Zebrafish are a small freshwater fish native to rivers in India, Bangladesh, and Nepal. They have become a popular model organism for biomedical research due to their low cost, short generation time, and optical transparency during development. Proper nutrition is important for zebrafish health and research validity, but their dietary requirements are not well defined. This article discusses the need to standardize zebrafish diets by determining nutrient requirements to improve research consistency and animal welfare.

1. Zebrafish (Danio rerio) are a small freshwater fish
belonging to the cyprinid family (Spence, 2006).
The species is native to warm water streams in the
Ganges and Brahmaptura River basins located in
India, Bangladesh, and Nepal (Barman, 1991; Laale,
1977). They are thought to be an annual species
that breeds during the monsoon season, when food
such as aquatic insects are most plentiful (Spence,
2006). Zebrafish are considered to be omnivorous
having been observed feeding throughout the
water column, from the surface to the benthos, on
a varied diet (Spence et al 2008).
Z
ebrafish have and continue to be a popular aquarium
fish thanks to their hardiness and low-cost but in recent
years the species has become of interest as a model
organism for biomedical, pharmaceutical, neurological,
eco-toxicological and genetics research. So much so, that zebrafish
are often coined as “the new laboratory rat”.
Many biological characteristics have contributed to their popular-
ity such as their high fecundity, short generation time, predictable
spawning and low cost of maintenance. Furthermore, approximately
70 percent of the human genome is similar to that of the zebrafish,
making it a viable model for human genetics research (Howe et al.
2013). Zebrafish are utilised throughout their life cycle but the early
developmental stages are particularly attractive to researchers as, unlike
mice, the animals produce an externally fertilised embryo that is trans-
parent, allowing its embryonic development to be observed simply by
placing it under a microscope.
Today these fish are cultured in most major biomedical research
facilities around the world including the United States (877 institutions),
Germany (359), England (180), China (255), France (219), Spain (138),
Taiwan (84), to name but a few (Kinth et al. 2013). Estimating the exact
numbers of fish used is almost impossible but millions, if not hundreds
of millions of zebrafish are now thought to be used in scientific research
every year (Reed & Jennings, 2010).
In 2010, the Research Animal Department of the British RSPCA
released figures detailing the number of scientific papers using zebrafish
published over recent years on the PubMed Database (Reed &
Jennings, 2010). Revisiting and elaborating upon these figures it is clear
that exponential growth in the use of zebrafish for scientific purposes
continues (Figure 1.).
Optimal culture conditions such as water temperature and water
chemistry values have been established for zebrafish, but our knowl-
edge on nutrition requirements has drastically lagged behind. Many
biomedical researchers are now asking for a standardised diet and
open-formulations for this important research animal (Lawrence 2007,
Penglase et al. 2012, Watts et al. 2012). This is not a new issue; a
standardised diet for rodent models was established almost 40 years
ago, followed by standardised diets for other models including guinea
pigs, rabbits, primates, and swine. At present zebrafish facilities feed
their stock a variety of different dry feeds, alongside live feeds. These
include flake intended for use by the aquarium hobbyist, pellet for
rearing larvae of marine fish and a select few commercially advertised
zebrafish diets.
Zebrafish nutrition
Zebrafish nutrition remains very much in its infancy, being mostly
limited to comparisons between commercially prepared feeds or
against live feed. Formulating appropriate diets is paramount to guar-
anteeing zebrafish are nutritionally satisfied and thus a healthy model
organism. At present poor nutrition and feeding practices has led to
variability among results from human disease, pharmaceutical, toxicol-
ogy, neurology and reproduction studies using zebrafish.
Meeting individual amino-acid requirements ensures that growth
of the animal is not compromised, but its importance extends to
the consideration that deficiencies can be of detriment to immune
and metabolic status. With some popular commercial zebrafish diets
containing up to 60 percent crude protein levels, over-formulation is
also of particular concern. Excessive supply of certain amino acids has
been suggested to incur similar effects to deficiencies triggering stress
responses, toxicity, interference with metabolic function and subse-
quently depressed growth (Choo, 1991).
However, this excess supply of protein is most likely to be of detri-
Nutrition for the new laboratory rat
18 | INTERNATIONAL AQUAFEED | May-June 2015
FEATURE
by Peter H. Bowyer, Plymouth University, UK and Marc Tye, University of Minnesota, USA

2. ment to water chemistry with elevated nitrogenous excretions placing
unnecessary strain upon maintaining optimal water quality param-
eters. Currently, quantitative dietary lysine and arginine requirement
research on juvenile and adult zebrafish is being conducted at the
University of Minnesota. Preliminary data suggests lysine and arginine
requirements are similar to that of common carp (Cyprinus carpio).
