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Squeezing females for Eggs

Four hours after the boost of hCG, the females are ready to begin laying eggs. The best test is to see eggs in the holding buckets. Also, the cloaca gets red and swollen like in X. laevis.

We recommend precoating the dishes that you will be putting eggs into with a little bit of L15/CS (see testis isolation section for description of L15/CS). This makes the manipulation and transferring of eggs and embryos much easier. You can use as little as 1/2 ml and then transfer that solution from dish to dish. Coat your transfer pipets (we use plastic ones) with the L15/CS as well. We use one dish per female so that we can keep each female's eggs separate in case an individual is producing poor quality eggs. As an alternative, you can coat your dishes with a thin layer of agarose (E. Amaya).

There are many ways to hold the females for harvesting the eggs, but we recommend the following. Hold the female so that the legs are pulled back with the cloaca at the vertex (see photo). Try to keep the cloaca as the lowest point so that the eggs fall out and land in your dish. The eggs are quite sticky and will run down your fingers and down her legs – anywhere but in the dish where you want them unless you keep the cloaca at the lowest point. Gentle pressure on the lower back and abdomen will expel the eggs. Examine the eggs to make sure that they look good with a nice pigmented animal pole and pale vegetal pole. There is no point wasting sperm on poor quality eggs. If the male being sacrificed for the fertilization is particularly valuable, you should check the quality of the eggs (either by a very small squeeze or checking the eggs present in the bucket) prior to sacrificing the male for testis removal.

Squeeze the female so that her cloaca is closet to the dish. This prevents the eggs from running down your fingers and her legs rather than in your dish.

After at least an hour of rest after the first squeeze, the females can be re-squeezed for a second fertilization if desired. A good female can expel over 1000 eggs in a squeeze, and with several squeezes, we can get 2000-3000 eggs. In a natural mating, the number of fertilized embryos can be over 3000.

Contributed by Tim Grammer and Mustafa Khokha