2.1.2 Artificial insemination†
126.96.36.199 On the day before of experiment†
- Separate healthy females spawning daily from males by keeping them in a tank without males.
- Healthy males are also kept separate from females.
188.8.131.52 On the day of experiment†
- In the morning, dissect a female, remove the ovary, and put it in the balanced salt solution for medaka (BSS) (Iwamatsu, 1983) in a 6-cm plastic dish.
- Remove a cluster of matured unfertilized eggs from the ovarian tissues using fine forceps under a dissection microscope.
- Separate single eggs from the cluster by cutting off their attachment filaments near the surface of the chorion of each egg using small scissors.
- Transfer the single eggs in a small amount of BSS in a watch glass. All of the eggs should be covered by BSS.
- Dissect a male, remove the testis, and put it in a drop of BSS on a hole slide glass.
- Cut the testis into small fragments using fine forceps under a dissection microscope preparing a sperm suspension.
- Add two to three drops of the sperm suspension to the single eggs in the watch glass and stir the BSS gently without damage to the eggs.
- Fertilization is confirmed by formation of perivitelline space (Iwamatsu, 2004) in 3 minutes after addition of the sperm suspension.
- Add enough volume of BSS to the watch glass containing the fertilized eggs and keep it at 26ºC for one hour to wait until the chorion become hard enough for handling.
- Transfer the fertilized eggs to 0.5ppm methylene blue solution in a 6-cm plastic dish and incubate them at 26ºC until the hatching stage.
- Iwamatsu, T. (1983) A new technique for dechorionation and observations on the development of naked egg in Oryzias latipes. J. Exp. Zool., 228, 83-89.
- Iwamatsu, T. (2004) Stages of normal development in the medaka Oryzias latipes. Mec. Dev. 121, 605-618.