Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage

- Start with a mating tank, a tank with a removable insert that allows eggs to fall through. Add two male and three female adult fish to a divided mating tank in the evening before embryo collection to acclimate to each other overnight.

The next morning, transfer the fish into a new mating tank containing system water and remove the divider. Wait for 15 to 30 minutes to give the fish time to mate and spawn.

Now collect the embryos and store them in a Petri dish containing Hank's embryo medium. Grow them at 28.5 degrees Celsius throughout the protocol. Examine embryos' morphology under a dissecting microscope.

You will observe several developmental features. Cleavage of the blastodisc gives rise to multiple cells. The blastoderm takes the shape of an inverted cup above the yolk cell. Paraxial mesoderm segments develop on the dorsal part of the embryo and so on.

Beyond 24 hours post-fertilization, simply sort embryos by total body length. In the example protocol, we will set up mating of transgenic mCherry reporter fish and sort the resulting embryos.

- To begin, culture embryos to three months of age, which is reproductive maturity. Segregate two adult male and three female fish from the desired strain into divided mating tanks of fresh system water on the evening before embryo collection. The following morning after the lights come on, remove the divider and allow the fish to mate naturally until embryos are observed in the bottom of the tank.

Collect embryos in 30-minute intervals and separate Petri dishes of embryo medium until the desired number are collected. To stage the embryos, culture them in groups of 50 to 75 per 10-centimeter Petri dish to promote consistent developmental timing of all embryos. Then keep the plates at 28.5 degrees Celsius.

Measure the embryo age using somite number after segmentation until approximately 24 hours post fertilization, or HPF, and separate the embryos based on developmental age.