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-

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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 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. review process, personnel training, and Aeromonas spp. occupational health and safety (OHS) are Aeromonad organisms are facultative anaerobic, Gram-negative rods, which can produce septicemia in infected fish. The species most commonly isolated is A. hydrophilia. It is found worldwide in tropical fresh examples of some unique applications we water and is considered part of the normal intestinal microflora of healthy fish. Humans infected with have developed for zebrafish users. Aeromonas may show a variety of clinical signs, but the two most common syndromes are gastroenteritis and localized wound infections. Again, infections are more common and serious in the immunocompro- mised individual. Protocol Review Other Bacteria and Protozoa Sections IV.C and IV.D in PHS Policy Below is a list of additional zoonotic organisms that have been documented in fish or aquarium water. and Chapter 1 in the Guide describe those Human infections are typically acquired through ingestion of contaminated water resulting in gastroen- components that need to be addressed in teritis symptoms or wound contamination. the animal care and use protocol or activi- Gram-negative Organisms: Plesiomonas shigelloides, Pseudomonas fluorescens, Escherichia coli, ty.Because many of our zebrafish Salmonella spp., Klebsiella spp., Edwardsiella tarda researchers perform identical animal use Gram-positive Organisms: Streptococcus, Staphylcoccus, Clostridium, Erysipelothrix, Nocardia procedures, have similar reasons for using Protozoa: Cryptosporidium zebrafish, use a centralized zebrafish facility

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ducing agents from fish to humans is low. variables such as water quality, food and minimum variation in size. For adult fish, In general, humans contract fishborne dis- feeding regimen, size, and age, and there- lack of mortality and the ease and repro- ease through ingestion of infected fish tis- fore may require optimization in each ducibility of breeding success define suc- sues or aquarium water, or by contamina- facility (see below). cessful husbandry. tion of lacerated or abraded skin. To mini- Zebrafish kept together for breeding Temperature and humidity: Zebrafish mize the possibility of exposure to one should have some means to escape from can tolerate a fairly wide temperature range. these agents, zebrafish handlers should more aggressive fish, in the form of either They have been kept for fairly long periods wash their hands regularly, wear gloves, more space or plantlike materials to be at 22–30˚C (71.6–86.0˚F) and can survive seek medical attention promptly if expo- used as hiding spaces. To prevent zebrafish temperatures of 18–32˚C (64.4–89.6˚F). A sure is suspected, and remember to inform from eating their eggs, the tank bottom widely used standard temperature for devel- their physician that they work with fish should be fitted so as to make the eggs opmental studies is 28.5˚C (or 83.3˚F). Our should they become ill. (∼1.0–1.5 mm diameter) inaccessible to the facility is frequently kept at this tempera- fish, yet easy to collect—for example, a ture. Heat-shock experiments have been Animal Environment, layer of marbles, an array of closely spaced carried out at temperatures above 30˚C (or Housing, and Management rods, mesh, or a box containing marbles or 86˚F). A gradual drop in temperature to covered with mesh. 22–23˚C (or 71.6–73.4˚F) to lower the Physical Environment Space recommendations: Zebrafish zebrafish metabolic rate is acceptable in Microenvironment and macroenviron- space requirements are usually given as emergencies, such as water system mechan- ment: The tanks or aquaria that contain numbers of fish per volume of water. As ical failures. the animals make up the microenviron- schooling fish, zebrafish can be kept at fair- Water temperature can be monitored ment, which predominantly comprises ly high densities. We use the following gen- by daily thermometer readings or by elec- water and the small air volume enclosed by eral guidelines. Starting with 20 eggs or tronic thermister readings. Automatic tem- the tank cover. The room housing the fish embryos per 100 ml water, the 20 fish can perature monitoring is preferable because tanks constitutes the macroenvironment. be kept in volumes ranging from 400 ml as problems can be detected immediately. There are many ways to link the micro and young larvae to 3 liters as the fish approach Thermostats are usually used to control the macroenvironments, depending