This document will provide general guidelines for zebrafish euthanasia in the washington university school of medicine basement facility. Euthanasia may be accomplished using one of the following methods. For zebrafish, that certain glow with its relatively small genome and its ability to produce mutations that are analogous to those in humans, the humble zebrafish is already a valuable tool in. Condition factor k, was measured by taking the length l and weight w of each zebrafish and using the following formula. Adult zebrafish are often difficult to section without first softening their skin and scales. Zebrafish embryos as an alternative to animal experimentsa. The zebrafish embryo offers unmatched opportunities to identify bioactive molecules in the context of an intact, complex organism. Welcome the same characteristics that make zebrafish a powerful system for basic research also make them an excellent tool for teaching. In addition, the zebrafish shares a high degree of homology with the human genome 5. The zebrafish is an undifferentiated gonochorist, implying that all individuals initially develop ovary. A toxicological study using zebrafish danio rerio as a model 14 toxicant exposure zebrafish embryos are collected and 45 embryos were placed in each well of a 24 well plate. Zebrafish represent a vertebrate model organism that has been widely, and increasingly, employed over the last decade in the study of developmental processes, wound healing, microbehost interactions, and drug screening. Tricaine methanesulfonate tms is an anesthetic that is approved for provisional use in some jurisdictions such as the united states, canada, and the united kingdom uk.
The schedule of biobide scientists this spring is taken by very relevant international events in the sector. In fish, the commonly used anaesthetic tricaine methanesulphonate, also known as 3aminobenzoic acid ethyl ester, metacaine or ms222, causes loss of. The toxicants used have been documented to induced. The lower two bars represent a control group n 32 compared with a group exposed to 20 ngl tb from egg stage to sexual maturity followed by a six. Hybridization, washing and incubation steps were done in. Behavioural effects of the commonly used fish anaesthetic tricaine methanesulfonate ms222 on zebrafish danio rerio and its relevance for the acetic acid pain test march 2014 plos one 93. Zebrafish nacre strain were bred and raised according to established protocols. The same characteristics that make zebrafish a powerful system for basic research also make them an excellent tool for teaching. Cyprinidae, which contains over 2,400 species, including the loveable goldfish. Characterization of housekeeping genes in zebrafish. In reality, the acceptance and use of zebrafish for toxicology lagged significantly behind for a number of important reasons.
Zebrafish international resource centers pathology services. Capturing tissue repair in zebrafish larvae with time. Behavioural effects of the commonly used fish anaesthetic tricaine methanesulfonate ms222 on zebrafish danio rerio and its relevance for the acetic acid. Two fish from the control group and one fish from the tricaine group had to be excluded due to methodological problems, leaving eight fish in the tricaine group and seven fish in the control group. Lp italiana spa, milan containing ethylenediaminetetraacetic acid edta. For zebrafish, that certain glow the new york times. Fish were euthanized in tricaine methanesulfonate ms222, 1. Fish anaesthetic tricaine methanesulfonate ms222 on zebrafish danio. Changes to extender, cryoprotective medium, and in vitro.
Animal use protocol zebrafish international resource center. Division of animal care washington university zebrafish. Tricaine mesylate an overview sciencedirect topics. Although realtime reverse transcription pcr qpcr, normalized to an internal reference housekeeping gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. The tricaine dose was based on the recommendation in the zebrafish book. A guide for the laboratory use of zebrafish danio brachydanio rerio. You can view a list of all subpages under the book main page not including the book main page itself, regardless of whether theyre categorized, here. This fullcolor atlas will aid experimental design and interpretation in these areas by providing a fundamental understanding of zebrafish. The day before processing, transfer the fixed fish to 5% trichloroacetic acid. With the enthusiasm around implementing zebrafish for human disease research, one might assume that the adoption of the model for toxicological studies would be easy and somewhat obvious. The embryos were exposed to toxicants by adding the toxicants to the water.
Rapid cooling has been suggested as an alternative to tricaine for repeated anesthesia of larval zebrafish up to 14 dpf chen et al. Using zebrafish as a model system for studying the. However, limited information is available on the endocrine disrupting effects of phthalates in aquatic organisms. The zebrafish is attracting increasing attention from the pharmaceutical industry with the aim of using the embryo as an alternative model that resembles the complexity of an adult animal. However, commercialization of nanoparticles might offer possible risks once they. Effectiveness of recommended euthanasia methods in larval zebrafish danio rerio. Tricaine mesylate is white powder used for anesthesia, sedation, or euthanasia of fish. They can be lifted out of the anesthetic with a plastic spoon. Mesab, ms222, tricaine, finquel solution of 4gl buffered to ph 7. More precisely, zebrafish belong to the single largest vertebrate family. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon. This category contains pages that are part of the the zebrafish in toxicology book. Evaluation of rapid cooling and tricaine methanesulfonate ms222.
Transplant recipient zebrafish were embedded in 1% lowmeltingpoint agarose containing 0. Eighteen male zebrafish abwt, date of fertilisation. Teaching stress physiology using zebrafish danio rerio. Anesthetize the male fish by immersion in tricaine see recipes, chapter 10. Guidelines for use of zebrafish in the nih intramural research program. Chapter 2 breeding embryo production by in vitro fertilization source.
