Avdesh, A., Chen, M., Martin-Iverson, M. T., Mondal, A., Ong, D., Rainey-Smith, S., et al. Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction. J. Vis. Exp. (69), e4196, doi:10.3791/4196 (2012).
This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.
Zebrafish housing and maintenance is easier and cheaper than traditional rodent models. Several thousand zebrafish can be housed in a small laboratory. As a result of this protocol, researchers will be able to manage a zebrafish facility which will provide healthy conditions to the zebrafish. In addition, the following illustrations will help identify fertilized eggs, adult zebrafish, and their food. An illustration of a male zebrafish (Figure 1A) and female zebrafish (Figure 1B & 1C) is shown to help researchers distinguish between a male and a female zebrafish for breeding purpose. Figure 2 depicts a microscopic view of; brine shrimps at 12X (Figure 2A), a single brine shrimp at 90X (Figure 2B), and an unfertilized brine shrimp egg at 90X (Figure 2C). This will help in understanding the difference between unfertilized and fertilized brine shrimps for proper feeding purposes. Fertilized and unfertilized eggs are shown in Figure 3. Figure 3A illustrates a microscopic view of fertilized and unfertilized embryos. Unfertilized embryos are generally opaque and/or with ruptured cell(s) inside the chorion (black arrow) whilst fertilized embryos appears intact and growing to the next cell division state (for detailed reading of different stages of zebrafish embryos see1). Higher magnification view of a fertilized and an unfertilized egg is shown in Figure 3B and 3C respectively. Figure 4 illustrates a female zebrafish's ovipositor to help researchers distinguish between a male and a female zebrafish.
Several critical problems that might occur in a zebrafish laboratory include blockage of water supply to individual/all tanks in the housing system, poor water quality, and leakage in pipes or reservoir of the circulating system. In addition, problems in obtaining embryos from breeding could be another concern. Troubleshooting of these issues is discussed below.
Figure 1. An illustration of a male zebrafish (A) and female zebrafish (B, C).
Figure 2. A microscopic view of brine shrimps at 12X (A), a single brine shrimp at 90X (B), and an unfertilized brine shrimp egg at 90X (C).
Figure 3. Microscopic view (16X) of fertilized and unfertilized eggs, where only two eggs are unfertilized, the unfertilized eggs are indicated with black arrows (A). Higher magnification view (90X) of a fertilized (B) and an unfertilized egg (C).
Figure 4. A female zebrafish's ovipositor (indicated with black arrow) illustration.
||50-150 mg/L CaCO3
||50-100 mg/L CaCO3
||300 -1,500 μS
Table 2. Water quality parameters. Water quality parameters. Optimum range of environmental parameters in the zebrafish aquarium11.
Zebrafish originate from the Ganges river in northern India and are becoming popular in research in both their adult and larval stages2, reviewed by Spence et al.3. Zebrafish possess several advantages over other animal models such as high fecundity, ease of maintenance, optical clearance of embryos, rapid embryonic development, and low maintenance cost. They are amenable to genetic manipulation6 and suitable for high-throughput drug screening4,5. Their fertilization is external which is advantageous for their use by developmental biologists. Due to these favourable characteristics, zebrafish are gaining popularity in genetics7, pharmacological8, and behavioural research9,10. There are a number of challenges to maintaining a zebrafish facility and zebrafish husbandry to obtain embryos. Herein, we describe our experiences and recommendations in addressing these challenges and outline a protocol for system maintenance, feeding, breeding and raising of the larvae.
