(3) Salmon trout

* This experiment will be conducted by two people.

I. Egg collection and fertilization

① Put a net in the pond to catch the parent fish, and confirm the sex of the parent fish.

② Wipe the body of female parent fish with a towel and wipe moisture.

③ One person (playing a military hand on a clever hand) holds the head of a female, the other person (playing a military hand on a non-clever hand) holds the tail, and the person on the tail side squeezes the belly and takes the egg into a monkey in a bowl. (not mixed with feces, small water, or water)

④ Wash the eggs with Ringer's solution for salmon trout and transfer them to a fog. Add Ringer to the surface of the egg further.

⑤ The male parent fish is similarly supported by two persons, and the stomach is squeezed to put sperm into a plastic cup. (Be careful not to mix small water.)

⑥ Take an adequate amount of egg into a monkey, cut Ringer and transfer it to another fog.

⑦ Place sperm on the unfertilized egg, mix the eggs well, add a little Ringer's solution (sperm activation), and then add river water (or well water) to half of the bowl. Place for a few minutes (fertilization).

⑧ Absent eggs, cut water. Once again, put river water (or well water) into the bowl, put the fertilized egg and wash it. Repeat several times to prevent sperm from remaining.

⑨ In case of breeding culture, disinfect with isodine solution and transfer to hatchery. When chromosome manipulation is performed, it is housed in a cell of a French press and subjected to pressure treatment.

II. Salmon egg membrane softening

① After fertilization, the eggs are placed in a colander, a kim-towel is placed under the colander to absorb water, and then the eggs are transferred to a container filled with acidic Ringer's solution (prepared at pH 1.8 with HCl). 15 minutes of acidic treatment is started. (Transfer should be completed within 1 minute after fertilization. (Transfer is completed within 1 minute after fertilization).

② During 15 minutes of acidic treatment, the pH of 0.2% Pancreatin-Ringer solution is prepared (by NaOH) to pH 11.5

③ When the acidic treatment was performed for 15 minutes, the acidic Ringer's solution was discarded and rinsed by Ringer's solution (pH no preparation)Subsequently, it is rinsed by basic Ringer's solution (prepared by NaOH to pH 11.5)

⑤ By immersing the fertilized egg in a pancreatin solution, a portion of the egg membrane begins to dissolve as early as 7 hours. Eggs whose removal of the egg membrane has been confirmed are quickly transferred into Ringer's solution (pH-free preparation).