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Regulations, Policies and Guidelines Pertaining to the Use of Zebrafish in Biomedical Research
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38 Regulations, Policies and Guidelines Pertaining to the Use of Zebrafish in Biomedical Research Samuel C. Cartner1, Earle Durboraw1, Amanda Watts2 1
Animal Resources Program, University of Alabama at Birmingham, Birmingham, AL, United States of America; 2 Institutional Animal Care and Use Committee, University of Alabama at Birmingham, Birmingham, AL, United States of America
Introduction The use of zebrafish as a biomedical research model of human and animal diseases has steadily increased since the first published report in 1951. Projects utilizing zebrafish are very diverse with the top five published subject areas being developmental biology (27%), biochemistry/molecular biology (18%), cell biology (14%), neurosciences/neurology (11%), and genetics (10%) (Kinth, Mahesh, & Panwar, 2013). In recent years, research using zebrafish has expanded to include using this experimental system to model many human and animal diseases (cardiovascular, cancer, obesity). The National Institutes of Health (NIH) has ranked zebrafish as the second most important animal model behind the mouse (Goldsmith & Solari, 2003). Following NIH’s 1998 program announcement “The Zebrafish as an Animal Model for Development and Disease Research,” the number of zebrafish publications in Clarivate Analytics’ Science Citation Index Expanded database rose by 149% between 1997 and 2006 compared to an increase of only 2.7% for animal research in general. And between 2005 and 2010 zebrafish research saw a sixfold increase in NIH funding and almost a fourfold increase in publications, with over 800 zebrafish research publications in 2010 representing almost $354 million in NIH funding alone (Dietrich, Ankeny, & Chen, 2014). The Zebrafish Information Network (ZFIN) Website maintains a database of laboratories using zebrafish. As of this writing, the list is well over 1000 (Howe DG et al., 2013). Some reasons zebrafish have become such an increasingly used biomedical model are that its eggs are laid and fertilized externally, embryos are transparent, and development of organs is rapid and may be observed
The Zebrafish in Biomedical Research https://doi.org/10.1016/B978-0-12-812431-4.00038-5
in vitro. Zebrafish are especially useful in genetic research as one breeding pair can produce over 100 embryos from a single mating and techniques for their genetic manipulation are well described (Harper & Lawrence, 2011; Westerfield, 2007). Zebrafish are also appreciated for containing research cost as they can be housed in large numbers, in less space, and more economically than mammalian animal models. This chapter will briefly review the applicable regulatory oversight of the use of zebrafish in biomedical research in the United States. Some of the challenges for institutional oversight of zebrafish research will be discussed.
Laws, Policies, and Guidelines (Anderson 2002; Bayne & Anderson, 2015, 2017) Researchers, funding agencies, and the general public expect that animals are cared for and used humanely and responsibly. The required regulations and oversight for vertebrate species contribute to the standardization of procedures and care, and thus, promote the production of reliable and consistent data. They also serve to ensure the humane care and use of research animals. As the use of the zebrafish model has become more common, several governmental agencies and nonprofit organizations have published documents to provide guidance for the specific and appropriate care and use of this animal.
United States Department of Agriculture (USDA) One of the most notable laws governing the use of animals in biomedical research in the United States is the
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Animal Welfare Act (AWA) signed into law in 1966. This law authorized the USDA to write and enforce the Animal Welfare Regulations (AWR). While these are often the most recognized by the general public due to their importance for the use of dogs, cats, nonhuman primates, and other warm-blooded animals (excluding birds and the genera Mus and Rattus), the AWR do not apply to the use of zebrafish. However, institutions receiving funds from the Department of Health and Human Services (DHHS) for research using the AWR covered species must comply with these regulations. So while the AWR do not apply to zebrafish, failure to follow AWR for other species could impact zebrafish research at an institution should program-wide restrictions/punishments be imposed for noncompliance.
Department of Health and Human Services (DHHS) In 1985, the U.S. Congress passed the Health Research Extension Act that mandated the Secretary of DHHS, acting through the director of the NIH, to establish policy for the proper care and treatment of animals used in biomedical and behavioral research. Much of the animal research performed in the United States is funded through the DHHS, including the Public Health Service (PHS) and NIH. Any institution receiving funds for animal research, training, biological testing, or animalrelated activities from the DHHS must comply with the PHS Policy on Humane Care and Use of Laboratory Animals and the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training (Table 38.1) (OLAW 2015). The NIH Office of Laboratory Animal Welfare (OLAW) enforces the PHS policy. NIH awardees must negotiate a letter of assurance with OLAW assuring that they meet the PHS Policy and that they use The Guide for the Care and Use of Laboratory Animals (The Guide) as the basis of their animal program development and management (Garber, Barbee et al. 2011). The Guide applies to all vertebrate animals, including zebrafish. One of the key requirements of the PHS Policy is that all animal research facilities must establish an Institutional Animal Care and Use Committee (IACUC) and that this committee includes members with specific qualifications. This is described in detail later in this chapter. The IACUC is responsible for reviewing all aspects of the institutional animal care and use program and assuring that all activities are conducted according to PHS Policy and The Guide. Another important NIH office with oversight of NIH funded research is the NIH Office of Science Policy (OSP). The OSP oversees a wide range of biomedical research areas, including biosafety, biosecurity, and emerging biotechnology. All NIH funded research involving the genetic manipulation of living organisms
TABLE 38.1 U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. I. The transportation, care, and use of animals should be in accordance with the Animal Welfare Act (7 U.S.C. 2131 et. seq.) and other applicable Federal laws, guidelines, and policies. II. Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society. III. The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid results. Methods, such as mathematical models, computer simulation, and in vitro biological systems should be considered. IV. Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals. V. Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents. VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure, or if appropriate, during the procedure. VII. The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be provided as indicated. VIII. Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals. Adequate arrangements shall be made for their inservice training, including the proper and humane care and use of laboratory animals. IX. Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly concerned but should be made, with due regard to Principle II, by an appropriate review group, such as an institutional animal care and use committee. Such exceptions should not be made solely for the purposes of teaching or demonstration.
must meet the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). The Guidelines require an Institutional Biosafety Committee (IBC) review and approval of any genetic additions, deletions, or changes done by recombinant engineering. Most IBC’s have broader purview, including the review and oversight of all biological hazard research involving infectious agents. The Center for Disease Control and Prevention (CDC), which publishes the Biosafety in Microbiological and Biomedical Laboratories, presumes that the institution (IBC and/or biological safety officer) conducts a biological risk assessment
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and reviews and approves experiments with infectious agents, including viral vectors and pathogens.
Institute of Laboratory Animal Research (ILAR) The Guide was published in 1963 and has been revised seven times through committees of ILAR, which report to the National Research Council of the National Academies. The eighth edition (2011) introduced expanded sections on aquatic animal care. Because the needs of fish and other aquatic or semiaquatic animals are diverse and species-specific, these expanded sections provide broad guidelines for the management of aquatic animal systems. Specific recommendations are made for environmental parameters, such as water quality, temperature, humidity, ventilation, lighting, noise, and vibration and for documenting monitoring and maintenance of appropriate water quality. The Guide also describes recommendations for appropriate housing and feeding; food preparation and storage; veterinary care, including health monitoring, anesthesia, and euthanasia; and colony and life system management, including social housing and environmental enrichment. Specifics from The Guide are described in later sections of this chapter.