To maintain zebrafish in a healthy condition, it is important to provide them with a clean environment in a properly functioning aquarium system. An important part of this is changing system filters regularly so that all the tanks receive proper water flow and clean water. It is vital to avoid failure of the cycling water supply to each tank due to blocked system pipes. The pipes can be cleaned using a higher than normal water pressure and flow if blockage does occur. Ideally, around 10% of the system's water should be replaced daily to maintain good water quality. Alternatively, water can be replaced while changing the Canister or Carbon filter. This ensures that dirt deposited in the pipes connecting these filters is removed. The quality of water should be checked on a regular basis. Parameters such as alkalinity, pH, temperature, hardness, ammonia, dissolved oxygen, salinity, and conductivity should be considered as important factors in representing the quality of the system water (see Table 2 for details). At the very least nitrate, pH, and temperature should be monitored on a regular basis to ensure good water quality for housing zebrafish. Ideal nitrate (NO3-) levels are <50 mg/L11; if high these levels can be reduced by replacing the water in the circulating system with the fresh system water. Occasionally, filters do not fit well and leak so it is recommended to check for any leaks after a filter change. If the water flow from the main reservoir is blocked either after changing the water pump or changing a filter, the water flow can be restored by either loosening or removing the filter for a few seconds to release any vacuum being generated in the pipes. The time required to change filters can vary depending on various factors such as total biological load on the system, cleanliness of other filters, and dirt deposited in the pipes. Hence, filters should immediately be changed if they appear dirty or if all the tanks are not receiving the correct water supply. It is also recommended that the fish net be cleaned with 70% ethanol, and rinsed in water to decontaminate it, and allowed to dry before being re-used. Drying ensures evaporation of ethanol which otherwise is toxic to fish.
Most of the zebrafish systems use de-chlorinated tap water; however, some systems use deionized water. It is important to keep the conductivity of the system water between 300 and ~1,500 μS as this reduces the energy the fish needs to maintain body salts. Therefore, zebrafish cannot be kept in deionised water unless salts are added to maintain the optimal conductivity levels. There is a risk of possible high copper concentrations in the system water if tap water is used because the carbon filter does not remove copper. Therefore, users should check for copper concentrations and avoid copper piping where possible.
Zebrafish should never be overfed as this may increase the nitrate level in the water, possibly affecting their breeding11, or viability, as some fish may die due to overeating. We recommend providing no more food during any one feeding than a tank of fish can finish within 10 min. It is very important to remove salt from the brine shrimps before feeding them to the zebrafish as excess salt concentration causes death. If more zebrafish eggs are required, fish can be fed three times a day. Cleaning the breeder fish tanks daily also improves levels of egg production.
When feeding on the Aquatic Habitats systems we usually turn off the water pump and air pump to allow the fish to eat the food for 10 min. This decreases the amount of food that is washed into the filters. However, users must be careful to remember to turn on these pumps again afterwards.
Zebrafish are usually at optimal breeding condition between ~3 and 18 months of age. Pairwise breeding should not be performed for two consecutive days11; however, in-tank breeding can be performed daily as a tank can hold many fish which reduces the chance of the same pair of fish being bred for two days in a row. Breeding should be undertaken at regular intervals even if eggs are not required. This process will ensure the breeding cycle of the fish is maintained. It is recommended that there are more females than males in a breeding set-up. Male zebrafish change their female partners on a daily basis12 which further supports this recommendation. Furthermore, within our laboratory we initially experienced problems with breeding, however, using more females than males in a breeding set-up helped solve the problem. Moreover, feeding with a high protein content diet and brine shrimp two-three times a day, mixing fish from different tanks (from different parents), maintaining the temperature of the breeding set-up between 27 and 28 °C, and squeezing the bellies of females with blocked ovary tubes using gentle massage further improved egg production. We recommend keeping a record of fish lines/ origins to avoid in-breeding between siblings. This further improves embryo production. Keeping a record of the number of embryos laid by fish from each tank is also recommended. This assists with keeping a track of the best breeding fish tanks and taking measures to improve breeding in the fish not laying eggs.
Raising of larvae
Feeding of larvae should commence from 5 dpf (days post fertilization). Young larvae can be fed with dry food of ~100 microns in size (e.g., ZM100) or live food such as paramecium and rotifers (which stimulates growth). The food size can slowly be increased to 200 microns (e.g. ZM200) or 300/400 microns (e.g. ZM300). A population of adult fish should be around 6-7 fish per liter of water. This practice is recommended for better maintenance of BOD (Biological Oxygen Demand) to the tanks.
No conflicts of interest declared.
The authors would like to thank Tammy Esmaili for her assistance in the management of laboratory consumables. AA is the recipient of a PhD Scholarship from the Centre of Excellence for Alzheimer's Disease Research and Care, School of Medical Sciences, Edith Cowan University. MC receives PhD funding support from the Rotary Club, Perth. AM, GV, KT, and RNM are funded by the McCusker Alzheimer's Research foundation.