Reprinted with permission from the authors, Nature Publishing Group, and Lab Animal. The definitive, original article (Matthews M, Trevarrow B, Matthews J (2002) A Virtual Tour of the Guide for Zebrafish Care and Users. Lab Animal. 31(3):34-40) is on the Nature Publishing Group (http://www.npg.nature.com) site Lab Animal (http://labanimal.com). This paper can only be downloaded from this AWIC publication for personal study use and cannot be copied for commercial purposes without the permission of the publisher. Contact information for Lab Animal is as follows: PO Box 5054, Brentwood, TN 37024-5054. Customer service #'s are 800-524-0384 or 615-377-3322. (http://www.labanimal.com). RESOURCE Volume 31, No. 3 Lab Animal March 2002 A Virtual Tour of the Guide for Zebrafish Users Monte Matthews, Bill Trevarrow, PhD, Although fish have long been used in bio- Because PHS Policy on the Humane Care and Jennifer Matthews, DVM, PhD medical research and testing, only in the and Use of Laboratory Animals9 (PHS last 30 years has the zebrafish (Danio rerio) Policy) defines animal as “Any live, verte- become an important research tool1.The brate animal used or intended for use in PHS-funded and AAALAC-accredit- explosion of developmental biology, neu- research, research training, experimenta- ed facilities are required to use the robiology, and genetics research2-4, as well tion, or biological testing or for related Guide as the basis for setting up a as environmental science, teratology, car- purposes,” institutions that receive PHS zebrafish care and use program. cinogenicity testing, and reproductive and funds or support must have a defined pol- behavioral studies3-6,have contributed to icy Assurance that describes the institu- The authors describe how they the rise in popularity of the laboratory tion’s compliance with the PHS Policy and accomplished this task at the zebrafish. the Guide. University of Oregon Zebrafish Several zebrafish qualities contribute to Although the Guide and PHS Policy do Facility. their suitability as models for biomedical not provide specific guidelines for the use research. First, they are easy to maintain in of zebrafish, the Office of Laboratory large numbers, readily reproducing under Animal Welfare (OLAW) states, “Many of laboratory conditions. Second, adult fish the principles embodied in the Guide, can be subjected to mutagenesis and hap- although not specifically addressing cold- loid embryos screened for mutations in the blooded vertebrates, generally can be first generation. Third, the zebrafish adapted to animal care and use programs embryo has few cells relative to other verte- for various kinds of amphibians, reptiles, brates, making it a “simple” model for and fishes10.” Detailed descriptions of the more complex vertebrates; moreover, the care and use of zebrafish are clearly beyond embryo is transparent and develops very the scope of this article, but we will rapidly and externally, permitting ready demonstrate in broad terms—based on observation of the events involved in dif- our experiences with zebrafish at the ferentiation of tissues such as the nervous University of Oregon—how the principles system. Fourth, direct access to the devel- in the Guide can be applied to the use of oping embryos permits such experimental zebrafish in animal research programs, manipulations as introducing foreign referring to each chapter in the Guide,with genetic material and labeling of cells. the exception of the physical plant descrip- Finally, their small size allows the large tion. numbers of zebrafish required for genetics The 1996 revision of the Guide,which studies to be easily maintained7. emphasizes performance-based standards, M. Matthews is Director, Office of Many of the institutions using zebrafish allows institutions to develop and define Veterinary Services and Animal Care, for research, testing, or teaching are funded their own goals, the methods for achieving B. Trevarrow is Zebrafish Facility Director by the Public Health Service (PHS) and/or those goals, and the means for evaluating and Interim Director of the Zebrafish accredited by the Association for them. This approach is particularly useful International Resource Center (ZIRC), Assessment and Accreditation of for zebrafish users who have few engineer- and J. Matthews is Research Associate Laboratory Animal Care International ing standards to follow8. and veterinarian, ZIRC, University of (AAALAC). Therefore, these institutions Although zebrafish are not covered by Oregon. Please send reprint requests to should use the Guide for the Care and Use United States Department of Agriculture M. Matthews at 5218 University of of Laboratory Animals8 (Guide) as a basis (USDA) regulations, the University of Oregon, Eugene, OR 