Wednesday, June 17, 2015

17/06/2015: Zebra fish: nutrition for the new laboratory rat
Image: kamujp
by Peter H. Bowyer, Plymouth University, UK and Marc Tye, University of Minnesota, USA

First published in International Aquafeed, May-June 2015

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 popularity 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 transparent, 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 knowledge 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 guaranteeing 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, toxicology, 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 subsequently depressed growth (Choo, 1991). 

However, this excess supply of protein is most likely to be of detriment to water chemistry with elevated nitrogenous excretions placing unnecessary strain upon maintaining optimal water quality parameters. 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 nutritional research to be conducted for zebrafish. With minimal socioeconomic 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 standardised 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 available. 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 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 somewhat 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 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 supplementation 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 particular 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 available through the gastrointestinal tract, deposition occurs predominantly 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 nutrient 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 community 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 particularly interested because they want a standardised diet to limit variation 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 community 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.

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