Includes content submitted by the community and imported from the zebrafish book, 5th edition. Euthanasia of zebrafish must be carried out by one of the following methods. Embryos were irradiated at 24 hpf and survival was scored every day up to 7 dpf. His most recent interests included the development of a zebrafish model for c. Lastly, the tetracycline tet or doxycycline doxinducible teton system 10,11 has been used in zebrafish to conditionally control tetresponsive transgene expression in specific cell. Behavioural effects of the commonly used fish anaesthetic. The zebrafish atlas contains 2 and 3dimensional, anatomical reference slides of zebrafish to support research and education worldwide.
In the current era, the commercial transition of nanotechnology from research level to industrial level is stimulating the worlds total economic growth. Adult zebrafish as a model organism for behavioural genetics. Recent research has demonstrated the suitability of adult zebrafish to model some aspects of complex behaviour. The fish were euthanized by rapid cooling and tricaine methanesulfonate ms222, based on the method of wilson et al.
Overdose of tricaine methane sulfonate ms222, 200300 mgl by prolonged immersion. Many hatcheries and research studies use tms to immobilize fish for marking or transport and to suppress sensory systems during invasive procedures. The zebrafish as a model system for analyzing mammalian and. Buffered tricaine methanesulfonate ms222 is the anesthetic typically used to provide surgical level anesthesia in zebrafish danio rerio, and most researchers use ms222 at a concentration of 164 mgl. Pdf anaesthetic tricaine acts preferentially on neural voltage. Rise of zebrafish as a model for toxicology toxicological. Functional behavior and reproduction in androgenic sex. Zfin community wiki general information zfin community wiki. Standard operating procedures chen laboratory zebrafish. Wholemount in situ hybridizations wholemount in situ hybridizations were performed essentially as described, with the following modifications.
Improper tms use can decrease fish viability, distort physiological data. Fish should be left in the solution for at least 10 minutes following cessation of opercular. The zebrafish atlas of macroscopic and microscopic anatomy the zebrafish danio rerio is avaluable and common model for researchers working in the fields of genetics, oncology, and developmental sciences. Tricaine ttbs western blot color development buffer. We generated two transgenic lines using the xenopus rhodopsin promoter to drive the reverse. This is where zebrafish researchers can share information with the rest of the community. The zebrafish in toxicologylinks wikibooks, open books for. Zebrafish danio rerio, the cyprinid schooling teleost, is an ideal model for studying genetics. Due to the covid19 pandemic, resource distribution has been suspended and services may be delayed. Zebrafish as a model system to study toxicology dai 2014. The isolation and molecular analysis of zebrafish behavioural mutants is now starting. Efficacy and safety of 5 anesthetics in adult zebrafish danio rerio. Effects of the proteasome inhibitors ps341 a, mg2 b and lactacystin c on radiation sensitivity of zebrafish embryos.
Anesthesia, analgesia, and euthanasia of the laboratory. Maintain breeding males and females on a daily schedule as described in the zebrafish breeding schedule for maximal embryo production page 2. All orders will be processed in the order received. Studies of reward behaviour, learning and memory, aggression, anxiety and sleep strongly suggest that conserved regulatory processes underlie behaviour in zebrafish and mammals. We developed a teton system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. The zebrafish as a model system for analyzing mammalian. Thus, the zebrafish is becoming a powerful model organism for studying genetics 68, development 911, environmental toxicology 1115, pharmacology 16, 17, dna damage repair 1820, cancer 4, 21, and other diseases 2224. Zebrafish photomotor response study july 2016 ntp update revision of the august 2015 update synopsis the national toxicology program ntp1 conducted a photomotor response study in zebrafish danio rerio to evaluate the toxicity of chemicals associated with the west virginia elk river chemical spill. Phthalates are extensively used as plasticizers in a variety of dailylife products, resulting in widespread distribution in aquatic environments. Large numbers of synchronously developing embryos can be obtained by in vitro fertilization. Tricaine methanesulfonate or ms222 is the most used anaesthetic in fish and is. Tricaine methanesulfonate ms222 application in fish anaesthesia.
B inhibitors alleviate and the proteasome inhibitor ps. Zebrafish also includes research with other aquarium species such as medaka, fugu, and xiphophorus. Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. Standard operating procedures chen laboratory zebrafish facility. Adult zebrafish as a model organism for behavioural. If a page of the book isnt showing here, please add text bookcat to the end of the page concerned. Rinse in fish water, and place belly up in a slit in a damp sponge. Danio rerio are among the smallest members of this family, with adults measuring 30 40 millimeters, or about 1. For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. Adult fish that require euthanization due to infertility, age, or experimental need should be anesthetized with lethal dose of tricaine 200300 mgl in system water or e3 solution containing 5 mm nacl, 0. Despite the progressively increasing use of zebrafish danio rerio in research, the most humane method of euthanasia for these fish has not been determined. The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. Preparation of medium for zebrafish embryios e3 medium.