97403-5218, or for designing, implementing, and evaluat- Oregon IACUC has chosen to adopt a sin- email: [email protected]. ing the program for zebrafish care and use. gle standard of care when dealing with ani- 34 March 2002 Lab Animal Volume 31, No. 3 RESOURCE mals that are not covered by both the PHS for husbandry and care, and have standard in the Guide,“An occupational health and Policy and USDA regulations. For instance, procedures for minimization of pain, dis- safety program must be a part of the over- we use USDA’s 12-hour rule for defining a tress, discomfort and injury, the University all animal care and use program. The pro- study area rather than PHS’s 24-hour rule of Oregon has adopted a template gram must be consistent with federal, state, for animal facility. Also, for painful or dis- Zebrafish Protocol Form7.This form still and local regulations and should focus on tressful procedures, we require the princi- requires the Principal Investigator to fill maintaining a safe and healthy work- pal investigator who works with zebrafish out all the animal care and use procedures place8”. An OHS program should be based to perform an alternatives search (USDA that are not described in the template pro- on the Guide, Occupational Health and requirement). tocol or the Zebrafish Book12, and are Safety in the Care and Use of Research unique to the study. Animals13, and Biosafety in Microbiological Institutional Policies and and Biomedical Laboratories14. Essential to Responsibilities Personnel Qualifications and the OHS program are hazard identification PHS-funded and AAALAC-accredited Training and risk assessment, personnel training, zebrafish facilities must have an Institutional All personnel who work with zebrafish personal hygiene, facilities, procedures and Animal Care and Use Committee (IACUC) must be adequately trained in the tech- monitoring, personal protection, medical to oversee the animal program, facilities, and niques described in the protocol or includ- evaluation, and preventive medicine. animal procedures, and to ensure that the ed in general zebrafish husbandry and care. Those who work with zebrafish at the institution’s program is based on the Guide Training or instruction must be made University of Oregon are required to par- and PHS Policy.The US Government available to all researchers, technicians, stu- ticipate in the OHS program. As part of Principles for the Utilization and Care of dents and other personnel involved in our training OHS program, we use a one- Vertebrate Animals Used in Testing, Research, zebrafish care or use. Most training at our page handout designed specifically for and Training11 form the basis of the Guide zebrafish facility is task-specific and ori- those working with fish that conveys infor- and can be used by IACUCs to evaluate their ented either to the individual or to small mation about zoonoses, personal hygiene, program and individual animal use proto- groups. and other hazards associated with animal cols. For example, regarding the minimiza- exposure. Participation in the OHS pro- tion of discomfort, distress, and pain, Occupational Health and Safety of gram is linked to the animal use protocol7. Principle IV states, “Unless the contrary is Personnel Aside from food poisonings, the overall established, investigators should consider As required by PHS Policy and as stated incidence of transmission of disease-pro- that procedures that cause pain or distress in human beings may cause pain or distress in Known and potential fishborne zoonoses. other animals.”Because little is known about Mycobacterium spp. zebrafish pain, distress, and discomfort, the Organisms in the genus Mycobacterium are nonmotile, acid-fast rods. There are multiple atypical (non- University of Oregon IACUC uses this prin- tuberculosis) species of Mycobacterium (M. marinum, M. fortuitum, M. chelonae, M. abscessus) that are recognized pathogens of laboratory zebrafish. Humans can be infected by contamination of lacerated or ciple when evaluating potentially painful or abraded skin with aquarium water or fish contact. A localized granulomatous nodule may form at the site stressful procedures. of infection, most commonly on hands or fingers. The granulomas usually appear approximately six to eight weeks after exposure to the organism. They initially appear as reddish bumps (papules) that slow- PHS Policy Section IV and Chapter 1 of ly enlarge into purplish nodules. The infection can spread to nearby lymph nodes. More disseminated the Guide describe general IACUC func- forms of the disease are likely in immunocompromised individuals. It is possible for these species of tions and responsibilities. The protocol Mycobacterium to cause some degree of positive reaction to the tuberculin skin test.