Association of Assessment and Accreditation of Laboratory Animal Care International (AAALACi) Many institutions seek to attain the highest standards in animal care and use and voluntarily participate in an accreditation program administered by the AAALACi. AAALACi accreditation is widely recognized as the gold standard for animal care and use programs. AAALACi is not associated with any governmental agency; however, it is recognized by OLAW as evidence of achieving a high standard of care. Accreditation is obtained after an animal care and use program is reviewed by professionals from accredited peer institutions for adherence to the standards of The Guide and other applicable regulations and guidelines. These professionals are appointed by AAALACi to carry out such reviews as a site visit. AAALACi accredited programs must undergo this extensive review at least every 3 years to maintain their accreditation. Since AAALACi accreditation is based on successful adherence to PHS Policy, The Guide, AWR, NIH Guidelines, AVMA Guidelines, and other regulations, the maintenance of AAALACi accreditation is interpreted by the funding agencies and others as evidence that the institution is meeting all requirements to receive PHS funding. OLAW assigns those institutions with AAALACi accreditation category 1. Such institutions do not have to submit their semiannual program
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assessment reports directly to OLAW, unlike unaccredited institutions, which are assigned category two and must submit the semiannual program assessments directly to OLAW. Institutions with DHHS funding may have other sources of funding that recognize OLAWassurance and AAALACi accreditation, such as the National Science Foundation and Department of Veterans Affairs. Many of these agencies have a memorandum of understanding (MOU) with OLAW that states that OLAW will apply PHS Policy and require PHS assurance for their funded projects. The MOU allows these projects using vertebrate animals to come under the purview of OLAW oversight.
American Veterinary Medical Association (AVMA) The AVMA is a nonprofit organization representing more than 91,000 veterinarians working in private, government, industry, academia, and uniformed services primarily in the United States (AVMA Website). The AVMA provides information resources, continuing education opportunities, and publications, and lobbies for animal-friendly legislation. The AVMA also produces policies in response to member requests and stakeholder interests. These policies are based on the best available scientific evidence. One of these policies is the AVMA Guidelines on the Euthanasia of Animals, intended for use by members of the veterinary profession who carry out or oversee the euthanasia of animals (Leary, Underwood et al. 2013). The overriding commitment of this policy is to provide veterinarians guidance in relieving pain and suffering of animals that are to be euthanized. Specific guidelines for the euthanasia of fish and other aquatic species are defined, and most regulatory bodies discussed in this chapter require recognition and adherence to this publication for euthanasia of zebrafish used in biomedical research.
Occupational Safety and Health Administration (OSHA) and Other Responsible Organizations Zebrafish facilities should comply with local and state regulations regarding workplace safety, general biosafety, environmental compliance, and waste management. For occupational hazards, some U.S. states regulate workplace safety at the state level, with the others falling under the Occupational Safety and Health Administration (OSHA). Laboratory and research safety differ from other workplace chemical safety concerns in several ways; this is why the United States has a separate OSHA standard for laboratories and research workplaces. Laboratory chemical safety in zebrafish research facilities falls under OSHA’s Occupational
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Exposure to Hazardous Chemicals in Laboratories standard (29 CFR 1910.1450) for OSHA-regulated states. State equivalents apply elsewhere: 8 CCR 5191 addresses laboratory chemical safety in the California Code of Regulations (OSHA 1990). Medical sharps or human blood or tissues that are used in the zebrafish facility may also be regulated under OSHA’s Bloodborne Pathogens standard (29 CFR 1910.1030) or a similar local equivalent (OSHA 1991). Testing laboratories should follow the Organization for Economic Cooperation and Development Guidelines for the Testing of Chemicals. The National Research Council’s Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards, Updated Version (2011) addresses laboratory chemical safety. This applies to everything from pH balancing chemicals to mutagens and teratogens used on zebrafish embryos (NRC 2011). Imaging or other biokinetics work with radioisotopes must meet local or federal, As Low As Reasonably Achievable (ALARA) principles using time, distance, and shielding in addition to complying with applicable state regulations, or Nuclear Regulatory Commission regulations in 10 CFR 20 for facilities that are not regulated by the 37 “Agreement State” regulations. The CDC and NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), provides guidance for work at biosafety levels (BSL) and animal biosafety levels (ABSL) one to four (CDC 2007). The American Society for Microbiology (ASM) “Biological Safety: Principles and Practices,” fifth edition, and ASM “Guidelines for Biosafety in Teaching Laboratories” also provide laboratory biosafety guidance (Emmert, Byrd, Gyure, Hartman, & White, 2012; Wooley & Byers, 2017). In addition to normal laboratory waste, which may be either solid waste or listed hazardous waste, zebrafish facilities may also encounter wastewater and medical waste disposal constraints based on local, state, or federal environmental regulations. For example, most larger zebrafish facilities in the United States will fall under the National Pollutant Discharge Elimination System (NPDES) point source regulations and will need to coordinate with the local wastewater publicly owned treatment works (POTW) for discharge pH and any possible additional chemicals in the discharge. Different cities and towns have different wastewater treatment capabilities, so this often varies by the sewer system. Local medical waste regulations apply to the handling and disposal of potentially infectious waste, which may include viral vectors for recombinant work or pathogens used in zebrafish. In the United States, these regulations vary by state but fall under the state EPA equivalent, with additional guidance by CDC, OSHA, and U.S. Food and Drug Administration (FDA). For ground transportation of biomedical waste, the United States. regulates untreated biomedical waste and samples as
Hazard Class 6, Division 6.2 infectious substances under the U.S. Hazardous Materials Regulations (49 CFR 173.134), and air transportation of samples fall under the International Air Transport Association (IATA). Hazardous Materials or Dangerous Goods Regulations (DGR) require medical wastes that may be Class 6, Division 6.2, Category A infectious material (defined as an “infectious substance in a form capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals”) to be treated before transport, typically either by autoclaving or chemical sterilization. Note that medical waste vendors frequently require zebrafish facility staff to complete IATA training every 2 years, or U.S. DOT hazmat training every 3 years. Those institutions receiving DHHS funding and creating, maintaining, and disposing of genetically manipulated zebrafish must meet the NIH Guidelines (see Section IIb NIH OSP) (RAC NIH OSP 2016). The NIH Guidelines require that the institutional IBC review and approve the creation of genetically modified zebrafish. Regulations differ between recombinant zebrafish and recombinant rodents; the NIH Guidelines exempt purchase, transfer, and certain types of recombinant rodent breeding in Section III-D-4-c. But the NIH Guidelines otherwise regulate recombinant animals, including zebrafish, under Section III-D-4, Experiments Involving Whole Animals. Researchers must coordinate with their IBC prior to creating knockout or transgenic zebrafish.
Institutional Responsibilities for Zebrafish Oversight Scientists The primary responsibility of any scientist conducting animal research is to assure the integrity and quality of the research performed and the welfare of the animals involved. The integrity of the research conducted begins in the planning stages of the study. Well-founded research designs and methods are key to the responsible conduct of animal studies and are supported and strengthened by extensive literature reviews and interactions with peers. Maintaining compliance throughout the study by adhering to approved study protocols and funding agreements can assure compliance with animal welfare regulations, laws, and standards. It is also important to apply ethical and responsible practices to both the conduct of research activities, and the collection, analysis, and presentation of data. Performing all aspects of the animal study with a conscientious regard for animal welfare and scientific integrity is key to the successful outcome of any animal research study. Due to the specialized nature of zebrafish care and the relative novelty of the model,
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primary care of the animals and management of the housing system or facility may fall partially or in whole to the investigator. In these circumstances, the investigator, who may possess unique knowledge and expertise, could be responsible for the day-to-day management of the animal model but should work closely with the program veterinary and care staff, as well as the IACUC, to assure that the needs of the animals, the research, and the program are aligned and being consistently met.
