Zebrafish Housing, Husbandry, Health, and Care: IACUC Considerations
George E. Sanders
Abstract
The articles in this issue review pertinent and specific information about infectious and noninfectious disease, housing
system design and function, husbandry practices and techniques, and Internet resources as they specifically relate to
zebrafish (Danio rerio). This article explores aspects that
members of institutional animal care and use committees,
husbandry care staff, physical plant personnel, veterinarians,
principal investigators, and research personnel should consider with regard to the appropriate and necessary care and
use of this unique fish model in a teaching, laboratory, or
research setting. This information is designed to enhance
understanding and facilitate collegial discussions to inform
decision making about zebrafish care and use at various institutions or facilities.
Key Words: Danio rerio; disease; housing systems; husbandry
and care; institutional animal care and use committee
(IACUC); resources; zebrafish
The Zebrafish Model and The Guide for
the Care and Use of Laboratory Animals
A
s indicated by various authors in this issue, zebrafish
(Danio rerio) have rapidly expanded from their humble
origins of exclusive use as a model for vertebrate development and genetics to a become an increasingly popular
biomedical research model with varying uses and applications. In contrast to the previous version of the Guide for the
Care and Use of Laboratory Animals (hereafter, the Guide;
NRC 2011), about which several articles have been written
to assist the users of zebrafish models with the incorporation
of this and other aquatic animal species into existing regulatory framework (Borski and Hodson 2003; DeTolla et al.
1995; Koerber and Kalishman 2009; Matthews et al. 2002;
Neiffer and Stamper 2009; Smith and Noll 2009; Sneddon
George E. Sanders, DVM, MS, Certified Fish Pathologist (AFS/FHS), is a
lecturer and Aquatic Animal Program Director in the Department of
Comparative Medicine in the School of Medicine at the University of
Washington and is the Veterinary Medical Officer of the US Geological
Survey’s Western Fisheries Research Center in Seattle.
Address correspondence and reprint requests to Dr. George E. Sanders,
Department of Comparative Medicine, University of Washington School of
Medicine, Health Science Center Box 357190, Seattle, WA 98195-7190 or
email [email protected].
Volume 53, Number 2
2012
2009), the 2011 version of the Guide incorporates aquatic
animals much more expansively and specifically. This article is intended to help correlate the regulatory landscape
described in the Guide (chapters 2–5) with the articles in
this issue as they relate to the use of the zebrafish model.
Information about the zebrafish model has become
widely available and is increasing at a tremendous rate. A
good source of this information is the Internet. In this issue,
Smith (2012) provides a resource for locating information
about methodology, techniques, and organizations associated with zebrafish.
Animal Care and Use Program
Although not exclusive to the use of zebrafish, this section of
the Guide has been discussed in previous issues of the Journal
and other publications (Borski and Hodson 2003; DeTolla
et al. 1995; Koerber and Kalishman 2009; Lawrence et al.
2009; Mason and Matthews 2012; Matthews et al. 2002;
Smith and Noll 2009) with great explanation and detail. As a
result and in the interest of brevity, I defer to those references.
Environment, Housing, Management,
and Physical Plant
In this issue, Lawrence and Mason (2012) explain that
zebrafish housing systems should function to (1) provide
a stable and favorable environment that produces and maintains healthy and productive fish and (2) support specific
research goals of investigative staff. Although a state-of-theart fish housing system is important, its functionality is maximized by the quality of the people who manage it. The rapid
expansion in the use of the zebrafish model system to diverse
applications in various fields may require the use of specialized housing equipment, especially when study designs call
for the evaluation of fish over long periods of time. Zebrafish
housing systems combining design principles from industrial aquaculture, laboratory rodent housing, and research
genetics are now commercially available from a number
of sources, and an increasing number of academic research
institutions are constructing large, centralized facilities to
support their growing zebrafish research programs.
Nasiadka and Clark (2012, in this issue) explain that
zebrafish breeding is not a simple process and is still being
elucidated. Mate choice and mating behavior depend on olfactory cues, visual stimuli, and social interactions, and spawning
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is affected by fish age and size, intervals of egg production,
light cycle, diet, and health status. Regarding line propagation and vigor, current breeding strategies avoid inbreeding
and focus on maintaining genetic diversity. However, this often results in a high degree of polymorphic variations, which
can affect the effectiveness of the research and its reproducibility. Because there are no zebrafish inbred lines in which all
individuals are identical and homozygous(as has been described for mouse isogenic lines), attempts should be made to
develop breeding schemes that yield relatively robust inbred
lines characterized by a high degree of genetic homogeneity.
In an article on larval rearing and husbandry, Wilson
(2012, in this issues) considers both published literature and
unpublished methods to describe current techniques. She
notes that there is, in fact, minimal knowledge about the
requirements of larval zebrafish; instead, their care tends
to be a function of the constraints and pressures of the facilities and laboratories in which the zebrafish are reared. Even
though many aspects of zebrafish early larval physiology,
anatomy, and genetics are well studied, this knowledge has
seldom been used to improve larval rearing and husbandry
techniques. Successful larval rearing is dependent on matching the biological and morphological needs of the larval fish
to the rearing protocol. Studies in this area need to be completed and published so that standardization in this field can
be based on sound scientific evidence.