on the juvenile stage. Recommended density for water temperature, either by heating the arrangement of the laboratory and water growing juvenile fish and holding adults is water directly or by heating the room air. system. five fish/liter. Growing fish are considered To maintain a given water temperature and Automatic water exchange can be to require more space than breeding compensate for evaporative cooling, one by either continuous or discontinuous adults, which require more space than should expect to heat the air a degree or flowthrough, or by recirculation. A flow- nonbreeding adults, but this requirement two warmer. through system introduces clean water into has not been critically tested for zebrafish. Ventilation: Proper ventilation for a the tank and discards outflow water. A recir- In small breeding tanks, a pair of fish can fish tank or facility depends primarily on culating water system processes the outflow be kept overnight in 1.5 liters or as many as sufficient oxygen (O2) supply and adequate water from the tanks by one or more filtra- six fish in 2.3 liters of water. carbon dioxide (CO2) removal. To facilitate tion steps to remove undesirable materials Depending on experimental parame- optimal O2/CO2 exchange, the surface area and compounds and restore desirable ones. ters, such criteria as survivability, growth and turbulence of the water in tanks can be

This treated water is then recirculated to the rate, and fecundity can be used to assess increased, bringing the O2-poor, CO2-rich tanks. Tanks maintained by manual water not only the adequacy of the space being water to the surface so that the diffusion of changes should be fitted with small filtration provided, but also the genetics of the fish, the gasses is driven by concentration differ- units that will continually remove undesir- the food quality, frequency of feeding, and ences. This can be accomplished in indi- able materials from the water. the water quality. For eggs and embryos the vidual fish tanks, centrally in a pump/fil- Housing: Zebrafish are usually kept in most appropriate criteria seem to be sur- tration area, or both, with aeration and gas transparent glass, Plexiglas (acrylic), or vival (80–95% is good) and final size exchange equipment. polycarbonate tanks or aquaria, permitting obtained (1.0–1.5 cm is good) over a stan- An adequate dissolved oxygen (DO) easy observation of the animals. Tank dard time period (0–21 days post fertiliza- reading is 6.0 p.p.m. (mg/l). Fish that drains should be screened to keep fish in tion in our facility). During the first few remain close to the surface of the water and the tank. Ideally, the drains should be days fish larvae should be kept in shallow appear to be gulping air are probably not designed so that they require no cleaning water, so that they can gulp some air to getting enough O2.Whether a given tank until replacement. Zebrafish space require- inflate their swim bladder15.For juvenile contains adequate oxygen depends on ments for physiological, behavioral, and fish, a useful standard consists of rapid many variables: number and size of fish, social interactions are affected by other growth to a breedable adult size, with a tank surface area, aeration in the tank,

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temperature (oxygen is more soluble at Activity: Species-typical behaviors To minimize contamination, prepared lower temperatures), and rate of water include swimming, feeding, mating, and foods should be purchased in small exchange if there is central aeration. If pos- social interactions. Human interaction amounts, stored only a few months, and sible, the fish rooms should be kept at with fish can be stressful. Habituation to kept frozen or refrigerated in sealed con- slightly positive pressures to exclude poten- the presence of humans may reduce or tainers. All food containers should be tial pathogens. eliminate the stress. labeled with date of production (if Illumination: Illumination is important known), date of receipt, and date opened. for breeding and minimizing stress and dis- Husbandry Live foods should be cultured on the ease. For these purposes, standard fluores- Food: The precise nutritional require- premises, derived from environments con- cent lamps are sufficient. Eliminating cer- ments of zebrafish have not been deter- sidered unlikely to harbor pathogens of tain wavelengths may inhibit algae growth, mined. There are two nutritional models freshwater fish (e.g.,brine shrimp cysts), or but this is not as critical for fish health as for zebrafish—warm-water and cold-water treated after collection to decontaminate the intensity (between 5 and 30 ft cd or commercial aquacultural