Production Of Haploid Embryos

Owned by Jonathan Knight
Last updated: Jan 17, 2018 by Anne Eagle

(Source: R. Andrews and T. Ellis from Zebrafish Book 5th Edition)

This method allows the production of homozygous haploid embryos for screening for recessive mutations.  Haploid embryos develop fairly normally for the first few days, but eventually develop bent tails and vacuolated body cavities. They are inviable.


Overview of haploid production

1.  Maintain fish according to the schedule for Embryo Production By In Vitro Fertilization, page 2.14.
2.  Obtain sperm as described for Embryo Production By In Vitro Fertilization (page 2.14) and transfer them (in the 0.5 ml drop of full strength Hank's saline) to a watch glass.
3.  Irradiate sperm with an ultraviolet light (UV, e.g. 29 cm from a 43 cm long Sylvania® germicidal tube) for 2 minutes.
4.  Proceed with in vitro fertilization as described.


Detailed procedure for UV sperm

This is a general procedure for making infertile sperm that can still activate eggs for production of haploid, early pressure (EP) or heat shock (HS) embryos.

Materials Needed:

Watch glass 

Large glass Petri dish 

Ice

Two Pasteur pipettes

Pipette bulb

Stopwatch

UV lamp

Latex kitchen gloves

Safety glasses

Small test tube with cork marked for UV sperm

Sperm in Hank's solution in a small test tube

Procedure:
1.   Fill the bottom of the glass Petri dish with ice.
2.   Put watch glass on top of the ice in the dish.
3.   Using a Pasteur pipette, transfer the sperm from the test tube onto the watch glass.  Be sure to get as much of the sperm into the pipette as possible without taking up any bubbles of air with it.  Also, do not bubble the sperm in the watch glass as you are expelling them from the pipette.  Expel sperm in a thin, even layer on watch glass.  DISCARD THIS PIPETTE.
4.   Cover the sperm with the top of the Petri dish.
5.   Put on the Latex gloves and safety glasses.
6.   Place the covered Petri dish under the UV lamp.
7.   Remove the cover of the Petri dish and start the stopwatch.
8.   Gently move the dish under the lamp in a swirling motion for 2 minutes.
9.   After exposing the sperm to UV light for 2 minutes, replace the cover on the Petri dish and remove the dish from under the lamp.
10.   Using the CLEAN pipette, remove the sperm from the watch glass and transfer them to the small test tube marked for UV sperm.
11.   Put the UV sperm test tube into the ice to be used later for fertilizing eggs.Â