This issue of the ILAR Journal explores the potential

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1 Testing the Waters: IACUC Issues Associated with Fish Stephen A. Smith and Laura E. Noll This issue of the ILAR Journal explores the potential for fish to perceive pain and experience distress. The articles describe challenges in assessing fish welfare, anatomical adaptations for pain perception in fish, morphological changes that occur in fish under stress, drugs used to minimize pain and distress in fish, and the use of behavioral responses to evaluate pain and distress in fish. This article addresses considerations for principal investigators (PIs) and the members of institutional animal care and use committees (IACUCs) in the presentation and review of protocols calling for the use of fish in a research, laboratory, or teaching setting, with a particular emphasis on minimizing the potential for the animals pain and distress. The information is intended to enhance the thinking and decision making of investigators, IACUC members, and institutional administrators concerning the use of these unique and highly diverse species. Challenges of Using Fish Researchers have described approximately 30,000 species of fish, representing an enormous diversity in morphology, behavior, nutrition, reproduction, and environmental habitats. In addition, morphological, physiological, and behavioral changes may occur in a particular species at different periods of its life. But only a fraction of fish species (about 100) are used for research, laboratory, or educational purposes, and specific information is available for an even smaller number (20 to 30), most commonly those of aquaculture importance (e.g., trout, catfish, carp, striped bass, goldfish, fathead minnows, zebrafish, medaka, guppies). It is therefore difficult to establish appropriate artificial environments and laboratory animal care procedures for most fish species (Casebolt et al. 1998; DeTolla et al. 1995; Westerfield 2000). In addition to the general lack of information about fish, most IACUCs and their members do not deal with fish or Stephen A. Smith, DVM, PhD, is Professor of Aquatic Medicine/Fish Health in the Department of Biomedical Sciences and Pathobiology at the VA/MD Regional College of Veterinary Medicine; and Laura E. Noll, BS, RLAT, ILAM, is IACUC Administrator in the Office of Research Compliance, both at Virginia Polytechnic Institute and State University in Blacksburg. Address correspondence and reprint requests to Dr. Stephen A. Smith, Department of Biomedical Sciences and Pathobiology, VA/MD Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Duck Pond Drive, Phase II, Blacksburg, VA or stsmith7@vt.edu. fish-related protocols on a regular basis, so the submission of a protocol for a fish species requires more time and attention by IACUC reviewers to ensure that all pertinent information is included. The protocol s author also should take additional time and care to make clear points that are unique to the species so that the reviewers are well informed about the species and the project. A Unique Feature of Fish: Water, Water, Everywhere One of the obviously unique features of fish, as opposed to terrestrial animals, is that they live entirely mate, spawn, and develop in water, which itself has particular qualities (e.g., temperature, salinity, ph, and dissolved oxygen). Most species have a preferred range for these parameters, but some require extremely specific conditions. Investigators must evaluate and establish the water quality needs for each species as well as limits for other aspects such as hardness, alkalinity, and waste products (e.g., ammonia, nitrites, and nitrates). Failure to establish parameters for the intended study species will likely result in at least some degree of distress for the fish. Fortunately, except for temperature, salinity, and dissolved oxygen, there are relatively few differences between species for the other water qualities (Fisher 2000). How often the parameters need to be evaluated to ensure optimum ranges for fish welfare depends on a number a factors including the species, water type, water volume and turnover rate, temperature, filtration, life stage, reproductive status, biomass, and feeding rate. Fish generally have an optimal temperature and an acceptable range of water temperatures; as poikilotherms, they are incapable of controlling their body temperature, which normally depends on the temperature of the surrounding water. It may therefore be necessary to adjust the temperature of the water or facility to ensure that it is appropriate not only for the species but also for its life stage as, for instance, the ideal temperature for the growth of a particular species may not be optimal for its reproduction (e.g., goldfish, yellow perch). Temperatures outside the tolerated range or abrupt changes in temperature can be stressful to fish (Harper and Wolf 2009, in this issue) and should be avoided. Fish generally fall into three categories according to whether their natural habitat is freshwater, saltwater, or a brackish environment, but some change salinity preferences as they develop, from freshwater to saltwater or vice versa. It is essential that the investigator know the species and life Volume 50, Number