fish. Neither is (e.g.,bleach-decapsulated brine shrimp 54–324 lux at the surface of the water) and perfect. Zebrafish are warm-water cypri- cysts). Live foods collected directly from the circadian light cycle (14 h light, 10 h naformes. The commercial fish with well- the wild in fresh waters should not be used dark). Unlike other fish, zebrafish do not studied dietary requirements are cold- as food for laboratory zebrafish. require a seasonal change in their day water cyprinaformes or rather distantly Water: Critical for successful zebrafish length to bring them into a breeding state. related warm-water fish. maintenance are control and monitoring Fluorescent lighting is relatively inex- Zebrafish are fed different foods of water quality. An understanding of the pensive and easy to maintain. Fixtures sim- depending on their age. Young zebrafish complex chemical cycles and the many ilar to stack lights in libraries or asymmet- require small-sized food items that can be dynamic components involved can help in rical wall-washing lights used in commer- swallowed whole. Larger juveniles can manipulating or controlling parameters cial displays will provide better directed swallow larger items but also require more through chemical additions and filtration light and thus better illumination levels. food. Newly hatched zebrafish can eat processes. Among the chemical cycles are Noise: Zebrafish react to loud sudden paramecia (800 µm × 80 µm), as well as a the nitrogen, carbon, oxygen, and carbon- noises. Their sensitivity to various vibra- variety of prepared foods, , and ate and buffering cycles. These processes tions or sounds like talking or music is rotifers. As they grow larger, zebrafish partially overlap and can interact exten- uncertain. Although they do not display hatchlings can add to their diet larger items sively. One reason for choosing zebrafish as any obvious reactions to such sounds, such as vinegar eels, microworms, or larger a model system is their broad tolerance of whether or not they are stressed has not prepared foods. Eventually they are large water conditions. been well investigated. Fish raised in a par- enough to eat Artemia nauplii (newly Water quality parameters can be moni- ticular environment may adapt to the stim- hatched brine shrimp), which have a high tored by hand or by instruments. uli commonly present there. Fish raised in protein content and can be hatched on Procedures can use colorimetric reagents, one environment may become stressed on demand in large numbers, but the eggs can electrodes of various kinds, or various being moved to an unfamiliar one. be very expensive. Alternative foods in this combinations of the two. Which parame- Common design considerations and man- size include cultured Moina and larger ters should be monitored and what they agement decisions are usually based on sizes of prepared foods. indicate are complex issues that involve a human health and comfort. Juvenile AB fish in our facility thrive on good understanding of your particular live foods, an observation that is supported water system. Among the most useful water Behavioral Management by results of other investigators. Those parameters to monitor are concentrations + – Structural environment: As shallow- studies indicate that some less inbred pet of ammonia, [NH3/NH4 ]; nitrite, [NO2 ]; – 2+ water schooling fish, zebrafish seem more store zebrafish do as well on either live or nitrate, [NO3 ]; calcium, [Ca ]; and mag- or less indifferent to environmental enrich- prepared foods, whereas the AB fish still do nesium, [Mg2+]; temperature; pH; buffer- ments, with the possible exceptions of such better on live foods than prepared foods. ing (or alkalinity); salinity (either as dis- environmental irregularities that become a Hence, facility-specific growth rate and solved solids or conductivity); and levels of focus of egg-laying during mating. survival tests should be used to optimize dissolved oxygen (DO). In addition, it is Social environment: Although their results. Adult-size fish can be fed adult pre- sometimes important to determine the schooling habits indicate they are somewhat pared foods ( flake foods, trop- chlorine concentration, [Cl2]—as distinct sociable, zebrafish may not require social ical fish micropellets, and ground trout from the chloride ion concentration, [Cl–], interactions. Indeed, zebrafish have been meal) and live adult brine shrimp. which is a component of salinity—and the kept in isolation for extended periods, after Alternative live foods include Artemia, total dissolved gases (to determine whether which they have still bred successfully. Moina, and Drosophila larvae. or not the water is supersaturated).

