Breeding and Culture of 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:

 

                  
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