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Are Zebrafish the New Mice?

Wed, 06/18/2014 - 8:31am
Dan Rothen, DVM, Szczepan Baran, VMD, MS, and Marcel Perret-Gentil, DVM, MS

Recent breakthroughs have propelled zebrafish from hobby to research standard.

The field of biomedical research has been using mice and rodents as the bulk of their animal populations for decades, with the first transgenic mouse having been created in 1980.1 When the mouse genome was first sequenced and published in 2002, it allowed for an explosion of mouse research related to genetic manipulation and the creation of transgenic lines of animals. Most research facilities today use some degree of transgenic mice in their research. The Zebrafish Genome Project began in 2001 by the Wellcome Trust Sanger Institute2 and what followed was similar to what was observed in mice. We are now starting to see similar growth in the number of zebrafish (Danio rerio) used in biomedical research. A recent paper examined the global growth of zebrafish research and showed a dramatic increase from 1996 to 2012 when the number of publications grew from only 226 to 1,929.3 This growth coincides with a groundbreaking paper written in 1995 by Kimmel, et al. about the embryonic development of zebrafish.4

Most people have seen zebrafish in pet stores as they are one of the most commonly produced and maintained tropical aquarium fishes. Millions of these wild type fish are transported domestically and internationally to satisfy the demands of aquarium hobbyists. These fish originate from the streams and waterways of India, Pakistan, Bangladesh, Burma, and many other Asian countries. Although there is still widespread collection of wild types, zebrafish are being produced domestically in vast numbers. The hobbyist can even purchase genetically-modified zebrafish in their local pet stores under the name GloFish®.5 These fish have had genes from biofluorescent jellyfish or corals inserted into their genome, and thus, future generations pass this gene on to the offspring and create a line of exotic transgenic fish available to aquarists. The company has expanded the same technology into other tropical species, but the original GloFish® is still a top-seller.

While the mouse continues to hold the title of most prevalent research animal, this new understanding of zebrafish genetics might change the balance in the near future. Zebrafish have numerous advantages over traditional and transgenic laboratory mice. These advantages involve husbandry management, research opportunities, and continued striving to fulfill the 3 Rs: Replacement, Reduction, and Refinement.

Husbandry Management
Zebrafish are relatively easy to maintain in a research facility as long as the management staff has a basic understanding of aquatic husbandry and disease. These small, yet hardy fish can be kept in containers as simple as a five gallon glass aquarium or as complex as a multi-thousand-dollar recirculating system. The needs of each laboratory determine the type of system and level of automation an institution will need. Most large-scale research is being done using large, recirculating systems and source water filtered by reverse osmosis.

Zebrafish are small, with adult fish reaching about two inches in three months. They are also a social species and can often be kept in high densities in small tanks, as long as water quality is properly maintained. Adult zebrafish can reproduce quite rapidly and in great numbers. It can take a pair of mice three weeks to produce 10 pups, but a pair of zebrafish can produce hundreds of embryos only three days post-fertilization.6 When scientists are digging for rare genetic backgrounds, having large numbers of second filial generation (F2) fish can make the process much faster and more efficient.

The costs associated with maintenance of a zebrafish colony are significantly lower compared to that of a mouse. Virginia Hughes wrote in Popular Science in 2013 that a tank of zebrafish costs 6.5 cents a day to maintain compared to 90 cents for five mice in a cage.6 Of course there are many individual factors involved in calculating these numbers for a particular institution, but the trend would point to zebrafish being more cost effective. One caveat is that there are potentially large up-front costs associated with creation of an aquatic facility whereas most institutions are already set up to maintain rodent species.

Pathogens for both rodents and aquatics have been well described. There are numerous laboratories offering clinical diagnostic and pathologic services for aquatic pathogens and Specific Pathogen Free (SPF) colonies have been created. Although there are many pathogens of concern, including some with zoonotic potential, pathogen control may be easier in an aquatic facility. There is little to no concern of wild/feral animals infecting a colony so long as the husbandry staff uses proper hygiene and appropriate personal protective equipment. The main concern would be introduction of pathogens from new imports or via the feed supply. One way to mitigate these concerns is to use multiple smaller and independent systems instead of one large communal system. A quarantine and sentinel system should be in place at all aquatics facilities to ensure optimum colony health.

Research Opportunities
The first article on zebrafish was published in 1951,3 long before their genome was manipulated. Regardless of genetic background, zebrafish have some distinct biologic advantages that make them attractive to researchers. One of the most favorable traits is that zebrafish have transparent embryos and larvae. This allows for in vivo imaging with a simple microscope and the ability to follow specific embryos throughout their development. In vivo imaging of embryonic rodents is a much more intensive and costly process with its own limitations.