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Wedemeyer 16,Moe17, and Spotte18 should in a recirculating system is typically paper and cardboard. Sodium borate treat- be consulted for a comprehensive review of exposed to UV light to kill bacteria in the ment is fairly effective against insects and is water-quality parameters. water, this treatment should not be expect- unlikely to harm the fish. Monitoring should be sufficiently fre- ed to provide 100% protection against any Emergency, weekend, and holiday care: quent to detect problems. Some parame- organism. The choice of which water-qual- An emergency or disaster plan should ters should be checked several times daily, ity parameters to monitor depends on the address all reasonably foreseeable situa- others once a day, some only once a week system design (e.g., in a flow-through water tions. With electrical power outages and or less. For example, mechanical systems, system using dechlorinated tap water, fre- mechanical equipment failures high on the such as pumps, should be continuously quent monitoring of chlorine levels is list of failure scenarios, minimal precau- monitored. Some water-quality parame- essential). tions include having backup equipment ters that change rapidly, like DO and salin- Sanitation: A good sanitation program and an electrical generator. ity, should be monitored daily. Other para- prevents buildup of waste products and Weekend and holiday care should pro- + – – meters, such as NH3/NH4 ,NO2 ,NO3 , potential cross-contamination between vide at least minimal feeding for adults and and calcium, can be monitored weekly. tanks. Holding and breeding tanks, and feeding and care for the baby fish. Adult These monitoring frequencies will depend their tops, can be washed with disinfectant fish can tolerate a few days without food on each facility's set-up and experience. or sterilized by autoclaving. Racks and but require daily feeding for optimal egg Several companies produce systems that rooms can also be sanitized periodically as production. Because larval fish require can automatically monitor, log data, trig- necessary. Nets used with fish can be steril- normal care daily, it may be advisable to ger alarms at certain thresholds, and dial- ized by autoclaving or disinfected by schedule fewer fish in the nursery during out to people who are “on call.” bleaching. Tank surfaces need regular holidays to reduce labor requirements. As outlined above, water systems can be cleaning to permit easy viewing of the fish. flowthrough or recirculating, and central- Waste disposal: Tank wastes, including Population Management ized or distributed. A flowthrough water uneaten food, fish feces, and dead, decayed Identification and records: Identifica- system maintains good water quality by fish, must be removed from the tank by tion labels placed on zebrafish tanks exchanging old water for new. Although all hand (siphoning), by the tank’s filter, or by should contain information about the water systems exchange some water, recir- a centralized (room-scale or rack-scale) genetic background, stock number, the culating water systems rely more heavily water system filter. Filters, if used, must also date of fertilization, and the researcher’s than flowthrough systems on maintaining be cleaned to remove filtered material from name. In larger facilities, these records may good filtration processes. A well-operated the water system. Tank wastes, as well as be kept in computer database files. A recirculating water system will provide euthanized fish and wastewater, will usually downloadable version of such a database greater control over the fish environment be disposed of through municipal sewer can be obtained from the University of and less sensitivity to external environ- lines. Important considerations include Oregon Zebrafish Facility website mental variables. Centralized water sys- preventing contamination of the local envi- (http://darkwing.uoregon.edu/∼zfish/). tems maintain water quality using a rela- ronment either with disease pathogens Some facilities also use barcodes to auto- tively small number of pieces of equip- (perhaps being used intentionally in the mate and reduce data entry error. More ment serving many fish tanks. More dis- laboratory) or with foreign organisms— sophisticated systems have their files on a tributed systems will involve fewer tanks recalling that zebrafish are native to water- dedicated server powered by an uninter- being served by a given set of equipment, sheds in and around India and Myanmar. ruptible power source (UPS), with auto- whereas more centralized systems general- Pest control: Pests most often encoun- matic backup. ly make more efficient use of labor in tered in fish facilities are arthropods: Genetics and nomenclature: Genetic maintenance and can cost less. Because roaches, silverfish, and lice. Control con- nomenclature guidelines have been estab- centralized water systems circulate essen- sists predominantly of prevention by elim- lished for zebrafish mutations and wild- tially the same water through many or all inating pest food, hiding places, and access. type lines. These guidelines are briefly tanks, a water quality parameter affecting Minimize fish food on the floor or the lid explained on the Zebrafish Information many tanks can be measured with one test of the tanks through careful feeding meth- Network (ZFIN) nomenclature page in a single location. By contrast, in a dis- ods that result in little spillage, as well as (http://zfish.uoregon.edu/). Zebrafish tributed water system, the test would have good sanitation in all areas. Control pest genetic composition is determined from to be repeated for each independently reg- access by tightly closing doors and by the records of the parents that generated ulated body of water. On the other hand, blocking conduits and other less obvious the fish, phenotypic classifications of the distributed water systems provide greater paths. Eliminate insect hideouts by treating fish and their siblings, and genetic and biosecurity because water is not shared spaces behind walls and between wall studs molecular tests to determine if fish carry among as many tanks. Even though water with boric acid and removing unnecessary particular recessive traits.

