This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.


Testosterone Treatment to Produce Males

(Source: C. Walker)

Young zebrafish of mixed sexes can be treated with testosterone to yield predominantly males. This is a useful procedure to ensure having enough males for breeding, particularly in lines which usually yield mostly females.


17-a-methyltestosterone; MW 302.5; Sigma #M-7252; final concentration = 1.7 x 10-7 M.

Stock solution:

150 mg testosterone in 50 ml Absolute Alcohol (very soluble). Keep in -20C freezer.

Treatment solution:

Add 10 µl of stock solution to 600 ml egg water slowly while stirring. Stir ~10 min.


1. Remove chorions from 24 h embryos
2. Add ~25 embryos to 50 ml fresh testosterone solution in a 250 ml beaker; add as little water as possible (i.e. 1 drop). Leave overnight.
3. The next day, add 50 ml fresh testosterone solution.
4. On each consecutive day, replace 1/2 of the testosterone solution with fresh testosterone solution.
5. Feed embryos normally.
6. Continue treatment until 15 days of development, moving fry from beakers to mouse cages at 9 days (see Raising Larvae in a Nursery, page 1).
7. At 15 days, remove most of the testosterone solution and add system water. Continue diluting during the regular daily cleaning.

CAUTION: Avoid contact with testosterone. Rinse well if you get it on your skin. Rinse glassware, etc. very well.

NOTE: Embryos need to be put in testosterone solution by 24 hrs. If treated later, some will develop as females.

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