This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.
Chapter 2 - Breeding
Detailed Methods for Breeding Over Marbles
(Source: S. Russell)
Fish and marbles
At the onset of the light cycle, zebrafish will generally initiate
breeding behavior that results in the laying and fertilization of eggs. In a tank
that has been marbled, the eggs collect between the marbles and escape predation.
However, when no marbles are in the tank, the fish will rapidly consume the
eggs. By eating the eggs, the fish are cycling the protein within the tank,
reducing the energy loss experienced in egg production and breeding behavior.
By marbling a tank, the egg protein source is denied and energy loss to a tank is
extensive, which is why fish should be put over marbles no more frequently than
once per week. On days when the fish are not over marbles, they should be fed
several times per day with protein rich foods to compensate for their energy
Marbles are used to cover the bottom of the tank the night before the user
expects to get eggs from the tank. By covering the bottom of the tank with a
layer of marbles, breeding fish are unable to eat the freshly laid eggs because the
eggs sink down between the marbles to safety.
Marbling a tank
The fish should be fed approximately 30 min before adding marbles
to give the fish sufficient time to consume the food. When the fish have stopped
feeding, it is important to siphon excess food and debris from the bottom of the
Add marbles to a freshly siphoned tank by slowly dropping them through
the water to the bottom of the tank. The tank bottom must be completely covered
with marbles. Approximately one liter of marbles is enough to cover the bottom
of a ten gallon tank properly. Marbles should be added to the tank slowly enough
so that the fish can avoid being hit by them.
Removing eggs from a tank
Remove eggs from the tank using a siphon (see Embryo Collection). To
siphon eggs, prepare a container to receive the eggs and water. Place the
container near the tank, and below the water line of the tank. To operate the
siphon, place the glass tube into the tank. Draw water by mouth into the siphon,
and quickly place the flexible tubing into the container. Water should flow into
the container. Siphon the bottom of the tank in a methodical manner to collect
Siphons are to be used once. When finished with the siphon, sterilize it
by bleaching and rinsing. Never use a siphon in more than one tank.
Removing marbles from a tank
Marbles should be removed from tanks promptly. After siphoning,
use a net to remove the marbles. Clean them with bleach and extensive rinsing.
Cleaning marble eggs
Clean eggs obtained from marble tanks either by rinsing with clean
system water to wash away debris or by removing the debris with a pipette. To
rinse, pour off dirty fish water with as much of the loose debris from the eggs as
possible. Add clean system water and repeat the process. With each addition of
clean water to the eggs, the debris is diluted and suspended in the water so that
it can be subsequently poured off. Repeat this process three to six times.
Finally, use a pipet to remove any remaining debris. This method is fairly quick,
but often does not completely free the eggs from debris. In addition, eggs can
sometimes be lost while pouring off the water.
An alternative is to use a pipette to remove debris. Place the eggs in a
small petri dish or a finger bowl. Using a squeeze bottle, deliver a stream of
system water into the petri dish so that the eggs and debris swirl around the dish.
Note that the eggs will gather in the center of the dish rather quickly, while most
of the debris will swirl around the dish for a longer time. Before it swirls in to
meet the eggs, use a pipet to remove the debris. System water can be squirted
directly on the eggs to dislodge stubborn debris. Repeat the process until the
eggs are clean. Finally, remove any heavy debris that survived the swirling with
Once the eggs are cleaned, transfer them into clean beakers full of system
water. Then place the beakers in a water bath or incubator. Records are kept for
each tank of marble fish. The number and quality of eggs collected are noted,
as well as the number of eggs that are used.
Fish used in producing marble eggs
Fish used for embryo production over marbles are for the most part
reliable at egg production until they are about 2 years old. Generally, marble fish
consist of 8 females and 4 males in a ten gallon tank.
Feeding marble fish
Because fish producing eggs over marbles expend more energy than
regular stock fish, it is important to provide them with a diet that compensates for
the energy lost in egg laying and breeding and that is ample enough to allow the
fish to develop the reserves from which new eggs will be produced and
In general, it is better to feed the fish several times (four to six) lightly
than to feed them once or twice heavily. Multiple light feedings allow the fish
better opportunity to utilize the food sources and preserves tank water quality by
minimizing the amount of food left rotting on the bottom of the tank. As a "rule
of thumb", fish should not be given more food than they can consume in five
minutes. If fish are still eating after five minutes, they are probably being
overfed, so reduce the amount next time. If, after five minutes, there is uneaten
food in the tank, and the fish are no longer feeding, the excess food should be
removed either with a siphon or a fine net.
Because marbles protect eggs from predation but also collect and
concentrate feces and uneaten food, it is wise to fast the fish while they are over
marbles. This reduces production of feces and provides a healthier environment
for eggs; therefore, the feeding information refers to times when the marbles are
not in the tank.
Example of Feeding Schedule
A.M. or early in the light cycle: Baby brine shrimp or OSU brood pellet food or Tetra Min staple food
Late A.M.: Repeat of above choices.
Early P.M.: Repeat of above choices.
Late P.M.: Repeat of above choices or OSU yellow food or adult brine shrimp.