Zebra Fish - Nutrition for the new laboratory rat

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).

Zebrafish 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 nutritionZebrafish 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

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 provisionAppropriate 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

SIZE 10MAXUM

80

160 0

NORGREN

MAX.

MIN.

393.31[9990]

391.31[9939]

48.00[1219]

52.19[1325]

DCCInlet

CYL.Disch.

End ofHead

BINInlet

53.25[1353]

57.69[1465]

102.13 [2594]

111.12 [2822]

2

18.00 [457]

1.93 [49]

256T060

1545

30

195.72 [4971]

F085

SHIM

PO

64.83[1647]

108.59[2759]

15.00[381]

36.91[937]

31.19[792]

29.19[741]

MAX.

MIN.

19.16[487]

30.00[762]

39.00[991]

Ă12.00[305]

2.00 NPT [WATER]

2.00 NPT [STEAM]

1.00 NPT [STEAM]

66.50[1689]

12.56 [319]

24.59 [625]15.88 [404]

284.00[7214]

1.00 NPT

2.00 NPT3/4 NPT

101.44[2577]

30.38[772]

88.00[2236]

108.28[2750]

278.03[7062]

199.38[5064]

269.88[6855]

67.28[1709]

15.00[381]

P.O. Box 8 100 Airport Road

Sabetha, KS 66534, USAPhone: 785-284-2153

Fax: [email protected]

www.extru-techinc.com

Engineered Pre-Kill ZonesOptimize Petfood Safety Food safety is rapidly changing the way the world looks at pet foods. Extru-Tech recently introduced “Advanced Features” to their line of Extruders that provide:

• Best in class design reduces horizontal surfaces and increases sanitation access under, on and around the unit

• Incorporates Advanced Venting Technology (AVT) for suppression of steam and renegade product mist

• Becomes the first line of defense in control/elimination of potentially dangerous pathogens.

• Completed an industry-first scientific validation study proving the kill/lethality step of the Extru-Tech extrusion system design

• All of these advanced features and validation in one extrusion system

With increased focus on food safety, Extru-Tech’s Advanced Feature Extrusion puts you well ahead of previous and current industry standards.

Contact a system specialist today at 785-284-2153 or visit us online at

www.extru-techinc.com.

Advanced Feature Extrusion

ET-261A.indd 1 2/12/15 4:52 PM

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

[email protected] www.evonik.com/animal-nutrition

DL-Methionine for Aquaculture™ The best choice for your aqua feed.

DL-Methionine for Aquaculture™ plays a central role in ensuring balanced nutrition and optimal growth of various fish species. Maximize your feed efficiency with our services AMINODat® Aqua and AMINOCarp®.

DL-Methionine for Aquaculture™

15-01-148 International Aquafeed Magazine AZ DL-Methionine for Aquaculture DIN A4 Mai 2015 .indd 1 13.04.15 14:10

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.

[email protected]

22 | INTERNATIONAL AQUAFEED | May-June 2015

FEATURE

May-June 2015 | INTERNATIONAL AQUAFEED | 23

FEATURE

Turning ideas into opportunities.PROGRESSIVE AQUAFEED PROCESSING

Imagine the possibilities

wenger.com

BElGIUm TAIwAN BRASIl ChINA TURkEy INDIA

Leave it to Wenger to redefine aquatic feed production via twin screw extrusion. Based on the proven Wenger Magnum twin-screw series, the new TX-3000 features barrel geometries that allow greater capacities than any other extruder in its class.

The combined features allow increased production capacity of up to 30 percent compared to previous and competitive aquatic machines — totally redefining cost/benefit. The TX-3000 can be equipped

with either the High Intensity Preconditioner (HIP) or the High-Shear Conditioner (HSC) to match specific process and capacity requirements, making it ideal for processing a full range of aquatic feed products.

Contact us now. With new concepts and visionary leadership, we’re ready to help you select the right tools for your extrusion and drying needs.

Our business in life is not to get ahead of others, but to get ahead of ourselves. —Stewart B. Johnson, Dutch Artist

TX-3000 RAISES ThE BAR ON AQUATIC FEED PRODUCTION

Wenger14.TX3000.Ad.210x147.indd 1 4/9/14 7:34 AM