2 stage of the fish being used and its salinity preference. This information is fairly well known for most species used for research and teaching, but there may be limited data about the range of tolerance (i.e., beyond which the animal experiences stress or ill health). The amount of dissolved oxygen in the water is another parameter that is commonly species dependent; coldwater fish usually require higher levels of dissolved oxygen in the water than warmwater species. Dissolved oxygen saturation fluctuates in response to environmental factors. It decreases with increasing temperature, salinity, and altitude, so requirements may vary according to both the species and the facility location. In addition, feeding may cause significant decreases in the amount of dissolved oxygen available by increasing oxygen use through increased activity and the metabolism of the fish. Supersaturation with gases (e.g., oxygen, nitrogen) may also result from the compression of water by circulation pumps or the infusion of water from deep sources. Concentrations of gases above atmospheric levels can cause clinical signs of gas bubble disease, chronic stress, or even death in fish. Because fish defecate and urinate in the same water they use for respiration and osmoregulation, it is essential to regularly monitor waste and nitrification products (e.g., ammonia, nitrite, and nitrate) in the water. Any system containing aquatic organisms must have adequate mechanical and biological filtration to remove these potentially toxic metabolic components. As noted above, it is also essential to maintain acceptable ranges of the water s ph, hardness, and alkalinity for the particular species to prevent stress in the fish. Housing, Lighting, Diet, and Other Considerations As with other species, housing design is an important consideration for fish. Options include static systems with in-tank filtration and recirculation systems with external filtration; glass, plastic, or fiberglass; and an assortment of sizes and shapes. In choosing a system, the investigator must consider the species, number, and size of the fish to be housed as well as their compatibility with the system. For instance, striped bass do not tolerate glass systems well and become stressed unless they have places to hide. Tilapia generally establish a social hierarchy and the dominant individual may become aggressive if the fish are maintained in small numbers (i.e., 3 to 4 per group). When dealing with multiple species of fish in aquaria, species compatibility is important so that less aggressive species are not in a constant state of anxiety from more aggressive species. Evidence of aggression (which may not occur during daylight hours) may present as submissive and avoidance behavior in individuals, posture and pigment changes, decreased feeding activity, weight loss, and physical injury. These can result in osmoregulatory deficiency, immunosuppression, and ultimately death. Again, the individual submitting the IACUC protocol needs to know the particulars of the species under study and be able to convey that knowledge to the IACUC members both to educate them and to ensure their confidence in the PI s ability to execute the study with minimal stress to the animals. Lighting (both photoperiod and intensity) can also be a source of stress in fish (DeTolla et al. 1995). Although 8 to 10 hours of light are generally adequate, this range does not allow normal reproductive cycling in many species, which may require more or less light to stimulate such cycling. Furthermore, lights that snap on in the morning and off in the evening are not natural and can startle fish and even cause individuals to jump out of uncovered tanks. A system that gradually increases the lighting in the morning and gradually decreases it in the evening can help reduce stress in fish. Another important aspect of fish maintenance that is often overlooked by investigators is diet. Not all flake or pelleted food is appropriate for all species; the nutritional requirements for zebrafish, for example, are quite different from those for trout, channel catfish, and sturgeon. Unfortunately, very little information is available about the dietary requirements of most species of fish, so the investigator must research the literature for whatever information is available. In addition, the correct presentation of the diet may be important so as to not stress or starve the fish. Some species do not readily feed from the surface, while others are hesitant to feed from the bottom; thus a pelleted diet should be provided in a floating, sinking, or mixed formulation depending on the species. Moreover, the pellet size needs to be appropriate for the species and size of fish and generally increased as the animal grows, as fish will not consume pellets that are too small or too large. Unconsumed food contributes to water quality degradation. Weighing fish periodically can help evaluate their physical condition and health, but normal growth curves are not available for most species and the weighing procedure can be extremely stressful for a fish. Finally, sources of fish, transportation methods, incoming health status reports, quarantine procedures, and sentinel surveillance may be quite different from those familiar to the veterinary and husbandry staff. Potentially extended quarantine and acclimation periods need to be factored into the study, and possibly into the housing equation as well since many species of fish continue to grow, unlike most traditional laboratory animal species. A long quarantine period followed by an acclimation period could yield fish radically different from those first acquired. Therefore, communication and coordination with IACUC members and with veterinary and husbandry personnel are crucial to ensure appropriate delivery of the animals, timing of the project, minimal stress during any movement of the animals, and the continued health of the fish. Fish Protocols and the IACUC Aquatic species represent challenges to the IACUC protocol submission (including the form design or adaptation) and 398 ILAR Journal

