Breeding and Culture of Zebrafish

 

Zebrafish (Danio rerio) Husbandry

                             Mujahidkhan A Pathan and Alok Kumar Shetty             

Fish Genetics and Biotechnology Division, ICAR-CIFE-Mumbai

 Introduction

The zebrafish has emerged over the past several decades to become an excellent mainstream animal model (Lawerence, 2011). Earlier it was used as an aquarium fish and sometimes studied in the laboratory (Laale, 1977), but now it is an omnipresent complement or alternative to the laboratory mouse. The growth in the usage of zebrafish in basic biomedical research has been driven by an ever-increasing array of advances in technology and molecular tools (Lawerence, 2011). The application of the zebrafish ranges from developmental genetics to the modeling of human disease, drug discovery, toxicology, and other diverse fields. Hence, due to large-scale use and economic investment in infrastructure, it is very apparent to have scientifically based standards for husbandry, management, and care of the fish.

 1. Breeding of  Zebrafish.

 Zebrafish are normally kept under laboratory conditions designed to replicate perpetual summer. Depending upon food availability and temperature they can breed all year round (Spence et al., 2006) with females generally producing eggs once every one to three days. Darkness allows the zebrafish to rest and the return of light will trigger fish to breed (Vargesson, 2007). A layer of marbles, closely spaced rods, or mesh can be used to cover part or the whole of the bottom of the tank to prevent the fish-eating their eggs once laid (Matthews et al 2002). Females consistently spawn more frequently and produce larger clutches of eggs with some males than others. A good clutch consists of between 70 and 300 eggs, of which at least 80% are fertilized (Brand et al., 2002).

 

A generalized description of the techniques and some of the equipment used in relation to the spawning process can be found in Lawrence (20

Typically: A small (typically <1L) plastic mating cage or box with a mesh or grill bottom is placed inside a slightly larger container that is filled with water; breeding pairs or small groups of fish are added to the box in the evening; when the fish spawn (usually the following morning), the fertilized eggs fall through the ‘floor’ of the inner box (which means the fish are prevented from eating them). It is possible for both males and females to reach sexual maturity within three months of hatching. Although establishments may begin using fish for breeding from this age (Kurtzman, 2010), initial batches of eggs from such young females may not be of optimal quality. The highest number of embryos are reported to be obtained from fish between 6 and 18 months of age (Vargesson, 2007). The mating behavior of zebrafish seems to be influenced by the exposure of mating partners to one another during the 24 hours before spawning begins (at sunrise) with males stimulated to perform courtship behavior by the detection of female gonadal hormones in the water (Delaney et al., 2002).

Source: Reed and Jennings, 2010

 2. Raising of larvae

Fertilized eggs are kept in an incubator (~28.5 °C) for 72 hr until the larvae are hatched The embryos are reared in embryo medium; a.k.a. EM3 (NaCl, 13.7 mM; KCl, 0.54 mM; MgSO₄, 1.0 mM; CaCl₂, 1.3 mM; Na₂HPO₄, 0.025 mM; KH₂PO₄, 0.044 mM; NaHCO₃, 4.2 mM) (Avdesh et al., 2012).

The different stages of the zebrafish life cycle (Reed and Jennings, 2010) has been broadly established as follows (Fleming, 2007):
·         0-72 hours post-fertilization - Embryos
·         72 hours to 13 days post-fertilization - Early larvae
·         14 days to 29 days post-fertilization - Mid larvae
·         30 days to 3 or 4 months - Juveniles
·         When sexually mature – Adults

 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 (Avdesh et al., 2012). The relationship between feeding frequencies and development in zebrafish is given below:

 

Relationship between feeding frequencies and development in zebrafish

(Source: Lawerence, 2011)

3. System Maintenance

 

Recirculatory system:

Zebrafish are kept in a circulating system that continuously filters and aerates the system water to maintain the water quality required for a healthy aquatic environment. The circulating system also helps to filter excess food and fish excreta. Different companies provide circulating zebrafish systems. A set of different kinds of filters are used in the system 120-micron filter pad, 50-micron canister filter, biological filter, active carbon absorption filter and UV disinfection filter. The filters need to be changed regularly (Avdesh et al., 2012).