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Veterinary Medical Care filled with packing chips. ness, redness, hemorrhage), changes in Animal Procurement and For international shipments, additional shape (weight loss, bloating, skeletal defor- Transportation paperwork may be necessary, including a mity, masses/swellings, exophthalmia), Specific zebrafish mutants and wild- letter to customs agents, a customs invoice, external lesions (ulcerations, fin erosion, type lines can be obtained from stock cen- and perhaps fish import permits. Forms gas bubbles, protruding scales), behavioral ters, other laboratories, or commercial that designate certain customs agents and alterations (rapid breathing, loss of equi- dealers. The ZFIN website lists many sup- specific airports can facilitate the shipment librium, lethargy, erratic movements, gath- pliers. handling, as well. Unlike domestic ship- ering at water surface), and increased mor- Stock centers and academic laboratories ments, international shipments are often tality. When moribund or dead fish are generally provide the expected mutations timed to be in transit during the weekend. noted, a clinical investigation is warranted and wild-type strains. Their facilities vary and a veterinarian trained in aquatic ani- somewhat in their health surveillance and Preventive Medicine mal medicine should be consulted. Water quarantine procedures. Some will ship out Quarantine, stabilization, and separa- quality and husbandry conditions should bleached eggs (facilitating the quarantine tion: All newly acquired zebrafish should be reviewed. Depending on the problem, procedures of the receiving institution). An be quarantined to avoid introducing dis- diagnostic workups can include skin scrap- academic institution’s shipment response ease into a colony. The health status of ings, fin biopsy, gill biopsy, necropsy, bacte- time can vary as a result of limited avail- incoming fish is rarely known, and the riology, virology, and histopathology. ability of rarely requested mutant strains. stress of shipping can often cause underly- Histopathology is a particularly useful In contrast, purchases from commercial ing diseases to become apparent. The pre- diagnostic technique, because the tiny dealers are usually genetically poorly ferred quarantine method is absolute, zebrafish can be fixed and sectioned whole, defined “wild types.” wherein only surface-sanitized embryos permitting the examination of all primary Zebrafish are frequently transported are introduced into a facility. An isolated organs on a single microscope slide. A use- between laboratory facilities. Transporting location and water system should be desig- ful tool for monitoring recirculating water fish over the short distances on a campus nated as the quarantine area. This can systems is the sentinel fish. Kept in a sepa- can be as simple as placing adult fish in a range from a simple individual aquarium rate tank that is fed with water from the bag in a box or, more commonly, in a small with a self-contained filter unit to an entire dirty or return water , these fish are covered plastic box (Tupperware® or poly- separate room with many tanks and a sampled periodically for disease investiga- carbonate filled with fish system water). flowthrough water system. Newly arrived tion (Fig. 1). The Zebrafish International Eggs or larvae can be transported in cov- fish should be equilibrated over an hour or Resource Center Pathology Service is one ered petri plates, beakers, or tissue culture two by floating the bag in the new tank and flasks. periodically introducing a small amount of Transporting zebrafish long distances to the new tank water into the bag. If the other facilities is more complicated. The shipment water is in poor condition, it may packing requirements are more stringent. be necessary to transfer the fish immedi- Adult fish should be double-bagged in a ately. Once introduced into their new tank, good-quality plastic fish shipping bag at a the quarantined fish remain there for three density of