3 review processes (including educating the committee as a whole and finding appropriately qualified reviewers) as well as husbandry practices, standard operating procedures (SOPs), and semiannual review documentation. It is the IACUC s responsibility to determine whether an investigator has the necessary education, experience, and proficiency in the appropriate procedures to undertake the study. And the PI considering development of a new project featuring a novel species such as fish should be aware of the impact of the new species and project for both the IACUC and the institution. Preparation of the Protocol Submission Forms The protocol review process truly begins in the study design process. The PI must consider the study thoughtfully and completely and present it cogently to the IACUC in the submission forms and throughout the review process. The PI, usually the expert in the field of study, is responsible for guiding and educating the IACUC members about the project. While this may seem obvious, some PIs have difficulty conveying information that has become second nature to them, and this may hamper their efforts to present an educational and informative protocol to the IACUC (especially when, for example, the PI arrives with fish projects from another institution). A pilot study may be an effective approach to refine husbandry and procedural techniques and to pave the way with the IACUC members and husbandry staff for a larger-scale project. It may be necessary to adapt the responses requested and explanations provided in the IACUC submission forms to accommodate the particulars of the fish species, project, or facilities required, as the answers provided will likely vary significantly from those for studies that involve traditional laboratory animal species. Such adaptations usually require the PI s close collaboration with the IACUC administrator (or someone in a similar position) to educate the individual about the particular needs of both the species and the study. It may also be useful for the PI to meet with the IACUC administrator, chair, and/or attending veterinarian before or during the development of the protocol submission. Education ahead of time for all concerned, including the PI, about how best to present the unique aspects of the study in the submission forms ensures that the process begins and remains as collegial, collaborative, and efficient as possible. Particular Considerations in the IACUC Review Unique aspects of aquatic studies that require particular attention in the protocol submission development and review phases may include specialized procedures and experimental techniques, state or federal permits, animal use numbers, animal husbandry methods and schedules, daily observations (both their quality and level of detail), feeding regimens (discussed above), treatment options for animals that become ill (before, during, or after the study), medications (see Neiffer and Stamper 2009 for a review), humane endpoints, euthanasia techniques, and disposal of remains. In short, every element requested on an IACUC submission form may present unique challenges to both the PI who writes the submission and the reviewers who read it. The following sections provide further discussion of some of these components of the IACUC protocol submission process. Permits The use of wild-caught specimens usually requires capture and holding permits or a letter from the appropriate agency or agencies stating that no such permits are required. The IACUC should withhold approval of any protocol until the necessary state and federal permit information is provided. Animal Numbers Accurate animal use numbers may be nearly impossible to provide in studies that involve wild fish populations, food fish production, fish embryos, schooling fish, or large tanks (whether inground or aboveground). This difficulty must be clearly conveyed to the IACUC members, who are probably accustomed to a strict accountability of animal use numbers. A large study population of rodents may number in the hundreds or even thousands over a 3-year protocol, whereas aquatic studies may include only a few individuals or may start in the thousands. Housing and Husbandry PIs should include and explain arrangements for the care and enrichment needs of the species, including shelters, adequate space for movement and exercise, social interaction, and housing (with appropriate measures to avoid injury resulting from aggression, especially when moving individuals from one social group to another), water quality parameters, tank size requirements (with provisions for projected growth rates and space requirements for the duration of the study), and any other species-specific requirements. It is important that the PI present all of this information clearly, especially when introducing a novel species to the IACUC, to assure the committee members that the project is well thought out and that the PI has the knowledge to carry out the project capably and successfully. Furthermore, some aquatic species require unusual feeding and cleaning schedules (and vastly different cleaning techniques and equipment) that do not align with traditional laboratory animal room charts and schedules. It is essential to educate not only the IACUC members about these differences so that they can read the room charts correctly but also the animal husbandry staff so that they complete tasks and charts effectively. Volume 50, Number