Veterinary Care and Animal Care Staff The health and well-being of research animals is the responsibility of all research staff but is the primary concern for veterinarians and animal care staff providing daily animal care and management. The best way to assure proper care and management of animals is to establish and adhere to daily care practices that support their physical and psychological health. These practices and standards should be designed to comply with the required standards and industry best practices, as well as to meet the individual needs of the institution’s research program. Practices should provide for the animals’ general care, and include methods for identifying, treating, and following-up on health issues and medical cases, as well as maintaining accurate and complete records documenting animal care. Beyond the immediate care of the animals, the veterinarians and care personnel are responsible for managing and maintaining a suitable environment for research animals and the facilities that support research activities. Facilities and established care practices should also be designed to promote and protect personnel safety to ensure a research program that provides consideration of animal welfare and the health and well-being of staff involved in animal care. In some instances, the investigator’s knowledge of proper care and management of the animals and their housing may exceed that of the program staff. In these cases, the veterinary and care staff are responsible for working closely with the researcher to assure the care of the animals supports their welfare and is in line with the industry-standard practices.
Institutional Official The authority to assure an institution’s compliance with PHS Policy falls primarily to the Institutional Official (IO). The IO is the administrator responsible, with the authority, to sign the institution’s Assurance with OLAW making a commitment that the requirements of the PHS Policy will be met. The IACUC, often the IBC, and other safety committees report to the IO. The IO must have authority to allocate resources to support
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the research program’s ongoing compliance with everchanging regulations and standards and evolving institutional needs. The IO must have a working knowledge of established safety committees, IACUC, and animal care activities, and the processes to promote and support the efforts of the research program. Frequent communications between the IO, the IACUC, and other unit administrators and staff about the needs and challenges of the program are crucial for preventing and managing noncompliance. Noncompliance incidents are reported to OLAW and other regulatory agencies through the IO. It is important for the IO to maintain open lines of communication internally to address any vulnerable or ineffectual areas of the program in order to avoid or minimize noncompliance, and to assure a culture of compliance within the institution.
Institutional Animal Care and Use Committee (IACUC) (Garber, Barbee et al. 2011, OLAW 2015) The IACUC is responsible for overseeing all aspects of the animal care and use program to ensure compliance with animal welfare regulations, standards, and recommendations. The committee represents the interests and needs of the animals in balance with the needs of the researchers and the institution, in accordance with all applicable laws and requirements. The IACUC is required to oversee the use of zebrafish by the PHS Policy, and The Guide. The committee must be appointed by the Chief Executive Officer and include at least five members. The members must have sufficient knowledge, experience, and ability to assess animal care, treatment, and practices in experimental research and represent society’s concerns regarding the welfare of the animals. The IACUC must include one member who is a doctor of veterinary medicine, one practicing scientist, one member whose primary concerns are in a nonscientific area, and one member who is neither affiliated in any way with the research facility nor an immediate family member of the research staff. One person can serve both roles of the nonscientific and unaffiliated members. However, the minimum number of members must be met, and not more than three voting members should be from the same administrative unit of the facility. The IACUC is responsible for reviewing all aspects of the institutional animal care and use the program, including specific research proposals, animal housing and procedures, facilities, personnel training and medicine programs. The IACUC must review program procedures and facilities at least every 6 months and report to the research facility’s IO. Before any animal research can be performed, a written proposal must be submitted, reviewed, and approved by the committee.
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The IACUC is authorized to require modifications of the proposal and suspend a research activity if it is determined that the activity is not being conducted according to the approved protocol. The research proposal must include the following information: (1) the species and approximate number animals to be used; (2) a rationale for the use of animals and justification of the species and number of animals to be used; (3) a description of the proposed use and procedures to be conducted with animals; (4) a description of procedures to minimize pain and discomfort; and (5) a description of any euthanasia method to be used.
Disaster Response Planning All research facilities either registered with the USDA, have assurance with OLAW and/or accredited by AAALACi are required to have a disaster response plan. Aquatic facilities are especially vulnerable to power outages and other support system failures. Please see the chapter on emergencies for important details to consider.
Personnel Qualifications and Training Principle VIII of the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (Table 38.1) requires all researchers and other personnel to be appropriately qualified and experienced for conducting procedures on living animals. Housing facilities for aquatic species, especially high volume zebrafish facilities can be challenging for personnel not accustomed to working in such environments. Institutions should take advantage of training opportunities for staff to obtain highquality instruction and experience.
Specific Challenges for Oversight of Zebrafish Research Many factors affect the applicability of the various regulations that may govern zebrafish use in research or teaching.
knowledge and experience is limited to those researchers or instructors who are also submitting their own proposals; therefore, leaving none or only a few individuals who do not have a potential or perceived conflict of interest. It may be possible to address this problem by using ad hoc reviewers from other institutions or providing additional training to IACUC members who do not have expertise with aquatic models in general or zebrafish in particular.
Performance Based Standards Zebrafish are relatively new animal models compared to mice, and the number of investigators and institutions using this novel model remains comparatively low to those using mice. Thus, the information about zebrafish husbandry in a research setting is sometimes extrapolated from other species or from commercial aquatic management practices. The Guide makes specific recommendations for the microenvironment, which includes anything with which the fish has immediate contact. Water quality is essential to the well-being of aquatic animals, and routine measurements of its characteristics are essential. Some important characteristics to monitor include pH, salinity, and levels of ammonium, nitrate, and nitrite. Monitoring should be documented with recorded data.
Transportation Many concerns about transporting terrestrial animals also apply to the shipping of zebrafish. These include the availability of commercial carriers, security provided by the transport containers, environmental control of transport vehicles and any loading or holding facilities, and time in transit. Transporting fish involves additional concerns of assuring appropriate oxygen levels are present in the water and avoiding any deleterious buildup of ammonia or other contaminants. In general, the longer the transport distance, the more serious the concern. There is also a need for care during intrainstitutional transportation between animal rooms and to or from procedure space, laboratories, or facilities. Please see additional information in the Chapter on Import/Export and Transportation of Zebrafish.
Knowledgeable IACUC Members Unless the institution has an established aquatics research program, it can be challenging to find IACUC members with the knowledge of issues and/or experience specific to the use of zebrafish to apply performance-based standards during project review or to assess the program and facilities. This is especially an issue when the pool of IACUC members with relevant
Animal Numbers IACUCs typically oversee and regulate the number of animals approved for use by requiring animal acquisitions from vendors, other institutions, or other investigators to be handled and recorded centrally. When animals are transferred between investigators within the institution, between protocols, or produced in breeding
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colonies, self-reporting, or other procedures for census monitoring are needed. It is relatively easy to count the number of adult fish present at any time or to estimate it based on the number and size of aquaria present and the average number of fish per aquaria of a specific size (density); however, it may be necessary to estimate the number of eggs collected and their average survival time to estimate the total number of animals used.
Counting of Zebrafish Onto Protocols and Pain Category Assignment OLAW provides guidance for accounting of larval forms of fish. Zebrafish hatch at approximately 3 days postfertilization (dpf) and must be included in an approved protocol at this stage and oversight provided by the IACUC (Bartlett & Silk, 2016). While fish at four to 7 dpf may require inclusion in a protocol, studies suggest that during this phase of development, the animals do not yet feel pain or distress. Thus, zebrafish do not need to be assigned a pain category until older than 7 dpf (NIH, 2016). Juvenile and adult fish, 8 dpf and older, must be assigned a pain category based on the proposed procedures. Institutions that are accredited through AAALACi must also follow recommendations for animal care and the oversight and guidance for aquatic housing and care, as outlined in The Guide. While The Guide does not refer to developmental stages in its application, it is generally accepted that The Guide standards be applied to zebrafish at and beyond the larval stage (>3 dpf).
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treatment, sanitation), reduce duplication of equipment, provide more efficient use of animal care personnel, and reduce transportation of animals. Decentralized facilities offer greater convenience to individual research teams. Decentralized facilities offer redundancy and may provide backup resources if there is a planned or unplanned disruption of support services in a facility. Populations and studies can also be isolated to address disease concerns or research needs.
