Zebrafish Basics

Zebrafish, Danio rerio, is a tropical fish belonging to the
Order – Cypriniformes
Family – Cyprinidae
Genus - DanioZebrafish belong to the same family as carp and minnow and are indigenous to areas within the Himalayan region, including Pakistan, Myanmar, Nepal, and India (1,2).  They are commonly found in slow moving water such as streams, canals, ditches, ponds, as well as rice fields and stagnant waters.  In their natural state, zebrafish feed on mosquito larvae as well as other insects.  Their breeding season is thought to be between April and August with egg laying occurring in the small pools of streams (2).Gender Identification
To the untrained eye all zebrafish may look the same, but there are subtle differences between males and females that stand out to well-trained zebrafish users.  Generally, gravid females have larger bellies than males and this distinguishing characteristic is enough to tell them apart.  Difficulties arise when females are not carrying very many eggs or when males are well fed and have bulging abdomens. Body color and fin color are used to differentiate between the sexes when differences in belly size are not obvious.  These color differences are affected by diet, age, and strain, so it is difficult to create simple descriptions of the color differences between sexes.  The best way to learn how to tell the difference between male and female zebrafish is to be trained by an experienced zebrafish user and then to practice separating males from females.

Water Quality
Zebrafish can tolerate a wide range of water quality parameters (3), although they do not handle rapid changes in short periods of time (4).

Chlorine is an important chemical for water purification at water treatment plant, but can be a fatal toxin to zebrafish (3).  At a minimum, tap water should be allowed to set for 24hr to allow all chlorine to evaporate before being used.  In many facilities where large volumes of water are needed, or there are a lot of impurities in the source water, reverse osmosis, deionized, or distilled water are used.  If a form of purified water is used, salts and mineral need to be added back to the water before it can be introduced to the zebrafish.

Temperature
Because zebrafish are native to tropical conditions, they require relatively warm water but can adjust to variable temperatures between 70°F -90 °F (5, 6) with an accepted optimum temperature of 83°F (6).  Although zebrafish can grow anywhere in this range, temperature is perhaps the most universally consistent environmental parameter in zebrafish husbandry and research (7).  Zebrafish raised in temperatures that deviate from the optimum temperature have shown skewed sex ratios (6) and similar fish have shown impacted performance of cellular function (9).

pH
Like most freshwater fish, zebrafish should be kept at a pH in the 7–8 range in order to promote good health of biofilters and stable water quality (7).  Because the stability of pH is more important than whether or not the pH is 7.4 or 7.8, most facilities tend to keep a pH that is the easiest to keep stable.  If possible, a stable pH on the lower end of the range is preferred; ammonia at any level should cause concern for overall health, but a lower pH shifts the ammonia equilibrium towards the less toxic ammonium (10).  Both ammonia and ammonium should be removed immediately upon detection and can be avoided with sufficient biofiltration and water changes.

Salinity
Salinity (or Conductivity) is the measure of total concentration of all dissolved ions in the water (11).  Salinity can have a considerable impact on survival, growth, and reproduction.  The general range of salinity for the husbandry of zebrafish is 0.25–0.75 ppt (7).

Nutrition
While the specific nutritional requirements of zebrafish are unknown, they are likely to be similar to those of other omnivorous warm water fish (i.e. goldfish, carp, shiners, minnows, etc…). Data for those species is available and can be used as a reasonable standard for comparison.  Most useful, however, is the experience and advice of those already successfully managing zebrafish colonies in a research setting.

Generally speaking, there are five major nutrient classes in fish diets, which can be delivered via two categories of feed.  The five classes of nutrients are proteins/amino acids, lipids, carbohydrates, vitamins and minerals.  Those components can be delivered via the two categories of feed: live diets or artificially prepared diets.

As zebrafish develop from fry to juveniles and finally mature adults, their nutritional requirements and their feeding strategies change.  Consequently, their diets must also change to match their needs and capabilities.  For example, dietary protein levels should be highest for juvenile fish and should decrease as fish enter adulthood.  Excess protein will increase the amount of ammonia that is produced as waste, which could negatively impact water quality, decreasing growth rates and reproductive success.  Dietary lipids are important at all life stages of zebrafish both as a source of energy as well as essential fatty acids required for normal growth and development.  While no dietary requirement for carbohydrates has been demonstrated in fish, the omnivorous zebrafish possess the enzymatic apparatus necessary to convert carbohydrates to energy.  It should be noted, however that diets with excess energy (in the form of lipids and carbohydrates) can decrease food consumption and lead to a reduced growth rate.  Vitamins are dietary essential organic compounds and are required in very small amounts by fish.  Although the precise requirements for zebrafish are unknown, most live foods are rich in vitamins and well formulated prepared diets will contain adequate levels of these compounds.  Minerals are inorganic elements required by fish in trace amounts for a number of biological processes, including ossification, osmoregulation, and nervous system function.  Many of these compounds are absorbed from the surrounding water through the gills, and required levels are likely provided by both live and formulated diets.