The other important features of zebrafish rearing system that needs due care for wellbeing of zebrafish are provided below:

Sl no	Features	Details
1	Lighting	A cycle of 14 hours light, 10 hours dark has been advised, and would appear to be common practice (Matthews et al., 2002, Brand et al., 2002). Ideally, where artificial lighting is used, a gradual brightening/dimming period of around 20-30 minutes in the morning/evening can be incorporated. Light triggers zebrafish to breed, so periods of darkness are important for allowing animals to rest (Vargesson, 2007; Brand et al., 2002). Francis (2008) states that one of the fastest ways to ensure fish will not lay eggs, is to leave the lights on all the time
2	Noise and other disturbances	It has also been suggested that spawning in these fish may be affected if it is very noisy or if there is a lot of nearby movement or activity (Vargesson, 2007).
3	Water Depth	Zebrafish are often described as surface-living fish, yet field studies show that they occupy the whole of the water column, with no significant difference in their distribution according to depth (Spence et al., 2006). It has been recommended that as long as tanks have a ‘relatively large surface area’ water depth does not have to exceed 25cm (Brand et al., 2002). Elsewhere it has been suggested that for spawning, just 10cm water depth in a 50-litre tank should be provided for three adult males and two females (Andrews, 1999).
4	Volume and population density	20 eggs/embryos per 100ml water. 20 young larvae per 400ml up to juvenile stage. Growing juvenile fish and holding adults - 5 fish per litre. For breeding, a pair can be kept overnight in 1.5 litres, or 6 fish in 2.3 litres of water (Matthews et al., 2002) 5 fish per litre in systems possessing filters and a biofilter, as long as there is good water exchange, good feeding regime and good water quality. For breeding purposes it is best to have less fish per tank (2-3 fish per litre). In a tank that does not have filters or a biofilter, the maximum number should be 1 or 2 fish per litre (Vargesson, 2007). In large-scale re-circulating systems, families of sibling adult fish are kept in serial tanks at densities of five adult fish per litre (60 fish/12 litres) (Brand et al., 2002). 25 fish in 45 litres (~10 gallons) (Westerfield, 2000)
5	Temperature	A widely used standard temperature for developmental studies is 28.5°C (Matthews et al., 2002) An ideal temperature for both breeding and development of the embryos is 28.5°C (Bilotta et al., 1999)
6	Cleaning	Standing water tanks Tanks maintained by manual water changes can be equipped with filtration units that will continually remove undesirable material from the water (Matthews et al., 2002). If a third of the water is replaced each day by siphoning up debris from the bottom of the tank, a separate tank filtering system should not be necessary. If a filter is used, around half the water will need to be changed at least once a week (Westerfield, 2000). Cleaning strategies should be designed to minimise disturbance and distress to the fish. Disinfectants should be used with extreme caution.
7	Tank material	Tanks used to hold zebrafish are usually made of polycarbonate, high-quality glass or acrylic (Matthews et al., 2002).
8	Colour and transparency	Glass and other transparent-walled containers have the advantage of allowing easy observation and monitoring of the fish, but a disadvantage in that movements of staff and equipment outside the tank can disturb them. On the other hand, opaque, or very dark colours can lead to hygiene problems since contamination may not be obvious (The Berlin Workshop 1994). A container colouration of medium blue is probably best. Consideration should be given to placing tanks on a dark surface which will prevent light emanating from below, as it is suggested that fish prefer this to light coloured surfaces (Brand et al., 2002).
9	Food type and feeding regime	Zebrafish larvae chase and catch their prey (e.g. Paramecium) in a process that appears to be predominantly visually guided (McElligott & O'Malley, 2005). Dry food alone is not sufficient to keep fish in good breeding conditions. Therefore it is necessary to supplement it with live or frozen food. The most commonly used additional live food is Artemia nauplii. Alternatively, or in addition to Artemia, Drosophila larvae or different types of frozen food that are available from aquaculture supply stores can be used. Live or frozen food (e.g. tubifex, Daphnia and Chironomus larvae) that has been harvested from freshwater systems that also harbour fish, should be avoided, as it may be a source of pathogens. On the other hand, salt-water-dwelling articulates are safe (e.g. frozen adult Artemia and krill). A typical feeding regimen is to feed adult fish tanks twice a day (once at weekends). Adult fish that have to be kept for longer periods of time without breeding require very little feeding (e.g. twice a week, preferably with live food). Two weeks of rich feeding will bring them back into breeding condition again (Brand et al., 2002). Newly hatched zebrafish can eat Paramecium (800μm x 80μm), as well as a variety of prepared foods, infusoria and rotifers (Matthews et al., 2002). Once fish reach one month of age: flake food supplemented with live food such as Artemia. Adult fish being prepared for breeding: live food (Howells and Betts, 2009).
10	Egg harvesting	There are a number of techniques associated with the procurement of eggs. The main ones are: Natural mating Manual expression (‘squeezing’) of eggs from females for in vitro fertilization A good clutch consists of between 70 and 300 eggs, of which at least 80% are fertilised (Brand et al., 2002). On the basis of current knowledge, a minimum interval of a week should usually be allowed between episodes of breeding in females.

