Pairwise Breeding of Individual or Natural Cross Fish

(Source: C. Walker from Zebrafish Book 5th Edition)

Embryos can be obtained from individual fish by pairwise breeding.  This method is especially well suited for screening individual fish for genes or mutations but is also useful for generating stocks of common fish or even for producing a modest number of embryos for routine work. Successful breeding can be obtained in relatively small containers (i.e. 500 ml plastic containers with lids). This method can be adapted for more than one breeding pair by using a slightly larger container (i.e. a 5 L mouse cage). 

The Natural cross is a method of choice because it produces less stress on the fish than in vitro fertilization.

The onset of light in the morning is a major stimulus for zebrafish to breed, however, the fish may be inspired to breed at other times such as when the lights go off at night.  Setting up fish in the late afternoon enhances the chance of them waiting until morning to breed.

The resulting embryos are quite clean and usually do not require sterilization because the containers and water are clean.  However, extensive washing of embryos after harvesting helps prevent mold.

Natural crosses over marbles

1.     Place pairs or groups of fish into clean plastic containers (i.e. mouse cages) with several inches of fish water and a layer of sterile marbles covering the bottom.

2.     Several hours after the lights come on the next day, remove the fish and return them to their tanks.

3.     Siphon the embryos from under the marbles (see Embryo Collection), either into a collecting net or into another container.

Natural crosses in false bottom containers

A simple container system can be fabricated for efficient transfers of fish and embryos*.  Obtain two containers, one a little smaller than the other so that the smaller one fits inside, but above the bottom of the larger container. Replace the bottom of the smaller container with stainless steel or plastic mesh that has holes smaller than the diameter of the eggs.  Provide clean plastic "grass" or netting as a hiding place for the fish.  This increases egg production.
1.     Place a clean smaller plastic container with a mesh bottom into one of the larger containers.
2.     Fill with enough fish water to cover the mesh with several inches of water.
3.     Transfer a pair or a group of fish into the container.
4.     Several hours after the lights come on the next day, remove the fish and return them to their tanks.
5.     Remove the top container and collect the embryos from the bottom container.
6.     Wash the plastic containers in a large animal cage washer.  Sterilize the marbles by bleaching.
           
Appropriate Uses of Natural Crosses

1. To identify mutation-bearing individuals.
2. To make outcross or intercross stocks of mutation-bearing fish.  (Most mutations are carried in heterozygous parents.)
   1. An outcross of a heterozygous fish bearing a mutation to a wild-type or clonal line would produce progeny that are 50% heterozygous for the mutation.
   2. An intercross between two mutation-bearing heterozygotes would produce ¼ diploid mutant offspring.  If the mutation is lethal as a homozygote, then ²/₃ of the surviving progeny are mutation-bearing heterozygotes; an enriched stock with an increasingly inbred background.
3. To make a stock of fish where a large number of parents is not required.
4. To produce embryos for study.

An average of 50-60% of fish set up for natural crosses will produce embryos.  Any handling of fish produces stress so it is good to allow a week's rest between natural crosses with the same fish.  Females can produce eggs in a natural cross shortly after being squeezed for eggs in an in vitro experiment.

*    This technique was devised in the Nüsslein-Volhard lab in Germany and the Driever lab in Boston.