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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\u2009mg\/L MS222; 20\u2009mg\/L propofol +100\u2009mg\/L lidocaine; 6\u2009mg\/L etomidate; 50\u2009mg\/L clove oil; and rapid cooling (water at 2\u00b0C\u20134\u00b0C). Two minutes after opercular movement ceased, animals were transferred into clean water for 20\u2009min 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.<\/jats:p>","DOI":"10.1089\/zeb.2022.0001","type":"journal-article","created":{"date-parts":[[2022,6,27]],"date-time":"2022-06-27T12:31:37Z","timestamp":1656333097000},"page":"148-159","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":21,"title":["Anesthesia Overdose Versus Rapid Cooling for Euthanasia of Adult Zebrafish"],"prefix":"10.1177","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8411-5950","authenticated-orcid":false,"given":"Jorge M.","family":"Ferreira","sequence":"first","affiliation":[{"name":"Laboratory Animal Science Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal."},{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, Porto, Portugal."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu\u00eds","family":"F\u00e9lix","sequence":"additional","affiliation":[{"name":"Laboratory Animal Science Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal."},{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, Porto, Portugal."},{"name":"Centro de Investiga\u00e7\u00e3o e de Tecnologias Agroambientais e Biol\u00f3gicas (CITAB), Universidade de Tr\u00e1s-os-Monte e Alto Douro, Vila Real, Portugal."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Jorge","sequence":"additional","affiliation":[{"name":"Laboratory Animal Science Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal."},{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, Porto, Portugal."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra M.","family":"Monteiro","sequence":"additional","affiliation":[{"name":"Centro de Investiga\u00e7\u00e3o e de Tecnologias Agroambientais e Biol\u00f3gicas (CITAB), Universidade de Tr\u00e1s-os-Monte e Alto Douro, Vila Real, Portugal."},{"name":"Inov4Agro, Institute for Innovation, Capacity Building and Sustainability of Agri-Food Production, Universidade de Tr\u00e1s-os-Monte e Alto Douro, Vila Real, Portugal."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"I. 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