INTERNATIONAL ZEBRAFISH AND MEDAKA COURSE European Zebrafish Resource Centre (EZRC) Karlsruhe Institute of Technology (KIT) Heidelberg University International Zebrafish Medaka Course IZMC International Zebrafish/Medaka Course (IZMC) European Zebrafish Resource Centre (EZRC) Karlsruhe Institute of Technology (KIT) Heidelberg University 1 International Zebrafish Medaka Course IZMC Synopsis Fish models have increasingly gained importance in biomedical research during the last few decades. Particularly two teleost species, zebrafish (Danio rerio) and medaka (Oryzias latipes), have become essential model systems for analyzing gene function in vertebrates. Their suitability for forward genetics especially their small size, the transparency of the embryos and the extra-uterine development made them ideal systems for systematic studies of developmental processes. In the meantime these fish have also provided many disease models that allow the study of pathophysiological processes in real time and at high cellular resolution in the intact embryo. The Directive 2010/63/EU of the European Parliament and of the Council of Europe demands that persons performing animal experimentation should be adequately trained. Today in Germany, a plethora of courses which follow the FELASA B concept is offered by universities and other research institutions. These courses mainly address research with mice and rats; however, work with fish is not covered. In order to train technicians, students, graduates, postgraduates and principal investigators in the use of zebrafish or medaka for biomedical research, the European Zebrafish Resource Center at the Karlsruhe Institute of Technology offers a course teaching all relevant topics. The course is held either in English or in German depending on requests of the participants. The theory element of this course is presented using online material accessible for participants via the ICMZ website. Theory topics include – amongst others –legal regulations and guidelines, ethical aspects of animal experimentation (3R, alternative concepts), biology, husbandry, breeding and feeding of zebrafish and medaka, as well as health monitoring and diseases. Other fish models in toxicology and biomedical research will be discussed. The practical elements of the course will include training in the handling, husbandry, breeding, anesthesia, fin clipping and euthanasia of zebrafish and medaka using the facilities of the EZRC and the associated KIT Institute of Toxicology and Genetics. The course will be completed with a multiple choice examination. Those who have passed the course and examination successfully will receive a certificate confirming their specific knowledge of zebrafish and medaka as well as their skills to work with these animals meeting the legal requirements. 2 International Zebrafish Medaka Course IZMC Table of Contents 1. History and Ethics of Animal Experimentation ..................................................... 5 2. Animal Welfare, the 3R-Concept and Alternative Methods .................................. 9 2.1 Recognition of pain, suffering and distress in fish ...............................................12 3. Legal regulations for animal experimentation .....................................................15 3.1 Animal welfare regulations for experimentation in Germany ...............................15 3.1.1 Legal framework………………………………………………………………………..15 3.1.2 Obligations of the establishment…………………………………………….. ..........16 3.1.3 Application procedures………………………………………………………………. .16 3.1.4 Projects requiring authorisation („genehmigungspflichtige Versuchsvorhaben“) .16 3.1.5 Project leader, substitute project leader („Leiter, Stellvertreter“)..………….. .......18 3.1.6 Staff……………………………………………………………………………............. 18 3.1.7 Projects requiring a formal declaration („anzeigepflichtige Versuchsvorhaben“) .19 3.1.8 Euthanasia of animals for scientific purposes……………………………….. .........20 3.1.9 Reservation on approval of genetically altered breeds with harmful phenotypes 20 3.1.10 Use of larvae……………………………………………………………………….. .....21 3.1.11 Record keeping requirement …………………………………………………………21 3.1.12 Annual report on laboratory animal use……………………………………….. .......21 3.1.13 National Animal Welfare Committee…………………………………………………21 3.1.14 Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes Introduction ........................................................................................22 3.2 Legal considerations for the shipping of fish, fish eggs and sperm samples ......... intended for scientific research ...........................................................................25 3.2.1 Additional regulations for shipment of fish (later than 5 days post fertilization) ...27 3.2.2 Additional regulations for shipment of fish that must be classified as genetically modified organisms…………………………………………………………………….28 4. Zebrafish and Medaka as experimental models .................................................31 4.1 The biology of zebrafish (Danio rerio) .................................................................33 4.2 Biology of Medaka (Oryzias latipes) ...................................................................41 5. Husbandry, breeding and feeding.......................................................................44 5.1 Zebrafish ............................................................................................................44 5.2 Medaka husbandry and breeding (Daniela Vallone, Felix Loosli).......................48 6. Anesthesia, analgesia and euthanasia in fish .....................................................51 6.1 Definitions ..........................................................................................................52 6.2 General considerations regarding the selection of the anesthesia procedure .....53 6.3 Stages of anesthesia ..........................................................................................57 3 International Zebrafish Medaka Course IZMC 6.4 Forms of anesthesia ...........................................................................................58 6.5 Dosing of inhalation anesthetics .........................................................................61 6.6 Non-chemical anesthesia ...................................................................................63 6.6.1 Hypothermia (cooling)…………………………………………………………………63 6.6.2 Electro-anesthesia……………………………………………………………….. .......64 6.7 Injection of anesthetics .......................................................................................65 6.8 Prolonged anesthesia and monitoring of anesthesia ..........................................66 6.9 Euthanasia .........................................................................................................67 6.10 Anesthesia and euthanasia in zebrafish .............................................................68 7. Health Monitoring and Diseases in Zebrafish (Danio rerio) and Medaka (Oryzias latipes) .................................................................................................70 7.1 Fish diseases ...................................................................................................70 7.1.2 Description of the most relevant diseases…………………………………… .........71 7.1.3 Treatment……………………………………………………………………........ .......74 7.1.5 Sample numbers for health monitoring……………………………………… ..........77 8. Statistical analysis ..............................................................................................80 9. Abbreviations .....................................................................................................83 10. Fish in toxicology and ecotoxicology ..................................................................86 10.1 Fish tests in waste water disposal ......................................................................86 10.2 Fish tests in chemical testing: acute toxicity testing with adult fish and the Fish….. Embryo Test as an alternative method ...............................................................87 10.3 Legal framework for fish tests other than acute toxicity tests ..............................91 10.4 Fish generally used in (eco)toxicology ................................................................98 10.4.1 Zebrafish (Danio rerio)………………………………………………………… ..........98 10.4.2 Japanese medaka (Oryzias latipes)…………………………………………. ..........98 10.4.3 Fathead minnow (Pimephales promelas) ...........................................................98 10.4.4 Rainbow trout (Oncorhynchus mykiss) .............................................................100 10.4.5 Sheepshead minnow (Cyprinodon variegatus) .................................................100 10.4.6 Guppy (Poecilia reticulata) ...............................................................................101 10.4.7 Golden ide (Leuciscus idus melanotus) ............................................................103 10.5 Diversity of fish species in alternative test methods ..........................................104 11. Zebrafish embryos as models in chemical biology and chemical genetics ........ 107 4 International Zebrafish Medaka Course IZMC 1. History and Ethics of Animal Experimentation (Almut Köhler, Jürgen Weiss) The development of animal experimentation as a model for humans has largely taken place in parallel with the development of medical science itself. The fundamentals of western medicine originate from ancient Greece. Indeed Greek Philosophers were