Environmental Enrichment The Guide recommends that all animals be given the opportunity to express species-specific behaviors, and this may be accommodated through the provision of environmental enrichment. For fish, this can be met in several ways. Fish are social animals and will school if housed in groups. If zebrafish must be individually housed for experimental reasons, they may be given enrichment, such as plastic plants to accommodate their being alone. Live food is often offered to zebrafish because of its nutritional advantages, but this provision also serves as environmental enrichment because it allows zebrafish to exhibit their natural predatory feeding behavior.
Primary Housing: Housing Density and Sanitation
The rule of thumb for assessing whether a procedure is painful has generally been “If a procedure is painful to a human, it is likely to be painful to a fish.” It is also important to consider what factors or conditions might be potentially distressful for a fish during its use. For instance, fish are social animals, and so it may be distressful for a fish to be housed in isolation. If it is necessary to house fish singly in a tank, the tank should be placed so that the single fish inside it can see other fish group housed nearby. In these circumstances, the singly housed fish may be seen to shoal in unison with the group in the adjacent tank, thus exhibiting a natural group behavior.
For many species, The Guide offers very specific information on the minimal acceptable amount of space allowed for each animal based on size and age. For fish, The Guide states that the number of animals in a location can vary based on the type and size of tank and age of fish, but the recommendation for adult zebrafish is a maximum of five zebrafish per liter. Many institutions have used performance standards to justify higher housing densities, which is allowable under PHS Policy if approved by the IACUC. There are also specific requirements for sanitation of tanks. This is in part based on the type of housing used, type of system used (static or recirculating), and water quality. Accumulation of debris or algae that would block the ability to view and monitor fish is not acceptable and should be removed and/or reduced. Fish tank lids may need to be cleaned regularly for removal of food debris.
Centralization of Zebrafish Facilities
Environmental Monitoring
Comparisons of the benefits of centralized versus decentralized facilities address many of the same considerations for zebrafish as for nonaquatic species. Centralized aquaculture facilities may cost less to build and maintain (ventilation, humidity, temperature, water
The macro environment is the physical environment of the secondary enclosure, generally the room in which an aquatic housing system is located. These conditions must also be regularly monitored and documented. Room humidity is closely monitored for other species,
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but is obviously not as critical for fish immersed in water and is often based on what is safe and comfortable for the staff. Similarly, the rate of air exchange in a room is not as crucial in fish facilities and may even be purposely lower than that required in rooms housing terrestrial animals in order to reduce evaporation from tanks. Some rooms housing aquatic species are kept at a similar temperature to that of the tank water to minimize water temperature fluctuations and to accommodate fish housed off the recirculating system in static tanks in the room. Illumination is also important, and because breeding is strongly affected by the room light cycle, the room photoperiod must be carefully regulated; the natural stimulus for breeding in zebrafish is the onset of daylight. Fish rooms should be maintained so that there is total darkness during the dark cycle, and the presence of even partial light during the dark period may disrupt breeding subsequently for days or even weeks.
Veterinary Care Zebrafish colony health should be regularly monitored and the monitoring documented. The Guide requires that trained personnel observe all animals for signs of illness, injury, or abnormal behavior at least daily. Professional judgment should be used to ensure that the frequency and character of the observations minimize risk to the individual animals and do not compromise the research. Unexpected deviations from the normal should be reported promptly to assure timely delivery of appropriate intervention when needed. Procedures for disease prevention, diagnosis, and therapy should be those currently accepted in veterinary and laboratory animal practice. Additionally, appropriate procedures should be in place to allow colony disease surveillance, diagnosis, and management. Health monitoring programs may include regularly scheduled submission of sentinel fish. Fish found in sump tanks may serve as sentinels. Alternatively, sentinel fish placed into the facility from a known specific-pathogen-free source and exposed to colony animals and subsequent diagnostic evaluation may be used. Sentinels may be placed in various locations, including on the system, in a separate tank, or into the sump or waste stream of a system. Sentinel fish should be submitted to a laboratory with aquatic disease diagnostic experience and capabilities. The Guide states that health monitoring programs should be designed based on the size and complexity of the facility, the species involved, and the institutional research focus. In addition, records of morbidity or sickness and mortality or death should be kept as part of the monitoring program.
Anesthesia, analgesia, and euthanasia of zebrafish are discussed in more detail in other chapters, but it is discussed briefly here, as it relates to PHS policy and The Guide. Anesthesia is necessary to minimize pain and distress to fish during procedures, such as for the collection of tissue samples for DNA isolation and genotyping, often done by fin clipping. Anesthesia may be used for other surgical procedures and to allow manual gamete removal for in vitro work. The most commonly used anesthetic technique is immersion in tricaine methanesulfonate (MS 222, Finquel). Investigators may discuss appropriate anesthetics and analgesics, dose and route of administration with veterinarians experienced with zebrafish research. The Guide encourages the use of pharmaceutical-grade drugs and chemicals whenever possible in order to prevent adverse effects caused by impurities, instability, or toxicity of compounds. The IACUC may approve the use of agents unavailable in pharmaceutical grade after appropriate consideration of purity, sterility, efficacy, and other relevant characteristics. Euthanasia of zebrafish used in research must comply with the regulatory requirements. In the United States these requirements are the AVMA Guidelines for Euthanasia 2013 (Leary, Underwood et al. 2013). Commonly used methods are immersion in the recommended concentrations of buffered MS 222, and rapid chilling in an ice water bath with the appropriate holding times. Embryonic and larval life stages <7 d postfertilization may be euthanized by immersion in 10% sodium hypochlorite solutions. Dead fish should be disposed of as medical waste. Water solutions contaminated with chemicals may require treatment before entering the public wastewater stream. Some states may require an alternative disposal method.
Acknowledgment The authors would like to acknowledge and thank the following colleagues for their contribution, careful review, and suggestions for this manuscript. Pam Bounelis, University of Alabama at Birmingham, Birmingham, AL 35294. Lauretta Gerrity, University of Alabama at Birmingham, Birmingham, AL 35294. Monte Matthews, University of Oregon, Eugene, OR 97403.
References Anderson, L. (2002). Laws, regulations, and policies affecting the use of laboratory animals. In J. Fox, L. Anderson, F. Lowe, & F. Quimby (Eds.), Laboratory animal medicine (pp. 19e33). Boston: Academic Press. AVMA Website, American Veterinary Medical Association. (2018). Retrieved 20181101 https://www.avma.org/Pages/home.aspx. Bartlett, D., & Silk, S. (2016). Office of laboratory animal welfare comments. Zebrafish, 13(6), 563e564.
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References
Bayne, K., & Anderson, L. C. (2015). Laws, regulations, and policies. Laboratory animal medicine (pp. 23e42). USA: Elsevier Academic Press. Bayne, K., & Anderson, L. C. (2017). Regulations and guidelines. Principles of animal research for graduate and undergraduate students (pp. 35e63). Academic Press. CDC, Centers for Disease Control and Prevention. (2007). Biosafety in microbiological and biomedical laboratories (BMBL). Washington, D.C.: Government Printing Office. Dietrich, M. R., Ankeny, R. A., & Chen, P. M. (2014). Publication trends in model organism research. Genetics, 198(3), 787e794. Emmert, E. A. B., Byrd, J., Gyure, R. A., Hartman, D., & White, A. (2012). Guidelines for biosafety in teaching laboratories. Washington: ASM Press. Garber, J., Barbee, R., Bielitzki, J., Clayton, L., Donovan, J., Hendriksen, C., et al. (2011). Guide for the care and use of laboratory animals. Washington, D.C.: Institute for Laboratory Animal Research. Goldsmith, P., & Solari, R. (2003). The role of zebrafish in drug discovery. Drug Discovery World(Spring). Harper, C., & Lawrence, L. (2011). The laboratory zebrafish. New York: CRC Press. Howe DG, B. Y., Conlin, T., Eagle, A. E., Fashena, D., Frazer, K., Knight, J., et al. (2013). ZFIN, the zebrafish model organism database: Increased support for mutants and transgenics. Kinth, P., Mahesh, G., & Panwar, Y. (2013). Mapping of zebrafish research: A global outlook. Zebrafish, 10(4), 510e517.
