item: Title: Monitoring of Discomfort Destination: subsubsection.2.2.4 Item: Title: Statistics and Data Presentation Destination: subsection.2.3 Item: Title: Results Destination: section.3 Item: Title: Discussion Destination: section.4 Children: Item: Title: Anesthetic Overdoses Destination: subsection.4.1 Item: Title: Electrical Stunning Destination: subsection.4.2 Item: Title: Hypothermic Shock Destination: subsection.4.3 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: A Multi-Site Assessment of Anesthetic Overdose, Hypothermic Shock, and Electrical Stunning as Methods of Euthanasia for Zebrafish (Danio rerio) Embryos and Larvae Author: おづちの〭ぐとどぬどばばづ〠きはっとは〬〠うぬはひどちの〠がちので〬〠ぇふどぬぬちふねづ〠ざちぬづのぴどの〬〠こラぢちびぴどづの〠あづつふ〬〠げはぢどの〠きっかどねね〬〠おふちの〠げちねはび〬〠すはぬちのつち〠こちちぶづつひち〠ごはひひづび〬〠こちひちと〠ぅ〮〠しとづちぴぬづべ〬〠おはびづばと〠えどででどのび〬〠きはぬぬどづ〠ぅ〮〠きどぬぬどのでぴはの〬〠ぐどち〠げづのでぴぶづつ〠がふのつづでちちひつ〬〠げふぢラの〠ぃとちねはひひは〠ざちぬぶづひつづ〬〠ざぬちびぴち〠おづのㄍどㄍ〠ちのつ〠かひどびぴどのづ〠ぶはの〠かひはでと Subject: 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. Keywords: zebrafish; Danio rerio; euthanasia; anesthesia; lidocaine; tricaine; benzocaine; clove oil; hypothermic shock; electrical stunning Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Apr 01 10:50:52 CEST 2022 ModDate: Fri Apr 01 10:53:14 CEST 2022 ID: 0x5db076f2d1ca5de603e2d6d0d3abec35, 0x5db076f2d1ca5de603e2d6d0d3abec35 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: BTFGED+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 178 FontDescriptor: FontName: BTFGED+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: VORBJF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: VORBJF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true 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