This research is the first known, albeit belated, quantitative nutri-
tional research to be conducted for zebrafish. With minimal socio-
economic or environmental sustainability considerations for dietary
protein provision in zebrafish diets, a wealth of ingredient options
seem available.
However, careful consideration will be required to provide sources
of protein that are readily available, highly digestible, nutritionally
consistent and clean. Fishmeal sources are, for the most part, a great
source of high-quality protein for fish; however varying macro and
micronutrient profiles could be a threat to the consistency of stand-
ardised diets. Plant protein sources are readily available from various
sources in various forms, but fluctuating protein contents as well as
the presence of anti-nutritional factors may also render them a risk.
Being the natural prey of zebrafish, perhaps one of the most attractive
options will be that of the various insect-derived proteins now avail-
able. Other avenues such as algae, marine invertebrate and single-cell
proteins may also be evaluated.
Lipid provision
Appropriate lipid provision is also integral to ensuring zebrafish
health. Essential fatty acids (EFA) play a crucial role as a metabolic
energy source in fish, with deficiencies and ratio imbalances leading
to depressed growth (Watanabe 1982). Exceeding requirements can
similarly decrease growth and lead to increases in mesenteric lipid
deposition (Du et al. 2006), with possible implications on biomedical
Figure 1. Publications on the PubMed database (www.pubmed.
gov) of the US National Library of Medicine, registering as a search
result using the keyword 'zebrafish' (Adapted from Reed & Jennings,
2010).
May-June 2015 | INTERNATIONAL AQUAFEED | 19
FEATURE
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3. studies in particular. Inappropriate dietary lipid levels may also lead to
disruption of lipostatic and endocrine systems.
But perhaps of most notable interest is the central role of EFA in
fecundity and reproduction (Watanabe 1982). As before mentioned,
much zebrafish work concentrates on early life stages, requiring high
fecundity, consistent spawning and healthy offspring as a solid base
of research, therefore health of broodstock is indispensable. Some
research has been conducted suggesting low n-3:n-6 fatty acid ratios
decrease growth and influence fecundity (Meinelt et al. 1999, Meinelt
et al. 2000) but quantitative requirements have yet to be determined.
Once this is achieved, the suitability of the many marine and vegetable-
derived oils can be assessed. Again of note is the high lipid content of
certain insect larvae, which could be an attractive option.
As an omnivorous cyprinid, it can be anticipated that zebrafish
are also able to utilise carbohydrates as an energy source relatively
efficiently. Although there is likely to be no specific requirement for
carbohydrates, evidence of decreased growth with low carbohydrate
levels has been demonstrated in the species (Robison et al. 2008). This
could be a preliminary indication that plant or algal-derived ingredients
should feature in diets to promote health.
Although characterising mineral requirements in fish can be some-
what problematic, efforts in zebrafish are indispensable due to the
large influence these micronutrients may have on the fields of research.
Mineral deficiencies can have profound effects on fish by causing,
biochemical, structural and functional abnormalities (Zhao 2014). A
highlighted area of concern among zebrafish facilities appears to be that
20 | INTERNATIONAL AQUAFEED | May-June 2015
FEATURE
Photo Nile Red stereoscopic view courtesy of ©James E. N. Minchin and ©John F. Rawls
from the Department of Molecular Genetics and Microbiology at Duke University
Photos courtesy of ©Peter H. Bowyer
and ©Ben Eynon, Plymouth University

4. animal-nutrition@evonik.com
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5. of spinal deformities, such as scoliosis. Ensuring the fish are provided
with adequate dietary mineral levels (e.g. calcium, phosphorous, zinc)
may help alleviate these occurrences.
On the other hand, excess provision and/or over supplementa-
tion can be just as great a threat to fish health by pushing tolerance
levels. With regards to macro minerals, high dietary calcium can
cause interference with other minerals and impede upon proper
digestion, whilst elevated phosphorous becomes an environmental
pollutant. Trace mineral excess is also of concern. Fishmeal in par-
ticular is known to contain relatively high levels of certain, potentially
toxic, minerals, as a consequence of bioaccumulation in the marine
food chain. One such example is methylmercury. Being readily avail-
able through the gastrointestinal tract, deposition occurs predomi-
nantly in the kidney and becomes a potent neurotoxin (Dórea et
al. 2008). With negligible cost limiting factors on fishmeal inclusion,
potential mineral toxicity or interference should be acknowledged
when formulating diets.
Overall, it is clear that varying or even unknown dietary mineral
concentrations could be jeopardising the consistency of research
findings, particularly from ecotoxicology, neurology, developmental,
and mineral metabolism studies.