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Leary, S., Underwood, W., Anthony, R., Cartner, S., Corey, D., Grandin, T., et al. (2013). AVMA guidelines for the euthanasia of animals (2013 Edition). NIH. (2016). Guidelines for use of zebrafish in the NIH intramural research program. Retrieved 20181128, 2018, from https://oacu.oir.nih.gov/ sites/default/files/uploads/arac-guidelines/zebrafish.pdf. NRC, National Research Council. (2011). Prudent practices in the laboratory: Handling and management of chemical hazards. Washington, DC: The National Academies Press. Updated version. OLAW, Office of Laboratory Animal Welfare. (2015). PHS policy on humane care and use of laboratory animals. Retrieved 20181101, 2018, from https://olaw.nih.gov/policies-laws/phs-policy.htm. OSHA, Occupational Safety and Health Administration. (1990). Occupational exposure to hazardous chemicals in laboratories. 29 CFR 1910.1450. OSHA. OSHA, Occupational Safety and Health Administration. (1991). Blood borne pathogens. 29 CFR 1910.1030. OSHA. RAC NIH OSP, Research Advisory Committee National Institutes of Health Office of Science Policy. (2016). NIH guidelines for research involving recombinant or synthetic nucleic Acid molecoules (p. 129). Bethesda, MD: NIH. Westerfield, M. (2007). The zebrafish book. Eugene, Oregon: University of Oregon Press. Wooley, D. P., & Byers, K. B. (2017). Biological safety: Principles and practices. Washington: ASM Press.
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38 Regulations, Policies and Guidelines Pertaining to the Use of Zebrafish in Biomedical Research Samuel C. Cartner1, Earle Durboraw1, Amanda Watts2 1
Animal Resources Program, University of Alabama at Birmingham, Birmingham, AL, United States of America; 2 Institutional Animal Care and Use Committee, University of Alabama at Birmingham, Birmingham, AL, United States of America
Introduction The use of zebrafish as a biomedical research model of human and animal diseases has steadily increased since the first published report in 1951. Projects utilizing zebrafish are very diverse with the top five published subject areas being developmental biology (27%), biochemistry/molecular biology (18%), cell biology (14%), neurosciences/neurology (11%), and genetics (10%) (Kinth, Mahesh, & Panwar, 2013). In recent years, research using zebrafish has expanded to include using this experimental system to model many human and animal diseases (cardiovascular, cancer, obesity). The National Institutes of Health (NIH) has ranked zebrafish as the second most important animal model behind the mouse (Goldsmith & Solari, 2003). Following NIH’s 1998 program announcement “The Zebrafish as an Animal Model for Development and Disease Research,” the number of zebrafish publications in Clarivate Analytics’ Science Citation Index Expanded database rose by 149% between 1997 and 2006 compared to an increase of only 2.7% for animal research in general. And between 2005 and 2010 zebrafish research saw a sixfold increase in NIH funding and almost a fourfold increase in publications, with over 800 zebrafish research publications in 2010 representing almost $354 million in NIH funding alone (Dietrich, Ankeny, & Chen, 2014). The Zebrafish Information Network (ZFIN) Website maintains a database of laboratories using zebrafish. As of this writing, the list is well over 1000 (Howe DG et al., 2013). Some reasons zebrafish have become such an increasingly used biomedical model are that its eggs are laid and fertilized externally, embryos are transparent, and development of organs is rapid and may be observed
The Zebrafish in Biomedical Research https://doi.org/10.1016/B978-0-12-812431-4.00038-5
in vitro. Zebrafish are especially useful in genetic research as one breeding pair can produce over 100 embryos from a single mating and techniques for their genetic manipulation are well described (Harper & Lawrence, 2011; Westerfield, 2007). Zebrafish are also appreciated for containing research cost as they can be housed in large numbers, in less space, and more economically than mammalian animal models. This chapter will briefly review the applicable regulatory oversight of the use of zebrafish in biomedical research in the United States. Some of the challenges for institutional oversight of zebrafish research will be discussed.
Laws, Policies, and Guidelines (Anderson 2002; Bayne & Anderson, 2015, 2017) Researchers, funding agencies, and the general public expect that animals are cared for and used humanely and responsibly. The required regulations and oversight for vertebrate species contribute to the standardization of procedures and care, and thus, promote the production of reliable and consistent data. They also serve to ensure the humane care and use of research animals. As the use of the zebrafish model has become more common, several governmental agencies and nonprofit organizations have published documents to provide guidance for the specific and appropriate care and use of this animal.
United States Department of Agriculture (USDA) One of the most notable laws governing the use of animals in biomedical research in the United States is the
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Animal Welfare Act (AWA) signed into law in 1966. This law authorized the USDA to write and enforce the Animal Welfare Regulations (AWR). While these are often the most recognized by the general public due to their importance for the use of dogs, cats, nonhuman primates, and other warm-blooded animals (excluding birds and the genera Mus and Rattus), the AWR do not apply to the use of zebrafish. However, institutions receiving funds from the Department of Health and Human Services (DHHS) for research using the AWR covered species must comply with these regulations. So while the AWR do not apply to zebrafish, failure to follow AWR for other species could impact zebrafish research at an institution should program-wide restrictions/punishments be imposed for noncompliance.
Department of Health and Human Services (DHHS) In 1985, the U.S. Congress passed the Health Research Extension Act that mandated the Secretary of DHHS, acting through the director of the NIH, to establish policy for the proper care and treatment of animals used in biomedical and behavioral research. Much of the animal research performed in the United States is funded through the DHHS, including the Public Health Service (PHS) and NIH. Any institution receiving funds for animal research, training, biological testing, or animalrelated activities from the DHHS must comply with the PHS Policy on Humane Care and Use of Laboratory Animals and the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training (Table 38.1) (OLAW 2015). The NIH Office of Laboratory Animal Welfare (OLAW) enforces the PHS policy. NIH awardees must negotiate a letter of assurance with OLAW assuring that they meet the PHS Policy and that they use The Guide for the Care and Use of Laboratory Animals (The Guide) as the basis of their animal program development and management (Garber, Barbee et al. 2011). The Guide applies to all vertebrate animals, including zebrafish. One of the key requirements of the PHS Policy is that all animal research facilities must establish an Institutional Animal Care and Use Committee (IACUC) and that this committee includes members with specific qualifications. This is described in detail later in this chapter. The IACUC is responsible for reviewing all aspects of the institutional animal care and use program and assuring that all activities are conducted according to PHS Policy and The Guide. Another important NIH office with oversight of NIH funded research is the NIH Office of Science Policy (OSP). The OSP oversees a wide range of biomedical research areas, including biosafety, biosecurity, and emerging biotechnology. All NIH funded research involving the genetic manipulation of living organisms
TABLE 38.1 U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. I. The transportation, care, and use of animals should be in accordance with the Animal Welfare Act (7 U.S.C. 2131 et. seq.) and other applicable Federal laws, guidelines, and policies. II. Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society. III. The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid results. Methods, such as mathematical models, computer simulation, and in vitro biological systems should be considered. IV. Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals. V. Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents. VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure, or if appropriate, during the procedure. VII. The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be provided as indicated. VIII. Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals. Adequate arrangements shall be made for their inservice training, including the proper and humane care and use of laboratory animals. IX. Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly concerned but should be made, with due regard to Principle II, by an appropriate review group, such as an institutional animal care and use committee. Such exceptions should not be made solely for the purposes of teaching or demonstration.
must meet the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). The Guidelines require an Institutional Biosafety Committee (IBC) review and approval of any genetic additions, deletions, or changes done by recombinant engineering. Most IBC’s have broader purview, including the review and oversight of all biological hazard research involving infectious agents. The Center for Disease Control and Prevention (CDC), which publishes the Biosafety in Microbiological and Biomedical Laboratories, presumes that the institution (IBC and/or biological safety officer) conducts a biological risk assessment
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and reviews and approves experiments with infectious agents, including viral vectors and pathogens.
