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As successively larger populations and more generations of zebrafish are maintained in the laboratory environment, it is becoming more incumbent on the community of people raising them, and those defining the standards for their care, to better define these ideal conditions. This effort is only beginning.

The use of the zebrafish as a laboratory animal model has exploded over the last decade. Indeed, the care and use of various aquatic animals (including medaka, Xenopus spp, fathead minnows, axolotls, urchins, and many more) in the laboratory setting has grown dramatically. This article will focus on the zebrafish, although the premise applies equally to these other prominent aquatic animal models. The primary reasons most often cited to explain the growth in the use of zebrafish are a comparison of the animal relative to mammalian models in its fitness for the purpose (the primary purpose being the description of human development and disease). One of the lesser cited advantages of this animal model, however, is its ability to be maintained and bred in a relatively wide range of environmental conditions. This hardy nature has allowed the science to move forward in advance of a concurrent effort to truly understand the ideal environmental requirements of the animal and its application in the laboratory setting.

The zebrafish was introduced as an animal model by George Streisinger in the 1970s when he sought an alternative model for the study of genetics and development in vertebrates. His decision was motivated, in part, based on the relative ease with which the animal could be maintained and bred. It is ironic that these attributes have allowed the research community to exponentially increase the use of the zebrafish as an animal model without a corollary increase in directed research of specific husbandry requirements. As the use of the zebrafish has increased throughout the world, individual institutions and labs have devised and evolved a variety of unique protocols for its care and use. Protocols for feeding, density, water quality, and breeding, among others, are often very different from laboratory to laboratory. Until recently, there was no apparent drive for consensus on the correct husbandry conditions and protocols for the animal. Complicating matters is the fact that there are a number of different stakehold-ers, from veterinarians, principal investigators, laboratory animal technicians, and others, with unique requirements and insights into the issue.

All of this being said, it is important to note that generalized care standards do exist. As vertebrates, the zebrafish fall under the revised Guide for Care and Use of Laboratory Animals.1This is the main body of work used by investigative authorities such as the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). An Institutional Animal Care and Use Committee (IACUC) will also follow such guidelines as a baseline. To date, however, a committee’s ability to properly assess care is left to interpretation of information presented by a particular research group. Outside of personnel safety and obvious detriments to the animal, a research group must simply provide and document criteria for housing and care. As mentioned above, these criteria vary greatly throughout the research community. A call to develop more rigorous standards appears to be rising in multiple places and from each of these stakeholder groups.

In the United States, a recent example of this rising call is the formation of the New England Zebrafish Husbandry Association (NEZHA). NEZHA was formally founded in 2006 in Boston, Massachusetts by a diverse group of industry stakeholders that shared the realization that a group effort to define standardized protocols for the care and use of zebrafish would benefit each of them. By organizing the exchange of information on a wide variety of topics, the group has begun to document the existing state of knowledge on animal husbandry and the gaps in that knowledge. Its nominal mission is to close the gaps in knowledge and establish best practices for zebrafish husbandry.

In the European Union, a more intense public interest environment and more stringent regulatory environment have led to more clearly defined care and use requirements. The Council of Europe’s European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes –Guidelines for Accommodation and Care of Animals (ETS 123) is a detailed body of information related to housing requirements for a wide variety of animals, with relatively more specifics for caging requirements of aquatic and amphibious animals than is presently available in the United States or other geographies. The standards in this set of guidelines were developed by an Expert Group using varied scientific and empirical information. The details of which are separately available from the Guidelines.

In both of the cases mentioned above, the results are not without continued and considerable debate and it stands to reason that the published guidelines from any groups will be subject to revision as additional, and hopefully shared, information becomes available and relative consensus can be achieved.

Further complicating the attempts at care and use standards are the many materials and methods used for housing these animals. At the outset, many systems for holding zebrafish were designed and implemented by the researchers or institutions themselves. Without a firm base in aquacultural or mechanical engineering, the result was often less than ideal from an operational, maintenance, and longevity perspective. Vendors of specialized caging systems have (in most cases) successfully employed lessons learned from the aquaculture industry, along with their own relevant experience, to house zebrafish. There is, however, no standard for these systems that ultimately effect key environmental (i.e. water quality) factors, including water turnover rates, method and amounts of disinfection, acceptable levels of mechanical filtration, etc. While water quality is an undisputed key to reduction in stress and therefore improved health and viability for any aquatic model, the ideal parameters are neither broadly agreed nor defined and in many cases are not documented adequately to support the research outcome.