about 10 fish to a half gallon of to four weeks. During this time, the fish are water. The bag should be about two-thirds observed closely for signs of disease and full of air or oxygen. Food should be with- treated, if necessary. The new fish are then held for a day before shipping so that fish bred and the embryos are surface-sanitized will produce less ammonia while confined. with a mild bleach solution (35 mg/liter FIGURE 1. Schematic of a sentinel fish Amquel (a commercially available ammo- sodium hypochlorite for 5 min). Only tank on a water system. A mixture of nia sequestrator) can be added to bind up these sanitized embryos are introduced potential pathogens are collected from all any ammonia that is produced. Zebrafish into the main aquarium facility. Upon con- the tanks served by the water system in the water flowing into the return water eggs and larvae are usually packed 200–300 firmation that the fish strain has been suc- sump. This untreated water (red) is then to a 250- to 500-ml tissue culture flask filled cessfully established from these embryos, pumped out of the sump to be filtered and ∼50–90% with water. Ideally, the sender the adults in the quarantine area are euth- UV-irradiated before it is returned to the tanks (black). A side stream (blue) is bleaches the eggs and places them in sterile anized. directed into the sentinel tank before any water. Methylene blue (0.5 mg/liter or 0.5 Surveillance, diagnosis, treatment, and filtration or irradiation treatment. The sen- p.p.m.) can also be added to the water to control of disease: Fish should be observed tinel fish thus provide a good, easily sam- pled system for detecting any pathogens reduce fungal growth. The packing box daily for signs of illness or injury. Signs of being shed by any of the fish served by should be insulated and any extra space disease in fish include color changes (pale- the water system.

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zeb.pdf. pathology service that offers complete diag- Because most procedures performed on 8. Institute of Laboratory Animal Resources, nostic services and consultations on zebrafish are very rapid, the need for a National Research Council. Guide for the Care and Use of Laboratory Animals zebrafish health and husbandry (see maintenance phase of anesthesia is usually (National Academy Press, Washington DC, http://zfin.org/zf_info/stckctr/health.html). not necessary. Maintenance anesthesia 1996). Most treatments for zebrafish disease doses would be lower (50-100 mg/L)19. 9. Office of Laboratory Animal Welfare. Public Health Service Policy on Humane Care have been adapted from the tropical pet During induction, spontaneous ventilation and Use of Laboratory Animals fish industry or food fish aquaculture. should be monitored closely and can be (Washington DC, 1996). 10. Potkay, S., Garnett, N., Miller, J.G., Pond, Among the many considerations involved used as an indicator to the depth of anes- C.L. & Doyle, D.J. Frequently asked ques- in initiating a treatment protocol are the thesia. tions about the Public Health Service diagnosis, the number of fish being treat- Policy on Humane Care and Use of Laboratory Animals. Contemp. Top. Lab. ed, the drug and route of administration, Euthanasia Anim. Sci. 36, 47-50 (1997); and the potential for toxicity to biological Zebrafish should be euthanized by http://grants.nih.gov/grants/olaw/refer- ences/faq_labanimals1997.htm#6 filtration. Before starting a generalized methods consistent with the 2000 Report of 11. Interagency Research Animal Committee. treatment, one should always test a drug the AVMA Panel on Euthanasia20.The US Government Principles for the on a few fish. method chosen depends on the researcher, Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training facility, or the intended use of the fish after (1985). Surgery euthanasia. An overdose of MS-222 is the 12. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish The most common surgical procedure most common method, using a slightly Danio(Brachydanio) rerio (University of done on zebrafish is a caudal fin clip to col- more concentrated solution (e.g., 200–500 Oregon Press, Eugene, 2000). 