Institute of Laboratory Animal Research (ILAR) The Guide was published in 1963 and has been revised seven times through committees of ILAR, which report to the National Research Council of the National Academies. The eighth edition (2011) introduced expanded sections on aquatic animal care. Because the needs of fish and other aquatic or semiaquatic animals are diverse and species-specific, these expanded sections provide broad guidelines for the management of aquatic animal systems. Specific recommendations are made for environmental parameters, such as water quality, temperature, humidity, ventilation, lighting, noise, and vibration and for documenting monitoring and maintenance of appropriate water quality. The Guide also describes recommendations for appropriate housing and feeding; food preparation and storage; veterinary care, including health monitoring, anesthesia, and euthanasia; and colony and life system management, including social housing and environmental enrichment. Specifics from The Guide are described in later sections of this chapter.
Association of Assessment and Accreditation of Laboratory Animal Care International (AAALACi) Many institutions seek to attain the highest standards in animal care and use and voluntarily participate in an accreditation program administered by the AAALACi. AAALACi accreditation is widely recognized as the gold standard for animal care and use programs. AAALACi is not associated with any governmental agency; however, it is recognized by OLAW as evidence of achieving a high standard of care. Accreditation is obtained after an animal care and use program is reviewed by professionals from accredited peer institutions for adherence to the standards of The Guide and other applicable regulations and guidelines. These professionals are appointed by AAALACi to carry out such reviews as a site visit. AAALACi accredited programs must undergo this extensive review at least every 3 years to maintain their accreditation. Since AAALACi accreditation is based on successful adherence to PHS Policy, The Guide, AWR, NIH Guidelines, AVMA Guidelines, and other regulations, the maintenance of AAALACi accreditation is interpreted by the funding agencies and others as evidence that the institution is meeting all requirements to receive PHS funding. OLAW assigns those institutions with AAALACi accreditation category 1. Such institutions do not have to submit their semiannual program
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assessment reports directly to OLAW, unlike unaccredited institutions, which are assigned category two and must submit the semiannual program assessments directly to OLAW. Institutions with DHHS funding may have other sources of funding that recognize OLAWassurance and AAALACi accreditation, such as the National Science Foundation and Department of Veterans Affairs. Many of these agencies have a memorandum of understanding (MOU) with OLAW that states that OLAW will apply PHS Policy and require PHS assurance for their funded projects. The MOU allows these projects using vertebrate animals to come under the purview of OLAW oversight.
American Veterinary Medical Association (AVMA) The AVMA is a nonprofit organization representing more than 91,000 veterinarians working in private, government, industry, academia, and uniformed services primarily in the United States (AVMA Website). The AVMA provides information resources, continuing education opportunities, and publications, and lobbies for animal-friendly legislation. The AVMA also produces policies in response to member requests and stakeholder interests. These policies are based on the best available scientific evidence. One of these policies is the AVMA Guidelines on the Euthanasia of Animals, intended for use by members of the veterinary profession who carry out or oversee the euthanasia of animals (Leary, Underwood et al. 2013). The overriding commitment of this policy is to provide veterinarians guidance in relieving pain and suffering of animals that are to be euthanized. Specific guidelines for the euthanasia of fish and other aquatic species are defined, and most regulatory bodies discussed in this chapter require recognition and adherence to this publication for euthanasia of zebrafish used in biomedical research.
Occupational Safety and Health Administration (OSHA) and Other Responsible Organizations Zebrafish facilities should comply with local and state regulations regarding workplace safety, general biosafety, environmental compliance, and waste management. For occupational hazards, some U.S. states regulate workplace safety at the state level, with the others falling under the Occupational Safety and Health Administration (OSHA). Laboratory and research safety differ from other workplace chemical safety concerns in several ways; this is why the United States has a separate OSHA standard for laboratories and research workplaces. Laboratory chemical safety in zebrafish research facilities falls under OSHA’s Occupational
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Exposure to Hazardous Chemicals in Laboratories standard (29 CFR 1910.1450) for OSHA-regulated states. State equivalents apply elsewhere: 8 CCR 5191 addresses laboratory chemical safety in the California Code of Regulations (OSHA 1990). Medical sharps or human blood or tissues that are used in the zebrafish facility may also be regulated under OSHA’s Bloodborne Pathogens standard (29 CFR 1910.1030) or a similar local equivalent (OSHA 1991). Testing laboratories should follow the Organization for Economic Cooperation and Development Guidelines for the Testing of Chemicals. The National Research Council’s Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards, Updated Version (2011) addresses laboratory chemical safety. This applies to everything from pH balancing chemicals to mutagens and teratogens used on zebrafish embryos (NRC 2011). Imaging or other biokinetics work with radioisotopes must meet local or federal, As Low As Reasonably Achievable (ALARA) principles using time, distance, and shielding in addition to complying with applicable state regulations, or Nuclear Regulatory Commission regulations in 10 CFR 20 for facilities that are not regulated by the 37 “Agreement State” regulations. The CDC and NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), provides guidance for work at biosafety levels (BSL) and animal biosafety levels (ABSL) one to four (CDC 2007). The American Society for Microbiology (ASM) “Biological Safety: Principles and Practices,” fifth edition, and ASM “Guidelines for Biosafety in Teaching Laboratories” also provide laboratory biosafety guidance (Emmert, Byrd, Gyure, Hartman, & White, 2012; Wooley & Byers, 2017). In addition to normal laboratory waste, which may be either solid waste or listed hazardous waste, zebrafish facilities may also encounter wastewater and medical waste disposal constraints based on local, state, or federal environmental regulations. For example, most larger zebrafish facilities in the United States will fall under the National Pollutant Discharge Elimination System (NPDES) point source regulations and will need to coordinate with the local wastewater publicly owned treatment works (POTW) for discharge pH and any possible additional chemicals in the discharge. Different cities and towns have different wastewater treatment capabilities, so this often varies by the sewer system. Local medical waste regulations apply to the handling and disposal of potentially infectious waste, which may include viral vectors for recombinant work or pathogens used in zebrafish. In the United States, these regulations vary by state but fall under the state EPA equivalent, with additional guidance by CDC, OSHA, and U.S. Food and Drug Administration (FDA). For ground transportation of biomedical waste, the United States. regulates untreated biomedical waste and samples as
Hazard Class 6, Division 6.2 infectious substances under the U.S. Hazardous Materials Regulations (49 CFR 173.134), and air transportation of samples fall under the International Air Transport Association (IATA). Hazardous Materials or Dangerous Goods Regulations (DGR) require medical wastes that may be Class 6, Division 6.2, Category A infectious material (defined as an “infectious substance in a form capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals”) to be treated before transport, typically either by autoclaving or chemical sterilization. Note that medical waste vendors frequently require zebrafish facility staff to complete IATA training every 2 years, or U.S. DOT hazmat training every 3 years. Those institutions receiving DHHS funding and creating, maintaining, and disposing of genetically manipulated zebrafish must meet the NIH Guidelines (see Section IIb NIH OSP) (RAC NIH OSP 2016). The NIH Guidelines require that the institutional IBC review and approve the creation of genetically modified zebrafish. Regulations differ between recombinant zebrafish and recombinant rodents; the NIH Guidelines exempt purchase, transfer, and certain types of recombinant rodent breeding in Section III-D-4-c. But the NIH Guidelines otherwise regulate recombinant animals, including zebrafish, under Section III-D-4, Experiments Involving Whole Animals. Researchers must coordinate with their IBC prior to creating knockout or transgenic zebrafish.