Buffered tricaine methanesulfonate ms222 is the anesthetic typically used to provide surgical level anesthesia in zebrafish danio rerio, and most researchers. Microrna expression in zebrafish embryonic development. Make up a 15 mm stock concentration of tricaineethyl 3aminobenzoate methanesulfonate salt as instructed in the zebrafish book 23. At the beginning of the trial, 20 fish were euthanized, ground into. The tricaine fish showed the following responses mean sd. Ethyl 3aminobenzoate methanesulfonate ms222 is the standard fish anaesthetic, widely used in zebrafish, but concerns have been raised regarding aversion and stress induction 6,7. Tricaine methanesulfonate, also called tricaine or ms222, is the only fdaapproved anesthetic for use in fish in the united states and is one of the most widely used anesthetic agents for poikilotherms worldwide. A toxicological study using zebrafish danio rerio as a model 12 introduction there has been an increase in the demand for new, robust, and costeffective ways to assess chemicals for their effect on human health, particularly early development. Two types of teton transgenic lines for doxycycline.
Holden was a member of the department of pathology at the university of utah school of medicine. Signal was detected using cdpstar chemiluminescent substrate roche. Dec 31, 2010 tricaine methanesulfonate tms is an anesthetic that is approved for provisional use in some jurisdictions such as the united states, canada, and the united kingdom uk. Confocal microscopy was performed for gfppositive cells using a zeiss lsm meta confocal microscope. This reversible nature of zebrafish feminization could be related to the developmental pattern in zebrafish.
As a local anaesthetic, ms222 may act more as a muscular blocking agent rather than as an anaesthetic 8, and it may reduce heart rate and causes high mortality under longterm sedation 9. The effects of tricaine methanesulfonate ms222, a commonly used fish anesthetic, on plasma nonesterified fatty acid levels nefa were examined in rainbow trout,oncorhynchus mykiss. A derivative of benzocaine, it has an additional sulfonate radical. We will contact you to arrange shipments when normal operations are resumed. Zebrafish is under the editorial leadership of editorinchief stephen c. Welcome zebrafish in the classroom is designed to be a resource for teachers and students who are using zebrafish in undergraduate courses. Tricaine can lead to a high level of mortality when zebrafish larval and adult are exposed over multiple days chen et al. This study evaluates the movement of zebrafish in response. Total nefa levels declined with increasing duration of exposure to ms222. The isolation and molecular analysis of zebrafish behavioural mutants is now starting, allowing the. A fresh look at zebrafish from the perspective of cancer. Evaluation of zebrafish kidney function using a fluorescent. With the increase in the laboratory use of zebrafish, several advantages, such as a high genetic homology to humans and transparent embryos, which allow clear disease. Euthanasia guidelines euthanasia of zebrafish must be carried out by one of the following methods.
Zebrafish as a model system to study toxicology dai. Ekker, phd, department of biochemistry and molecular biology, mayo clinic. This material is from the 4th edition of the zebrafish book. The upper two bars show the sex ratio of zebrafish danio rerio in a control group con.
Zebrafish embryos as an alternative to animal experiments. Males were anesthetized by immersion in 168 mgl tricaine methanesulfonate solution ms222. The zebrafish as a model system for analyzing mammalian and native. Anesthetize the embryos and consequently immobilized them to perform the microinjection procedure. Immobilization by submersion in ice water 5 parts ice1 part water, 04. Nov 12, 2008 research using the zebrafish model has experienced a rapid growth in recent years. Efficacy and safety of 5 anesthetics in adult zebrafish. Jun 01, 2018 males were anesthetized by immersion in 168 mgl tricaine methanesulfonate solution ms222. Includes antibodies submitted by the community and imported from zfin. The aim of the present study was to examine whether exposure to mono2ethylhexyl phthalate mehp, the hydrolytic metabolite of di.
Evaluation of rapid cooling and tricaine methanesulfonate. Tricaine methanesulfonate ms222 as methods of euthanasia in zebrafish danio rerio. Presently, nanotechnology is a multitrillion dollar business sector that covers a wide range of industries, such as medicine, electronics and chemistry. Thyroid endocrine disruption in zebrafish larvae after. Adult fish were fixed in 4% paraformaldehyde overnight. Zebrafish get their name because they resemble zebras. Tms is the only anesthetic licensed in the united states for fin fish that are. A toxicological study using zebrafish danio rerio as a model. The protocol for zebrafish use and maintenance was approved by the research animal resources center of the university of wisconsinmadison, which follows the national institutes of health guide to the care and use of laboratory animals protocol no. The zebrafish, danio rerio, has become another popular model organism with which to study fundamental biological questions. Wildtype zebrafish were used for northern blot analysis and microarray analysis. This study greatly promoted the development of genetic analyses in zebrafish 1. A new anaesthetic protocol for adult zebrafish danio. Zebrafish embryos and larvae were handled in strict accordance with good animal practice as approved by the appropriate committee mdi biological laboratory animal core iacuc number 20.
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