Behind the Scenes of Zebrafish Research

Today we have the 2nd in a series of articles by Jan Botthof, a PhD Student at the Cambridge University Department of Haematology and the world renowned Wellcome Trust Sanger Institute. Following his first article “Zebrafish: the rising star of animal models”, Jan discusses here how Zebrafish used in scientific research are housed, cared for and bred.

Today I am going to look at some of the things that have to happen in the background to allow scientists to carry out their research. These things include the rules and regulations covering zebrafish use in research, general care and daily work in the fish facility.

Regulations
Zebrafish research in the UK is covered by the same laws that govern research on all other vertebrates, as outlined in the Animals (Scientific Procedures) Act (ASPA), originally instated in 1986 and recently revised to implement the provisions of the new EU directive. This means that the standard of care is just as high for fish as for mammals. All institutes housing fish need a licence ensuring that standards are met, every research project is evaluated for possible harm to the animals and all of the people involved in research or care for the fish receive mandatory training in order to ensure that the fish are treated correctly. Everyone takes utmost care to ensure that the fish lead a comfortable life in the zebrafish facility.

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish housing
Now that we have covered the basic legislation, let’s talk about essential zebrafish care. Nowadays, fish are usually housed in special rooms, unlike the beginning of their use in research back in the 1970’s, when they were commonly just kept in a few tanks on a shelf in the lab. These rooms are designed to keep a constant temperature (between 24 and 28°C depending on the institution) and the lights are programmed to give a constant light-dark cycle to simulate the sun (usually around 16 hours of light and 8 hours of darkness).

Various commercial fish housing systems exist, but most of their features are very similar. The basic components of such a system are the fish tanks, racks to hold them, an integrated water supply, as well as water filtration and monitoring components.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

The tanks are designed to allow a constant inflow of fresh water, easy removal from the rack and convenient access for feeding. These tanks used to be made of glass, but currently different kinds of plastics are much more popular due to the lower weight, making it much easier to handle them. Unless a procedure requires identification or separation of a specific fish, they are always kept in groups, not only for practical reasons, but also because zebrafish are very social animals and need interaction with other fish.

The water filtration and monitoring system ensures that the water is free of contaminants, has the right pH, salinity, hardness, enough dissolved oxygen and does not contain too much nitrite and nitrate stemming from waste products (i.e. fish excretions, excess food). Apart from the constant flow of fresh water, tanks are cleaned regularly to prevent the accumulation of waste products, as well as microbial and algal growth.

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

A separate quarantine room is also very important. This is where incoming fish from outside facilities are kept on a separate water system to prevent the introduction of parasites and diseases into the main facility. These fish are preferably received as early embryos, which are disinfected before shipping to kill any germs.

Diet
Just like there are different housing systems, there are different possible food sources, ranging from commercially available dry fish flakes to adult or larval brine shrimp. This diet is often supplemented with paramecia (small single celled organisms) to achieve optimal growth and survival rates when the fish are raised to adulthood. Exactly which diet is chosen depends on the individual facility. At the Sanger Institute, fish are fed adult brine shrimp, which are very rich in protein, soft and easily digestible (especially compared to brine shrimp cysts) and they are able to survive and swim even in fresh water. This is much closer to the natural diet that the fish would obtain in the wild than most commercially available diets.

Brine Shrimp. Image: Hans Hillewaert

Brine Shrimp. Image: Hans Hillewaert

Disease prevention
During the daily cleaning and feeding tasks, all tanks are monitored for diseased or injured fish, which are then humanely euthanized to minimize suffering. Euthanasia is usually carried out using an overdose of a common fish anaesthetic followed by destruction of the brain to ensure the death of the animal. Detailed records are kept to identify recurrent problems, such as potential parasitic infections. Dead fish are also removed from the tanks and their data recorded. This monitoring is especially important for fish that have been treated with drugs or that carry mutations likely to cause disease.

Breeding
One essential component of working with fish is setting up matings between them. This is essential if you want to obtain embryos for studying them, or when crossing different genetically modified lines and many other procedures. Fish are placed in small mating tanks in the late afternoon before the actual mating, as zebrafish begin to mate right after sunrise in the wild. These mating tanks have a removable insert between the fish and the floor of the tank, so the fish cannot consume their own eggs, which they would otherwise do.

It is also possible to tilt the separation between the floor of the tank and the fish to further stimulate the fish, as they prefer shallow water for egg laying. If you need the embryos at a specific stage you can use tanks with a separator between the male and female, so you can control the time of the mating. An occasion when you would need to do this is when using the gene editing technique CRISPR to modify zebrafish genes, a process which requires injections of the Cas9 enzyme and appropriate guide RNAs during the first stage of development. Matings can be done in small groups or in pairs. It is very important to be able to correctly identify the sex of the animals – not only do you obviously need a male and a female to have a successful mating, but you also need to know this when you combine different transgenic lines. Here you would take a male from one line and a female from another, so you can put them back in the correct tank after the mating, as it is otherwise nearly impossible to identify individual fish.

Zebrafish mating tank with removable separation before and after assembly.

Zebrafish mating tank with removable separation before and after assembly.

Once the eggs have been laid and fertilized, you can collect them in a sieve, and place them in petri dishes containing water with some salts and minerals essential for development. Embryos are then raised at 28.5°C. Here at the Sanger, the zebrafish larvae are placed in nursery tanks when they are five days old and the yolk that feeds them during early development has run out. These fry reach sexual maturity within three months, from which point on they are considered adults and housed in the main facility. Zebrafish in the lab can live about two to three years, but usually we use younger fish for breeding, as they  lay more eggs.

In summary, a lot of work needs to be done before any actual research can be carried out. Moreover, a lot of effort is put into ensuring the health and welfare of all laboratory animals. The next time you read about some exciting new discovery made using animal research, try to picture how much effort was needed before any actual science was done!

Jan Botthof

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