Institutional Responsibilities for Zebrafish Oversight Scientists The primary responsibility of any scientist conducting animal research is to assure the integrity and quality of the research performed and the welfare of the animals involved. The integrity of the research conducted begins in the planning stages of the study. Well-founded research designs and methods are key to the responsible conduct of animal studies and are supported and strengthened by extensive literature reviews and interactions with peers. Maintaining compliance throughout the study by adhering to approved study protocols and funding agreements can assure compliance with animal welfare regulations, laws, and standards. It is also important to apply ethical and responsible practices to both the conduct of research activities, and the collection, analysis, and presentation of data. Performing all aspects of the animal study with a conscientious regard for animal welfare and scientific integrity is key to the successful outcome of any animal research study. Due to the specialized nature of zebrafish care and the relative novelty of the model,
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primary care of the animals and management of the housing system or facility may fall partially or in whole to the investigator. In these circumstances, the investigator, who may possess unique knowledge and expertise, could be responsible for the day-to-day management of the animal model but should work closely with the program veterinary and care staff, as well as the IACUC, to assure that the needs of the animals, the research, and the program are aligned and being consistently met.
Veterinary Care and Animal Care Staff The health and well-being of research animals is the responsibility of all research staff but is the primary concern for veterinarians and animal care staff providing daily animal care and management. The best way to assure proper care and management of animals is to establish and adhere to daily care practices that support their physical and psychological health. These practices and standards should be designed to comply with the required standards and industry best practices, as well as to meet the individual needs of the institution’s research program. Practices should provide for the animals’ general care, and include methods for identifying, treating, and following-up on health issues and medical cases, as well as maintaining accurate and complete records documenting animal care. Beyond the immediate care of the animals, the veterinarians and care personnel are responsible for managing and maintaining a suitable environment for research animals and the facilities that support research activities. Facilities and established care practices should also be designed to promote and protect personnel safety to ensure a research program that provides consideration of animal welfare and the health and well-being of staff involved in animal care. In some instances, the investigator’s knowledge of proper care and management of the animals and their housing may exceed that of the program staff. In these cases, the veterinary and care staff are responsible for working closely with the researcher to assure the care of the animals supports their welfare and is in line with the industry-standard practices.
Institutional Official The authority to assure an institution’s compliance with PHS Policy falls primarily to the Institutional Official (IO). The IO is the administrator responsible, with the authority, to sign the institution’s Assurance with OLAW making a commitment that the requirements of the PHS Policy will be met. The IACUC, often the IBC, and other safety committees report to the IO. The IO must have authority to allocate resources to support
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the research program’s ongoing compliance with everchanging regulations and standards and evolving institutional needs. The IO must have a working knowledge of established safety committees, IACUC, and animal care activities, and the processes to promote and support the efforts of the research program. Frequent communications between the IO, the IACUC, and other unit administrators and staff about the needs and challenges of the program are crucial for preventing and managing noncompliance. Noncompliance incidents are reported to OLAW and other regulatory agencies through the IO. It is important for the IO to maintain open lines of communication internally to address any vulnerable or ineffectual areas of the program in order to avoid or minimize noncompliance, and to assure a culture of compliance within the institution.
Institutional Animal Care and Use Committee (IACUC) (Garber, Barbee et al. 2011, OLAW 2015) The IACUC is responsible for overseeing all aspects of the animal care and use program to ensure compliance with animal welfare regulations, standards, and recommendations. The committee represents the interests and needs of the animals in balance with the needs of the researchers and the institution, in accordance with all applicable laws and requirements. The IACUC is required to oversee the use of zebrafish by the PHS Policy, and The Guide. The committee must be appointed by the Chief Executive Officer and include at least five members. The members must have sufficient knowledge, experience, and ability to assess animal care, treatment, and practices in experimental research and represent society’s concerns regarding the welfare of the animals. The IACUC must include one member who is a doctor of veterinary medicine, one practicing scientist, one member whose primary concerns are in a nonscientific area, and one member who is neither affiliated in any way with the research facility nor an immediate family member of the research staff. One person can serve both roles of the nonscientific and unaffiliated members. However, the minimum number of members must be met, and not more than three voting members should be from the same administrative unit of the facility. The IACUC is responsible for reviewing all aspects of the institutional animal care and use the program, including specific research proposals, animal housing and procedures, facilities, personnel training and medicine programs. The IACUC must review program procedures and facilities at least every 6 months and report to the research facility’s IO. Before any animal research can be performed, a written proposal must be submitted, reviewed, and approved by the committee.
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The IACUC is authorized to require modifications of the proposal and suspend a research activity if it is determined that the activity is not being conducted according to the approved protocol. The research proposal must include the following information: (1) the species and approximate number animals to be used; (2) a rationale for the use of animals and justification of the species and number of animals to be used; (3) a description of the proposed use and procedures to be conducted with animals; (4) a description of procedures to minimize pain and discomfort; and (5) a description of any euthanasia method to be used.
Disaster Response Planning All research facilities either registered with the USDA, have assurance with OLAW and/or accredited by AAALACi are required to have a disaster response plan. Aquatic facilities are especially vulnerable to power outages and other support system failures. Please see the chapter on emergencies for important details to consider.
Personnel Qualifications and Training Principle VIII of the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (Table 38.1) requires all researchers and other personnel to be appropriately qualified and experienced for conducting procedures on living animals. Housing facilities for aquatic species, especially high volume zebrafish facilities can be challenging for personnel not accustomed to working in such environments. Institutions should take advantage of training opportunities for staff to obtain highquality instruction and experience.
Specific Challenges for Oversight of Zebrafish Research Many factors affect the applicability of the various regulations that may govern zebrafish use in research or teaching.
knowledge and experience is limited to those researchers or instructors who are also submitting their own proposals; therefore, leaving none or only a few individuals who do not have a potential or perceived conflict of interest. It may be possible to address this problem by using ad hoc reviewers from other institutions or providing additional training to IACUC members who do not have expertise with aquatic models in general or zebrafish in particular.
Performance Based Standards Zebrafish are relatively new animal models compared to mice, and the number of investigators and institutions using this novel model remains comparatively low to those using mice. Thus, the information about zebrafish husbandry in a research setting is sometimes extrapolated from other species or from commercial aquatic management practices. The Guide makes specific recommendations for the microenvironment, which includes anything with which the fish has immediate contact. Water quality is essential to the well-being of aquatic animals, and routine measurements of its characteristics are essential. Some important characteristics to monitor include pH, salinity, and levels of ammonium, nitrate, and nitrite. Monitoring should be documented with recorded data.
Transportation Many concerns about transporting terrestrial animals also apply to the shipping of zebrafish. These include the availability of commercial carriers, security provided by the transport containers, environmental control of transport vehicles and any loading or holding facilities, and time in transit. Transporting fish involves additional concerns of assuring appropriate oxygen levels are present in the water and avoiding any deleterious buildup of ammonia or other contaminants. In general, the longer the transport distance, the more serious the concern. There is also a need for care during intrainstitutional transportation between animal rooms and to or from procedure space, laboratories, or facilities. Please see additional information in the Chapter on Import/Export and Transportation of Zebrafish.