4 Observation and Monitoring The monitoring of aquatic populations presents a variety of unique challenges. While zebrafish and similar species kept in small groups in somewhat traditional aquaria lend themselves to cageside observations, many species, study types, and housing conditions do not allow for such observations. Also, inappetence, an often-used indicator of ill health, can be very difficult to monitor in fish due to large populations and the fact that many feeds dissolve if uneaten, limiting the utility of this indicator. The housing of fish in large populations and enclosures (rather than individual tanks) increases the difficulty of identifying individual fish and of capturing an individual that needs to be examined or treated. In addition, the stress that accompanies the capture attempt may render the individual completely unrecognizable in the colony thus making it very difficult to capture when attempting to net or otherwise isolate it, due to potential masking of overt clinical signs (e.g., lessening of clamped fins, changes in opercular activity, diminished abnormal coloration) or may further negatively affect the animal s well-being. Health and Medical Treatment Aquatic studies often have specialized procedures, medications, and techniques that may have to be explained to the IACUC members so that they have the information necessary to perform accurate protocol reviews. For example, anesthesia methods used in the laboratory may not be appropriate for field studies. The PI should provide information to explain the need for the methods indicated, including, for example, a description of any unique anatomical features. Literature citations, when available, are required by most IACUCs and are usually of great assistance in this area. Narratives of consultations with experts in the field may also be of benefit but must be sufficiently detailed to provide the IACUC with a level of confidence similar to that afforded by citation of a peer-reviewed journal. As with other species that have not been truly domesticated, aquatic species typically disguise overt signs of ill health. But an experienced eye can often detect the animals subtle signs, so it is important to train all husbandry, veterinary, and research staff who will have responsibility for health observations, and to document the training for the IACUC to ensure proper protocol review and adequate animal care. Even when clinical signs of disease are present, treatment options may be limited either because of the nature of the study or because fish are an understudied species and so inadequate information is available. Humane Endpoints Investigators and IACUC members alike have a legal, ethical, and moral obligation to ensure that the use of all types of animals involves a minimum of pain and distress. But the evaluation of pain and distress in a species that commonly shows no obvious behavioral signs can be challenging and the identification and establishment of appropriate endpoints problematic. Indeed, humane endpoints are difficult to quantify in many aquatic species, especially when compared to traditional laboratory animal species (Sneddon 2009). As noted, aquatic species often hide signs of ill health, making it difficult for researchers to describe in the IACUC submission the clinical signs that may be used to define humane endpoints. The IACUC and PI must work together to ensure proper animal welfare and to establish Criteria and [a] process for timely intervention, removal of animals from a study, or euthanasia if painful or stressful outcomes are anticipated (NRC 1996, 10). In many cases euthanasia may be the only prudent option for clinically ill fish, and this must be presented to the IACUC in a manner that demonstrates both compassion and the need for this seemingly limited strategy of animal care. Investigators working with fish must consider species differences in euthanasia methods and agents. They should provide information about unique anatomical features or physiological parameters if the protocol includes methods of euthanasia not commonly encountered in traditional laboratory animal medicine, and justification of any deviations from recommended euthanasia procedures. Disposition of carcasses or animals not euthanized as part of the study is likely to entail alternative means that will require explanation in the protocol submission, especially in studies that include wild-caught specimens (Nickum 1988). In particular, some species may be covered by the Endangered Species Act (1973) or the National Invasive Species Act (1996), under the authorities of the US Fish and Wildlife Service and National Oceanic and Atmospheric Administration; investigators and IACUC members should check to ensure adherence to these regulations. Conclusion It is important for IACUC members and PIs to work collaboratively toward the common goal of promoting high-quality research. Depending on the nature of research conducted at an institution, aquatic species may represent a minor but extremely diverse subset of protocols or they may represent a large number of protocols concerning a limited range of species. In all cases, mutual education and collegiality are the keys to dealing with these nontraditional species. The IACUC must educate the PI about its requirements and regulations, and the PI must educate the IACUC about the nature of the study and the many facets of the variety of species involved. References Casebolt DB, Spear DJ, Horney BS Care and use of fish as laboratory animals: Current state of knowledge. Lab Anim Sci 48: DeTolla LJ, Srinivas S, Whitaker BR, Andrews C, Hecker B, Kane AS, Reimschuessel R Guidelines for the care and use of fish in research. ILAR J 37: ILAR Journal

5 Fisher JP Facilities and husbandry (large fish models). In: Ostrander GK, ed. The Laboratory Fish. San Diego: Academic Press. p Harper CL, Wolf J Morphologic effects of the stress response in fish. ILAR J 50: Neiffer DL, Stamper MA Fish sedation, analgesia, anesthesia, and euthanasia: Considerations, methods, and types of drugs. ILAR J 50: Nickum JG Guidelines for use of fishes in field research. Fisheries 13: NRC [National Research Council] Guide for the Care and Use of Laboratory Animals. Washington: National Academy Press. Sneddon LU Pain perception in fish: Indicators and endpoints. ILAR J 50: Westerfield M The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish Danio (Brachydanio) rerio. Eugene: University of Oregon Press. Volume 50, Number