The Reproductive Biology and Spawning of Zebrafish in Laboratory Settings - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 5

odors of nonrelated, unfamiliar (reared and maintained separately) males over those
of unfamiliar brothers for mating. The underlying genetic basis of this preference is
unknown, but may be the major histocompatibility complex (MHC) genes that are
important in kin recognition in other fish species (Apanius et al., 1997).
1.3 SPAWNING TECHNIQUES AND TECHNOLOGY
1.3.1 In-Tank Strategies
One general approach to breeding zebrafish in the laboratory is to simply provide a
spawning site or substrate directly in holding tanks, while fish remain “on system” or
in flow. This type of technique relies on the “natural” production of fish kept in mixed
sex groups with minimal manipulation of individuals. Another important feature of
this basic approach is that because fish remain on flow, water quality is regulated and
maintained throughout breeding events. Finally, it also largely minimizes the
handling of fish, which can be a stressful event (Davis et al., 2002).
The first formally described technique for breeding laboratory zebrafish is the
most basic example of an in-tank breeding method. In this approach, glass marbles
are placed at the bottom of holding tanks to provide a spawning substrate for the
animals. Fish spawn over the marbles, and the eggs drop into the spaces in between,
preventing egg cannibalism and facilitating their subsequent collection by siphon-
ing (Westfield, 1995; Brand et al., 2002). While this method may be effective to
some extent, it is generally impractical for use in large culturing facilities with
hundreds or thousands of tanks. Despite its shortcomings, it is still frequently cited
in the methods sections of zebrafish papers, and is often used by investigators
breeding zebrafish for the first time.
A slightly more advanced in-tank approach involves placing a breeding box or
container in holding tanks that fish will spawn over during breeding events (Fig. 1.2a).
A common feature of this method is that the box/container will have a mesh-type top
through which spawned eggs drop and are subsequently protected from cannibalism.
The box will also typically have some plastic plants affixed to it to make it more
attractive as a spawning site. This type of method is more facile than the marbles
technique, as boxes can be moved freely in and out of holding tanks as desired. It also
better facilitates the collection of staged embryos from groups of fish, and can also be
used for breeding pairs. This method is utilized relatively infrequently, and thus no
commercially fabricated equipment of this type is available. When this method is
chosen, the box/container must be custom-made to fit with the needs of the particular
facility in which it is being utilized.
Another form of in-tank breeding involves the use of a specially manufactured
crossing cage that is designed to fit inside holding tanks. The fish to be crossed are
netted out of holding tanks and transferred to the crossing cage. Eggs are collected
after breeding takes place by siphoning or after removal of the fish from the tank. This
method allows for production of time-staged embryos because it can include a divider
to separate males and females until eggs are needed for experiments. This technology