Veterinary Care
In terms of their effects on veterinary care, biosecurity, and
research model integrity, yet-unidentified viral agents pose a
significant threat to zebrafish models. According to Crim
and Riley (2012, in this issue), no naturally occurring viral
infections have been reported in the zebrafish. However,
because many aquatic viruses can infect multiple species,
zebrafish are likely to be susceptible to viral pathogens that
have been identified in other fish species. It is imperative that
naturally occurring viruses of zebrafish be identified and
characterized so sensitive diagnostic tests can be designed
and adequate health monitoring can be implemented. This
process is crucial to the improvement of zebrafish health,
reduction of unwanted variability, and continued development of the zebrafish as a model organism. Furthermore,
continued research to elucidate the specific pathogenesis and
transmission of each virus will be necessary to determine
which pathogens are of concern for various areas of research
and will aid in the design of biosecurity protocols.
Similarly, the impacts of subclinical disease in zebrafish
are of considerable concern. In this issue, Kent and colleagues
(2012), Whipps and colleagues (2012), and Sanders and colleagues (2012) report that the two most common infectious
diseases of zebrafish in research laboratories are mycobacteriosis and microsporidiosis, which present with both clinical
and subclinical signs.
For mycobacteria, a combined diagnostic approach is
the most informative, with histology and special staining as
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a first-level screening tool, polymerase chain reaction for
rapid detection and species identification, and the slower
culture method reserved for strain differentiation. However,
complete elimination of mycobacteria from a large facility
supplied with recirculating water is probably not feasible
given the ease by which mycobacteria can colonize or recolonize such systems.
For microsporidia, a combined spore identification diagnostic approach includes wet mount preparation from infected tissues, histology with special staining, and polymerase
chain reaction for more rapid confirmation. Because of the
horizontal and potential vertical transmission of this parasite
by spores that are resistant to the standard method of embryo
and egg surface disinfection used for zebrafish, the most
effective method for prevention is avoidance. Routine morbidity, mortality, and disease and pathogen surveillance is a
very important tool not only for the control of mycobacteriosis and microsporidiosis but also for the detection of other
pathogens and the monitoring of the overall health of zebrafish colonies.
Intestinal parasitism by the nematode Pseudocapillaria
tomentosa is less frequently observed (Kent et al. 2012, in
this issue). This disease usually exhibits clinical signs that
are associated with the level of parasitism. A myxozoan parasite is occasionally found in the common mesospheric
ducts but does not appear to be associated with any clinical
disease. A common condition that may or may not have an
infectious etiology is egg-associated inflammation in reproductively mature females.
Noninfectious diseases that are often clinically unapparent
in zebrafish include hepatic megalocytosis, bile and pancreatic
ductal proliferation, and neoplasms of the ultimobranchial
gland, gastrointestinal tract, and testis. Most of these lesions are
only evident histopathologically and normally do not display
clinical signs unless the lesions are severe.
Facilities should monitor stocks of zebrafish for the presence
of these concurrent diseases, and be aware of their existence
when interpreting study results. Understanding the cause,
modes of transmission, and distribution of these pathogens
can provide useful information for the development of control, prevention, and possibly treatment strategies.
With the increasing maintenance of adult zebrafish for
longer periods of time, neoplasia is becoming a more common finding in these populations. Spitsbergen and colleagues
(2012, in this issue) explain the importance of considering
spontaneous tumors in diagnostic cases and retired broodstock as well as carcinogenesis bioassays and mutant tumor
models because neoplasia has been observed in a wide variety of histologic types and has affected nearly every organ
and most cell types in zebrafish.
Data regarding dietary influences on neoplasia in zebrafish
are not yet published. Both diet and husbandry system type
have shown a strong influence on tumor incidences in
zebrafish. Thus one of the most urgent issues in the rapidly
growing field of cancer research using the zebrafish model is
the need to optimize husbandry systems and diets to eliminate or
minimize the natural carcinogens and possible tumor promoters
ILAR Journal
that confound research in many recirculating systems and
commercial diets. Some studies have a shown a clear link
between these, whereas others have not. The role of infectious
disease agents in the development and types of neoplasia observed in zebrafish is an ongoing topic of concern.
Finally, Matthews and Varga (2012, in this issue) describe standard anesthesia and euthanasia agents and doses
for zebrafish. They also discuss why anesthesia and euthanasia of the different stages of zebrafish can be challenging.
As per the Guide, the specific agents and methods chosen
for zebrafish anesthesia and euthanasia depend on several
host factors, professional veterinary judgment, and the needs
or scientific objectives of research protocols. Because
zebrafish, like most other animals, cannot communicate
states of (dis)comfort, distress, or pain directly and subjectively, it is necessary to rely on human interpretation and
knowledge of zebrafish’s species-specific behavior to assess
their condition. Typically the difference between sedation,
anesthesia, and euthanasia is closely related to drug/agent,
dose, and/or contact time. However, current techniques are
limited at best and depend on an interpretation of behavior or
a measuring of physiological responses, both of which add a
layer of complexity.
Conclusions
The articles in this issue elucidate standards set forth in the
updated Guide for the Care and Use of Laboratory Animals.
In addition, it is critically important that all individuals associated with the care and use of zebrafish at a given institution
or facility work collaboratively toward the goal of promoting
excellent research with this aquatic animal model. It is essential that everyone understand their particular role(s) in the
process and strive to do the best they can to contribute in a
collegial and constructive manner to meet the established
goal(s). Mutual education and communication among all groups
are imperative for success.
Volume 53, Number 2
2012
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