Adequate dietary provision
As fish are not able to synthesise vitamins, ensuring adequate
dietary provision is also indispensable to animal health. Vitamin
requirements in fish are well documented and provide a good
basis from which to begin defining those of zebrafish. The merits
of vitamin supplementation, to extend beyond basic requirements,
may also be considered in this case given the objectives of zebrafish
culture and the limited risk of exceeding requirement.
For example, it is well documented that ascorbic acid (vitamin
C) supplementation can provide significant benefits to growth,
reproduction, stress response, immunity and bone integrity (Li and
Robinson, 2008); all pertinent topics in optimising zebrafish culture.
Overall, it is clear that inappropriate or inconsistent dietary nutri-
ent levels may be of severe detriment to the solidity of findings from
research using zebrafish as a model organism. Due to our distinct lack
of knowledge on nutrition, the harsh reality is that unsuitable diets
are being fed extensively to these fish in facilities around the world.
These animals are then used in studies seemingly at the forefront
of increasing our scientific knowledge of human health, genetics.
The key goal in zebrafish nutrition at this stage is to define nutrient
requirements so that we can move towards standardising diets.
Priority should be placed upon consistent, clean, quality ingredients
so that requirements and optimum animal health standards can be
reliably met. Due to the low feed consumption of zebrafish, this
can be achieved irrespective of feed cost, unlike commercial finfish.
Achieving these criteria will allow researchers to use zebrafish as a
robust model in confidence, for the benefit of the scientific com-
munity and general public alike.
Zebrafish nutritional research is unique in that it is of interest to
several different scientific fields that traditionally do not collaborate.
The biomedical, ecotoxicology and pharmacology fields are particu-
larly interested because they want a standardised diet to limit vari-
ation in research. Those involved in veterinary medicine also have
an interest as it is their responsibility to care for and enforce health
standards of zebrafish at research facilities. The aquaculture sector
may also play a part through the potential of using the zebrafish
as a model for food or ornamental species. This diverse commu-
nity means that funding for research could be accessed from many
avenues. It is time for fish nutritionists to take up the challenge and
utilise their expertise, in order to contribute to scientific knowledge,
rigor and integrity in scientific research outside of aquaculture.
References available on request
Zebrafish Husbandry Association
The Zebrafish Husbandry Association (ZHA) is a non-profit organi-
sation devoted to promoting and developing zebrafish husbandry
standards through education, collaboration, and publication. ZHA’s
membership is comprised of a wide range of people interested in
zebrafish culture; including research and veterinary professionals,
facility managers, technicians, fish culturists, aquaculture engineers,
and representatives from various aquaculture supply and biomedical
companies.
The ZHA was originally created by zebrafish researchers in the
Boston area in 2005 and was called the New England Zebrafish
Husbandry Association (NEZHA). Membership in the NEZHA
began to grow rapidly and included many individuals outside of the
New England area so the group changed its name to ZHA in 2007.
Today it has members from around the world who are dedicated
to developing zebrafish husbandry standards. The associations first
working group project titled “The Effect of Stocking Densities on
Reproductive Performance in Laboratory Zebrafish” was published
in the journal Zebrafish in 2011. Since then, various working groups
are working on subjects such as larval rearing, reproduction and
spawning, water quality, health management, welfare, and nutrition.
The ZHA provides valuable information on the latest husbandry
methods through its new website (www.zhaonline.og) and from
its quarterly webinar series which includes topics ranging from
disease treatment and prevention to cryopreservation to expansion
of infrastructure. Along with the website, where the latest job
opportunities are posted, the ZHA has a presence on social media
including a discussion forum on LinkedIn, a Facebook page and a
new Twitter handle (@zhaonline) where you can find updates on
the latest ZHA news and upcoming events. The quarterly newslet-
ter “Stripes” provides information on the ZHA Board meetings,
introduces members to industry vendors, and has a semi-annual
“Featured Facility”.
Much of the latest research in the field of zebrafish husbandry is
presented at the ZHA annual workshop that is held in conjunction
with Aquaculture America. The zebrafish special session has two
full-days of oral presentations that range in topics from facility design
to zebrafish fertility to the latest nutritional research. The zebrafish
special session has been one of the more highly attended sessions
at the annual conference, with over 100 people attending many of
the lectures in 2014.
The ZHA is an affiliate with the World Aquaculture Society
(WAS) as well as the American Association for Laboratory Animal
Science (AALAS). This year the ZHA will be hosting its inaugural
seminar titled “Zebrafish Husbandry and Veterinary Care: Multiple
Perspectives” at the AALAS National Meeting in Phoenix, AZ.
ZHA has a presence at many national and international confer-
ences including Aquaculture America, Canadian Association for
Laboratory Animal Science Symposium, International Conference
on Zebrafish Development and Genetics, and the European
Zebrafish Conference.
admin@zhaonline.org
22 | INTERNATIONAL AQUAFEED | May-June 2015
FEATURE

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