The Bateson Centre Aquarium Facility holds over 750 different zebrafish families called lines. Six of these lines are wild-type strains, which are similar to the Zebra Danio fish that you can buy in most tropical fish shops. The remaining lines are all genetically modified by genetic engineering to incorporate a transgene or genetic alteration in their genome. These lines enable advanced biological analysis of life course biology.
We keep stringent records of all fish in the facility including the date of birth, numbers in each tank and the tank location of where they are kept. We use a database to hold these records that is updated regularly with each change that happens.
Here at Sheffield the aquarium team not only perform the daily husbandry for the fish but we also maintain the majority of the lines we hold. We identify heterozygous fish by phenotype (their physical appearance) and also provide a genotyping service (to view their genetic appearance).
Zebrafish are past their optimum breeding age by the time they are 2 years old and so to maintain the line we breed new generations at around 14 months of age. They are usually fully mature and breeding well by the age of 6 months so this ensures that we always have a healthy breeding stock.
The majority of genetically modified fish are kept in heterozygous form (asymptomatic) and are crossed to wild-type to maintain good genetic variation. As only 50% of the offspring will carry the genetic change we often need to in-cross the fish and examine the offspring for the required relevant phenotype. Some of these lines do not produce a phenotype and so will require a small amount of the caudal fin to be taken and the DNA sent off for analysis, this is called genotyping.
Transgenic fish are more often kept in homozygous form ( also asymptomatic) and are in-crossed to maintain the line, to stop these fish becoming unhealthy due to inbreeding they are also out-crossed to wild-type fish every 2 or generations. Transgenic fish can usually be selected before they reach the age of protection, at 5 days past fertilisation, and so do not need to be identified at a later stage.
We breed fish in 2 different ways.
One method is to put a large box with a mesh insert into a shoaling tank of more than 3 fish. On top of the mesh we place marbles, as these resemble small pebbles that are often found in the natural habitat of the zebrafish. The lights coming on in a morning triggers the fish to begin breeding, the females will lay her eggs onto the marbles and the males will then swim over and fertilise them. Once the eggs have fallen through the mesh that adult fish cannot reach them, as there is a tendency for the eggs to be eaten. We can then take the spawning box out of the tank, remove the mesh and marbles and collect the eggs.
The other method is useful if you want to know who the exact parents are or you want the fish to breed at a particular time. A male and female are placed into a breeding box that has a grid at the bottom. This grid acts both to separate the adults from the eggs and also to resemble the natural habitat. A divider is placed between the 2 fish, this acts not only to stop any fighting that may occur overnight but also means that the fish will not breed until it is removed, although they can sense each other through the transparent divider. Once the divider is removed, after the lights have come on, breeding will commence.