Source: Reed and Jennings, 2010

            To decontaminate the fish net, spray with 70% ethanol, rinse in water, and let it dry before re-using. It should be noted that UV filter disinfection dose rate is ~110 mJ/cm² at the beginning of the lamp life and the dose rate decreases over the course of time, hence it is necessary to replace the globe even when it appears to still be functional (Avdesh et al., 2012)

 

4. Water Quality Parameters

                        The optimal water quality parameters for raising zebrafish are provided below:

 

Sl no	Parameter	Optimum Range
1	Alkalinity	50-150 mg/L CaCO3
2	pH	6.8-7.5 (6.0-8.5 tolerated)
3	Temperature	26-28.5 °C
4	Hardness	50-100 mg/L CaCO3
5	Unionized ammonia	<0.02 mg/L
6	Nitrate	50 mg/L
7	Nitrite	<0.1 mg/L
8	Dissolved Oxygen	>6.0 mg/L
9	Salinity	0.5-1 g/L
10	Conductivity	300 -1,500 μS

                   Source: Avdesh et al., 2012

 

5. Zebrafish Health

            A good understanding of zebrafish biology and behaviour, including diseases, clinical signs and treatments, is necessary to minimise suffering or death. Zebrafish should be regularly monitored for signs of ill health. Some of the important resources for information on diseases in zebrafish are (Reed and Jennings, 2010):

·         Zebrafish International Resource Center - Disease Manual

http://zebrafish.org/zirc/health/diseaseManual.php

·         Laboratory Animal Medicine (2002) (Second edition)

American College of Laboratory Animal Medicine Series

·         The Laboratory Fish (2000) - Gary K. Ostrander (editor)

Academic Press, San Diego

            The maximal recorded life-span of zebrafish in the laboratory is 5½ years, though an average of 3½ years has been reported (Gerhard et al., 2002). In laboratories, these animals are routinely only kept for 18 months to two years, after which they are considered to be of lower reproductive value. In the wild, there is little evidence that individuals survive more than a year or two. This may be due to predation or parasites (Spence, 2007).

            While the growth of the use of zebrafish in basic biomedical research has been characterized by innovation, the methods and tools for fish husbandry, management, and care have been slow to evolve beyond those conceived during the initial establishment of the model system. While these approaches and technologies have certainly served the purposes of the field, they must now be improved to better match the widening scope and scale of research being done in fish. Such advances are made possible by applying new scientific information to the development of more sophisticated approaches for fish husbandry and management, and by considering the lessons learned during the establishment of the rodent model system (Lawerence, 2011).

The following resources will be of use to those using and caring for zebrafish:

·         Zebrafish Husbandry Association (ZHA)

www.zhaonline.org

·         British Association for Zebrafish Husbandry (BAZH)

www.bazh.co.uk

·         Zebrafish Information Network - the zebrafish model organism database (ZFIN)

http://zfin.org/zf_info/dbase/db.html

·         Zebrafish International Resource Center (ZIRC)

http://zebrafish.org/zirc/home/guide.php

The following book will also be of interest:

·         The Laboratory Zebrafish (2010)

     Claudia Harper & Christian Lawrence; CRC Press, Boca Raton, USA.

About the Author

          

                                  

Dr. Mujahidkhan. A. Pathan, is a geneticist and fish breeder. He belongs to Agricultural Research Service and is working as scientist at ICAR-Central Institute of Fisheries Education, Mumbai. He has seven years of experience in fish genetics and biotechnology. His research areas include developing zebrafish inbred models, zebrafish model to evaluate carbon nanotube toxicity, genetic evaluation of common carp in inland saline environment, recombinant protein production etc. He teaches quantitative genetics, fish breeding, research methodology, genetics in commercial aquaculture at the university.

 

Mr Alok Kumar Shetty is pursuing his doctoral studies at ICAR-Central Institute of Fisheries Education, Mumbai. His research is on the synthesis, characterization of carbon nanotubes and evaluation of developmental toxicity of CNTs in zebrafish model.

References:

                        The information presented in this article is collated from various sources and is not the original work of the authors.

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Please cite the article as follows:

Pathan, Mujahidkhan., Shetty, Alok, Kumar. 2019. Zebrafish Husbandry. SDP on Zebrafish as a Vertebrate Model for Biological studies (13-23 Aug 2019). ICAR-CIFE, Training manual. Pp 14-23

            Zebrafish (Danio rerio) Husbandry