13. Committee on Occupational Safety and lect tissue for DNA isolation and PCR mg/liter) than is typical for anesthesia. Fish Health in Research Animal Facilities, analysis. The technique permits screening are left in the MS-222 solution for 5–10 Institute of Laboratory Animal Resources, for fish carrying mutations in a particular min following the cessation of opercular National Research Council. Occupational Health and Safety in the Care and Use of gene. With practice one can do the proce- movement. A second method of euthaniz- Research Animals (National Academy dure very rapidly and cause no bleeding. ing zebrafish is immobilization by submer- Press, Washington DC, 1996). 14. US Department of Health and Human No presurgical cleansing of the caudal fin sion in ice water followed by cranial con- Services, Centers for Disease Control and should be necessary, but gloves should be cussion and decapitation using an in-sink Prevention & National Institutes of Health. worn and the surgical area should be clean. garbage disposal. Another method, useful Biosafety in Microbiological and Biomedical Laboratories 4th edn (US Government Before doing surgery, small (500 ml) indi- when tissues must be preserved, is anesthe- Printing Office, Washington DC, 1999). vidual tanks containing clean fish water for sia with MS-222 followed by quick freezing 15. Goolish, E.M. & Okutake, K. Lack of gas bladder inflation by larvae of zebrafish in anesthesia recovery and holding should be in liquid nitrogen. the absence of an air-water interface. J. set up. Individual fish should be identified Fish Biol. 55, 1054-1063 (1999). and isolated until the PCR analysis is com- Received 1/10/02; accepted 2/1/02. 16. Wedemeyer, G. Physiology of Fish in Intensive Culture Systems (Kluwer plete. Another surgical manipulation of Academic Publications, Boston, 1996). zebrafish is tattooing. 17. Moe, M.A. Jr The References Reference: Systems and Invertebrates 1. West, R. Big splash. Oregon Quarterly (Green Turtle Publications, Plantation, Pain, Anesthesia, and Analgesia Spring, 22-26 (1996). 1989). 2. Klontz, G W. in Animal Models for 18. Spotte, S. Captive Seawater Fishes: Although it is generally accepted that Biomedical Research Vol. 4 27-30 Science and Technology (Wiley- fish do experience pain, it can be difficult (National Academy of Sciences, Interscience, New York, 1992). to assess. Signs of pain or distress could Washington DC, 1971). 19. Mashima, T.Y. & Lewbart, G.A. Pet fish for- 3. Laale, H.W. The biology and use of mulary. Vet. Clin. North Am. Exotic Anim. include escape behavior or frantic move- zebrafish, Brachydanio rerio in fisheries Prac. 3, 117-130 (2000). ments, increased respiration (rapid move- research—a literature review. J. Fish Biol. 20. 2000 Report of the AVMA Panel on 10, 121-173 (1977). Euthanasia. J. Am. Vet. Med. Assoc. 218, ment of opercula), and blanching of color. 4. Vascotto, S.G., Beckham, V. & Kelly, G.M. 669–696 (2001). To decrease overall stress, fish are anes- The zebrafish’s swim to fame as an experi- thetized for all procedures that could cause mental model in biology. Biochem. Cell Biol, 75, 479-485 (1997). pain and distress or that require temporary 5. Travis, J. Gone fishing! Scientists use immobilization. The most common anes- mutant zebrafish to learn how vertebrate embryos develop. Sci. News 150, 360-361 thetic agent used is MS-222 (tricaine (1996). methanesulfonate) in an aqueous solution. 6. Bunton, T.E. The use of an aquatic bioas- Fish are induced rapidly following immer- say in carcinogenicity testing. Lab Anim. (NY) 19, 46-47 (1990). sion in a solution containing MS-222 (100- 7. Animal use application to the Institutional 200 mg/L)12,19 and are recovered by return- Animal Care & Use Committee (IACUC) Univeristy of Oregon: Zebrafish Form, ing them to fresh, well-aerated water. http://zfin.org/zf_info/zfbook/chapt1/anim-

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