It is important to point out that the goal of improving the standards in care and use should be to reduce and eliminate potential suffering by the animal. Although the term suffering has a typically negative connotation, as used here it reflects a positive and responsible idea of use. David Morton, Professor of Biomedical Science and Ethics, University of Birmingham UK was quoted by Andrew Moore2as saying “ethics shouldn’t be a separate issue for researchers, it should part and parcel of their daily activities…Always assume animals will suffer, rather than the reverse.” Mr. Moore himself goes on to point out that the reduction in suffering improves the cost:benefit ratio of an experiment and makes it ethically more defensible, but it also increases the quality of the science, because pain and other forms of suffering can strongly influence physiological responses and hence scientific outcomes.²

This last point is particularly noteworthy as the use of zebrafish in the laboratory setting is expanding into more advanced uses in the fields of toxicology and disease research. Further the term “researcher” should broadly apply to include all involved with the research process rather than the primary investigators alone. Each of the stakeholders can contribute and benefit from the evolution of standards; the primary investigator in data obtained, the husbandry staff in ability to provide care, the equipment provider in the ability to design appropriate equipment, and the veterinary staff in its ability to provide appropriate medical care. All participate in a portion of the research and should therefore actively participate in the positive outcome.

As we continue to work towards the appropriate answers for optimum care and use of zebrafish as a research model, it is important to accept and discuss our basic differences in applied theories. The lack of elaborated animal welfare requirements in relation to aquatic animals has complicated the development of standards.3For instance there remains great debate surrounding the idea of pain perception in fish, an idea that is at the heart of many ethical debates. Do we understand “pain” as it relates to the zebrafish? Is it enough to accept the reduction of quantifiable stress as a reduction in “pain”? The idea of using animals in research is generally accepted, and the use of poikilo-thermic aquatic animals such as zebrafish has many distinct advantages.2,4The advantages of lower physiological complexity, shorter generational cycles, greater density per square foot of laboratory space, and the relative ease with which these animals can be maintained in laboratory settings shows potential but does not give a clear mandate for care.4It is not enough to impart our beliefs as humans to a model such as the zebrafish. Does environmental enrichment have merit for the zebrafish housed in a laboratory setting or simply satisfy a perceived need from our human viewpoint? To responsibly act in the interest of the research model as well as the health of the research industry we must first assess the impact on the animals of housing them in a laboratory setting. Is thezebrafish a sentient being aware of its environment and are we basing this on science or human assumption? Can we house them in a laboratory in a manner that provides near ideal conditions?

We have much to gain from standardizing zebrafish care. Standards will provide a number of benefits. They will provide for a better assessment of the costs of holding the animals relative to other common laboratory animals along with a concomitant improvement in the ability to make proper funding and space allocation decisions. They will allow generalized protocols to be developed that improve animal health, reduce maintenance costs, and make the skills of laboratory technicians more transferable between laboratories. They will allow those charged with the care of the animals a clear understanding of the requirements of audits and accreditation. By continuing to apply the 3Rs (replacement, reduction, and refinement) in relation to the zebrafish, both the zebrafish as a laboratory animal and those who use them will benefit by further definition of its ideal husbandry conditions and how to achieve them.

References

1. Guide for the Care and Use of Laboratory Animals. National Research Council (NRC), National Academy of Sciences, 1996.
2. Moore, A. What’s in store for animal research in the EU? European Molecular Biology Organization (EMBO) Reports 6 (7): 606-609, 2005.
3. Lund, V. Ethics and Welfare. www.aquamedicine.no, 2004.
   http://www.aquamedicine.no/fag. asp?fag=10&meny=6
4. MacMillan, J.R. Comments on AAH March 2006 Reports. National Aquaculture Association, 2006. http://www.national aquaculture.org/pages/documents/AnimalWelfare June262006OIE.pdf

For more information: • www.aaalac.org • www.zfin.org

Steve Aldrich is the President of Marine Biotech, an engineering and manufacturing company providing products and services for aquatic animal life support. Prior to his involvement with Marine Biotech, Steve has held positions in financial and market analysis for Raytheon and Intel Corporation. Steve holds an MBA from Duke University's Fuqua School of Business and can be reached at 978-927-8720 x117 or steve.aldrich@marinebiotech.com.

Chris Obenschain, LATG is the Business Development Manager of Marine Biotech. With over 10 years of experience in the aquatic animal research industry including program operation management at both the National Institutes of Health and Charles River Laboratories, Chris brings a variety of expertise supporting the industry on areas of facility design and program development and implementation. Chris holds a BS in Animal Science from the University of Maryland and can be reached at 301-651-3633 or at chris.obenschain@marinebiotech.com.