Knowledgeable IACUC Members Unless the institution has an established aquatics research program, it can be challenging to find IACUC members with the knowledge of issues and/or experience specific to the use of zebrafish to apply performance-based standards during project review or to assess the program and facilities. This is especially an issue when the pool of IACUC members with relevant
Animal Numbers IACUCs typically oversee and regulate the number of animals approved for use by requiring animal acquisitions from vendors, other institutions, or other investigators to be handled and recorded centrally. When animals are transferred between investigators within the institution, between protocols, or produced in breeding
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colonies, self-reporting, or other procedures for census monitoring are needed. It is relatively easy to count the number of adult fish present at any time or to estimate it based on the number and size of aquaria present and the average number of fish per aquaria of a specific size (density); however, it may be necessary to estimate the number of eggs collected and their average survival time to estimate the total number of animals used.
Counting of Zebrafish Onto Protocols and Pain Category Assignment OLAW provides guidance for accounting of larval forms of fish. Zebrafish hatch at approximately 3 days postfertilization (dpf) and must be included in an approved protocol at this stage and oversight provided by the IACUC (Bartlett & Silk, 2016). While fish at four to 7 dpf may require inclusion in a protocol, studies suggest that during this phase of development, the animals do not yet feel pain or distress. Thus, zebrafish do not need to be assigned a pain category until older than 7 dpf (NIH, 2016). Juvenile and adult fish, 8 dpf and older, must be assigned a pain category based on the proposed procedures. Institutions that are accredited through AAALACi must also follow recommendations for animal care and the oversight and guidance for aquatic housing and care, as outlined in The Guide. While The Guide does not refer to developmental stages in its application, it is generally accepted that The Guide standards be applied to zebrafish at and beyond the larval stage (>3 dpf).
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treatment, sanitation), reduce duplication of equipment, provide more efficient use of animal care personnel, and reduce transportation of animals. Decentralized facilities offer greater convenience to individual research teams. Decentralized facilities offer redundancy and may provide backup resources if there is a planned or unplanned disruption of support services in a facility. Populations and studies can also be isolated to address disease concerns or research needs.
Environmental Enrichment The Guide recommends that all animals be given the opportunity to express species-specific behaviors, and this may be accommodated through the provision of environmental enrichment. For fish, this can be met in several ways. Fish are social animals and will school if housed in groups. If zebrafish must be individually housed for experimental reasons, they may be given enrichment, such as plastic plants to accommodate their being alone. Live food is often offered to zebrafish because of its nutritional advantages, but this provision also serves as environmental enrichment because it allows zebrafish to exhibit their natural predatory feeding behavior.
Primary Housing: Housing Density and Sanitation
The rule of thumb for assessing whether a procedure is painful has generally been “If a procedure is painful to a human, it is likely to be painful to a fish.” It is also important to consider what factors or conditions might be potentially distressful for a fish during its use. For instance, fish are social animals, and so it may be distressful for a fish to be housed in isolation. If it is necessary to house fish singly in a tank, the tank should be placed so that the single fish inside it can see other fish group housed nearby. In these circumstances, the singly housed fish may be seen to shoal in unison with the group in the adjacent tank, thus exhibiting a natural group behavior.
For many species, The Guide offers very specific information on the minimal acceptable amount of space allowed for each animal based on size and age. For fish, The Guide states that the number of animals in a location can vary based on the type and size of tank and age of fish, but the recommendation for adult zebrafish is a maximum of five zebrafish per liter. Many institutions have used performance standards to justify higher housing densities, which is allowable under PHS Policy if approved by the IACUC. There are also specific requirements for sanitation of tanks. This is in part based on the type of housing used, type of system used (static or recirculating), and water quality. Accumulation of debris or algae that would block the ability to view and monitor fish is not acceptable and should be removed and/or reduced. Fish tank lids may need to be cleaned regularly for removal of food debris.
Centralization of Zebrafish Facilities
Environmental Monitoring
Comparisons of the benefits of centralized versus decentralized facilities address many of the same considerations for zebrafish as for nonaquatic species. Centralized aquaculture facilities may cost less to build and maintain (ventilation, humidity, temperature, water
The macro environment is the physical environment of the secondary enclosure, generally the room in which an aquatic housing system is located. These conditions must also be regularly monitored and documented. Room humidity is closely monitored for other species,
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but is obviously not as critical for fish immersed in water and is often based on what is safe and comfortable for the staff. Similarly, the rate of air exchange in a room is not as crucial in fish facilities and may even be purposely lower than that required in rooms housing terrestrial animals in order to reduce evaporation from tanks. Some rooms housing aquatic species are kept at a similar temperature to that of the tank water to minimize water temperature fluctuations and to accommodate fish housed off the recirculating system in static tanks in the room. Illumination is also important, and because breeding is strongly affected by the room light cycle, the room photoperiod must be carefully regulated; the natural stimulus for breeding in zebrafish is the onset of daylight. Fish rooms should be maintained so that there is total darkness during the dark cycle, and the presence of even partial light during the dark period may disrupt breeding subsequently for days or even weeks.
Veterinary Care Zebrafish colony health should be regularly monitored and the monitoring documented. The Guide requires that trained personnel observe all animals for signs of illness, injury, or abnormal behavior at least daily. Professional judgment should be used to ensure that the frequency and character of the observations minimize risk to the individual animals and do not compromise the research. Unexpected deviations from the normal should be reported promptly to assure timely delivery of appropriate intervention when needed. Procedures for disease prevention, diagnosis, and therapy should be those currently accepted in veterinary and laboratory animal practice. Additionally, appropriate procedures should be in place to allow colony disease surveillance, diagnosis, and management. Health monitoring programs may include regularly scheduled submission of sentinel fish. Fish found in sump tanks may serve as sentinels. Alternatively, sentinel fish placed into the facility from a known specific-pathogen-free source and exposed to colony animals and subsequent diagnostic evaluation may be used. Sentinels may be placed in various locations, including on the system, in a separate tank, or into the sump or waste stream of a system. Sentinel fish should be submitted to a laboratory with aquatic disease diagnostic experience and capabilities. The Guide states that health monitoring programs should be designed based on the size and complexity of the facility, the species involved, and the institutional research focus. In addition, records of morbidity or sickness and mortality or death should be kept as part of the monitoring program.
Anesthesia, analgesia, and euthanasia of zebrafish are discussed in more detail in other chapters, but it is discussed briefly here, as it relates to PHS policy and The Guide. Anesthesia is necessary to minimize pain and distress to fish during procedures, such as for the collection of tissue samples for DNA isolation and genotyping, often done by fin clipping. Anesthesia may be used for other surgical procedures and to allow manual gamete removal for in vitro work. The most commonly used anesthetic technique is immersion in tricaine methanesulfonate (MS 222, Finquel). Investigators may discuss appropriate anesthetics and analgesics, dose and route of administration with veterinarians experienced with zebrafish research. The Guide encourages the use of pharmaceutical-grade drugs and chemicals whenever possible in order to prevent adverse effects caused by impurities, instability, or toxicity of compounds. The IACUC may approve the use of agents unavailable in pharmaceutical grade after appropriate consideration of purity, sterility, efficacy, and other relevant characteristics. Euthanasia of zebrafish used in research must comply with the regulatory requirements. In the United States these requirements are the AVMA Guidelines for Euthanasia 2013 (Leary, Underwood et al. 2013). Commonly used methods are immersion in the recommended concentrations of buffered MS 222, and rapid chilling in an ice water bath with the appropriate holding times. Embryonic and larval life stages <7 d postfertilization may be euthanized by immersion in 10% sodium hypochlorite solutions. Dead fish should be disposed of as medical waste. Water solutions contaminated with chemicals may require treatment before entering the public wastewater stream. Some states may require an alternative disposal method.
Acknowledgment The authors would like to acknowledge and thank the following colleagues for their contribution, careful review, and suggestions for this manuscript. Pam Bounelis, University of Alabama at Birmingham, Birmingham, AL 35294. Lauretta Gerrity, University of Alabama at Birmingham, Birmingham, AL 35294. Monte Matthews, University of Oregon, Eugene, OR 97403.
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