For a researcher, zebrafish are a wealth of genetic information. Derek Stemple told The Observer in 2013 that humans and zebrafish share 70% of their genome;7 even more interesting, he said that genes involved in genetic diseases in humans are 84% shared between the two species. A literature search shows transgenic zebrafish span all areas of biomedical research. Some common areas include: developmental biology, biochemistry, toxicology, cell biology, neuroscience, genetics, zoology, and ecotoxicology. There are also studies being conducted at Duke University,6 the University of Miami, and other academic centers where researchers are sequencing blood from children with rare genetic disorders and creating lines of zebrafish with analogous genotypes to monitor clinical development and specific gene involvement.

Another attractive benefit of zebra- fish is the ease at which one can apply chemicals for toxicology, mutagenicity studies, or high throughput screening for drug discovery. In mammalian species, the majority of chemicals are manually injected into individual animals. In zebrafish, chemicals can be added to the water supply, which are absorbed through the skin, and thus reach every fish. This allows a researcher to treat hundreds of fish at a time, reducing labor costs.

Spirit of the 3 Rs
As laboratory animal professionals, we are charged with fulfilling the goals of Replacement, Reduction, and Refinement, as first described in 1959 by William Russell and Rex Burch.8 With the advent of the transgenic zebrafish, there is great potential to make a giant leap in fulfilling the 3 Rs. Currently and for many decades, a lot of genetics research has been performed using fruit flies (Drosophila melanogaster). These animals have been tremendously helpful in the understanding of genetics, but they are not vertebrates and do not share many of the same physiologic characteristics as their vertebrate counterparts. That’s where the laboratory mouse comes in. It has served as the basic vertebrate model for human disease for decades.

Zebrafish have entered the research arena as a middle ground between the two. Zebrafish are vertebrates with similar physiologic characteristics to humans, while at the same time being a less sentient species. By using zebrafish, we can reduce the numbers of mammals used by replacing them with non-mammals. In certain fields, such as developmental biology, it can be argued that their use is a refinement. The transparency of zebrafish embryos allows for visualization techniques that could otherwise be impossible with intra-uterine embryos.

Summary
The zebrafish has been used extensively in the aquarium fish trade for many years. Although a small number of papers were published from 1951 until the 1990s, it was a paper in 1995 that helped propel this fish from hobby to research standard. Their basic biology allows them to be good candidates for developmental biology and toxicology studies among other fields. The zebrafish genome became the focus of a multi-institutional effort starting in 2001 with more and more discoveries taking place every year. The zebrafish has become a critical tool in biomedical research, not only because they are easy to maintain but because of the massive collaborative work done to sequence their genome and perform genetic manipulations. The first transgenic mouse was created in 1980 and the mouse genome was first published in Nature in 2002.9 Since then, transgenic mice have become the backbone of biomedical research. It is only reasonable to anticipate that a new less sentient species, less costly to maintain and reproduce, and one which provides new imaging and research opportunities will follow the same trajectory and become an irreplaceable player in the field of biomedical research.

References

  1. Mobraaten, L.E., Sharp, J.J. Evolution of Genetic Manipulation of Laboratory Animals. Proceedings from the 50th Annual Meeting of the American Association for Laboratory Animal Science. Chapter 17:129-135, 1999.
  2. http://www.sanger.ac.uk/resources/zebrafish/genomeproject.html
  3. Kinth, P., Mahesh, G., Panwar, Y. Mapping of Zebrafish Research: A Global Outlook. Zebrafish. 2013; (10)4:510-517.
  4. Kimmel, C., Ballard, W., Kimmel, S., et al. Stages of Embryonic Development of the Zebrafish. Developmental Dynamics 203:253- 310 (1995)
  5. www.glofish.com
  6. Hughes, V., Will This Fish Transform Medicine? Popular Science Online, 2013.
  7. McKie, R., How the Diminutive Zebrafish is having a Big Impact on Medical Research. The Observer Online, 2013.
  8. Russell WMS, Burch RL. The Principles of Humane Experimental Technique. London: Methuen; 1959.
  9. Mouse Genome Sequencing Consortium. Initial Sequencing and Comparative Analysis of the Mouse Genome. Nature 420:520-562, 2002.

Dan Rothen, DVM, Division of Veterinary Resources, University of Miami Miller School of Medicine, 1501 NW 10th Avenue, Miami, FL 33176. He can be reached at drothen@med.miami.edu.

Szczepan Baran, VMD, MS, Veterinary Bioscience Institute, 200 East 1st Street, Winston-Salem, NC 27101. He can be reached at sb@vetbiotech.com; www.vetbiotech.com.

Marcel Perret-Gentil, DVM, MS, Laboratory Animal Resources Center, The University of Texas at San Antonio, 1 UTSA Circle, San Antonio, TX 78249. He can be reached at marcel.perret@utsa.edu.

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