Live diets for zebrafish can include a number of zooplankton species including Artemia, rotifers, and Paramecium.  The qualities that make a suitable live food item include amenability to mass-culture, balanced nutritional profiles, digestibility, and attractiveness/acceptability.  Artificial or prepared diets are designed to replace live diets, and are formulated using biological materials.  The primary reason for using prepared diets is economic; in most instances, using prepared feeds represents a cost-savings over live feeds (reduced labor and production costs).  In addition, prepared diets can be sterilized thereby reducing the probability of bacterial contamination.  However, since the exact nutritional requirements of zebrafish are unknown it, is not recommended to completely replace live prey items with artificial diets.

When selecting diets for zebrafish, it is important to consider the life stage that you are planning on feeding.  Zebrafish larvae begin exogenous feeding at about 5 days post fertilization (about the same time that they open their mouth, inflate their swim bladder, and development is complete on their digestive tract).  Over the next 3-4 weeks their energy demands are higher than at any other developmental stage in their life.  Live diets are suitable for fish at this stage as long as they are small enough to be consumed without getting stuck in the fish’s mouth (approx. 150-200 µm at this stage).  Paramecium, rotifers and a wide variety of artificial diets are appropriate, but Artemia are typically a bit too large.  Live diets tend to have well rounded nutritional profiles and artificial diets for fry should contain up to 45-60% protein, 6-10% fat and less than 5% carbohydrate.

Zebrafish juveniles still require relatively high levels of proteins and lipids, but carbohydrates can now be used to “spare” proteins for growth.  The size of the particles they can ingest is increased to the range of 400-600 µm thereby allowing for Artemia nauplii to be fed.  If a manufactured diet is preferred, choose one with a slightly higher lipid content (6-15%).

Once zebrafish reach adulthood, their dietary needs shift from supporting growth and development to gamete production.  Although they can ingest prey items in excess of 600 µm, it is best to keep particles in the range of 400-600 µm to facilitate digestion.  Appropriate nutrient profiles will resemble 45-55% protein, 10-15% lipid, and less than 5% carbohydrate.  It should also be noted that adult zebrafish not being used for gamete production have much lower nutrient demands and can be fed at greatly reduced densities and frequencies (12).

Zebrafish Quarantine Procedures
A quarantine method is highly recommended when setting up a zebrafish facility, as the potential for bringing in harmful pathogens such as Mycobacterium exists when introducing fish and embryos from other sites.  A room separate from the main system with a flow-through or recirculating system is best to eliminate the spread of pathogens.  Persons entering and exiting this room should be kept at a minimum.  Dress in procedures such as shoe covers, lab coats, hair bonnets, and gloves should be donned before entering the room and after exiting the room hands should be scrubbed with soap or, if possible, a chlorohexadine scrub brush to minimize any transmission from the quarantine system to the main facility.

All fish and embryos received should be immediately taken to the quarantine room upon arrival to the facility.  Only embryos < 36 hours post fertilization can be bleached upon receipt and introduced to the main system without quarantine.  If the embryos are > 36 hours post fertilization or have hatched, they must be raised in the quarantine room.

Quarantine fish should be observed closely for signs of illness or disease for 2 weeks.  Depending on experimental needs, it is recommended that sick fish be euthanized to minimize the further spread of unwanted bacterial contaminants.

As soon as fish have reached sexual maturity, they may be bred for embryos, which are then bleached and introduced to the main system.  Once an established line can be maintained in the main room, the fish in quarantine can be euthanized (13).

References
1. http://en.wikipedia.org/wiki/Zebrafish#Taxonomy
2. Zebrafish in the World:  A Review of Natural History and New Notes from the Field; Zebrafish.
2007; 4: 1.
3. Detrich W, Westerfield M, Zon LI, The Zebrafish: 2nd Edition Genetics, Genomics, and
Informatics. Elsevier Academic Press, San Diego, CA, 2004, p. 686.
4. “Working with Laboratory Zebrafish.” AALAS Learning Library © 2005 American Association for
Laboratory Animal Science.
5. Cortemeglia C, Beitinger TL, Temperature tolerances of wild-type and red transgenic
zebra danios. Trans. Am. Fish. Soc. 2005; 134: 1431–1437.
6. Westerfield M, The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio)
(3rd edition). University of Oregon Press, Eugene, OR, 1995, p. 385.
7. Lawrence C, The husbandry of zebrafish (Danio rerio): a review. Aquaculture 2007; 296: 1-20.
8. Uchida D, Yamashita M, Kitano T, Iguchi T. An aromatase inhibitor or high water temperature
induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic
female zebrafish during sex-reversal. Comp. Biochem. Physiol. 2004; A137: 11–20.
9. Place SP, Hofmann GE. Temperature interactions of the molecular chaperone Hsc70 from the
eurythermal marine goby Gillichthys mirabilis. J. Exp. Biol. 2001; 204: 2675–2682.
10. Emerson K, Russo, RC, Lund, RE, Thurston, RV. Aqueous ammonia equilibrium calculation
effect of pH and temperature. J. Fish. Res. Bd. Can. 1975; 32: 2379-2383.
11. Buttner JK, Soderberg RW, Terlizzi DE. An introduction to water chemistry in freshwater
aquaculture. Northeastern Regional Aquaculture Center Fact Sheet. 1993; 70.
12. Adapted from training materials created by Christian Lawrence, Aquatic Resources Program
Manager, Children’s Hospital, Boston, MA.
13. Zirc Quarantine Recommendations.