Preparation of the Host and Donor Embryos

1. Incubations: Quail and chick eggs are incubated with their long axis horizontal during the time necessary to obtain the stages adequate for the experiments: 36-48 h for the experiments described here (see Note 4).

2. Environmental conditions of the experiments: The experiments have to be made under relatively sterile conditions. Eggshells are cleaned with 70% alcohol. Instruments are sterilized in a dry oven (1.5 h at 120°C). Sterile physiological liquids are supplemented with antibiotics (10-20 IU/mL). Experiments are performed in a clean separate room, but never under forced-air apparatus (see Notes 6 and 12).

3. Opening the eggs: The blastoderm develops on the top of the yolk against the shell membrane. Before opening a window in the shell, a small quantity of albumen (about 0.3 mL) is removed at the small end of the egg, using a syringe in order to separate the blastoderm from the shell membrane. Another method consists of perforating the eggshell at the level of the air chamber and then rolling the egg horizontally several times. These manipulations are sufficient to loosen the blastoderm from the shell membrane and allow a window through the shell without damaging the embryo. The small holes through the eggshell are obturated with a piece of tape or a drop of paraffin (see Note 5).

4. Contrasting the embryos in ovo: India ink, diluted 1/1 in PBS or Tyrode supplemented with antibiotics, is injected under the blastoderm (donor or recipient) using a glass micropipet mounted with a plastic tube for mouth use (see Note 10).

5. Gaining access to the embryos in ovo: The vitelline membrane, which covers the embryo, is slitted out with a microscalpel just at the place where the microsurgery will be made.

6. Explanting the donor blastoderms: In certain cases, the donor blastoderm is cut out from the egg with Pascheff-Wolff scissors, washed free of vitellus in PBS or Tyrode supplemented with antibiotics, tranferred onto a dish with a black plastic base, and pinned out before dissection.

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