① Female rearing water is replaced by new water in the morning of the day before egg collection. (environmental stimulation)
② hCG was injected intraperitoneally into males and females in the evening of the previous day (from 5 o'clock to about 10 o'clock). (Females 1-10 units/gBW, males 0.1 to 1unit/gBW)
③ Check ovulation from morning on the day
④ Males were anesthetized, semen was collected in hematocrit capillaries, diluted 50-100-fold with artificial seminal plasma, and stored in a refrigerator. (Artificial seminal plasma :NaCl 5.61 g/L, KCl 5.23 g/L, CaCl2 · 2H2O 0.33 g/L, MgCl2 · 6H2O 0.22 g/L, NaHCO3 0.2 g/L, pH8.0)
⑤ The females are anesthetized and the ovulations are squeezed out on saran wrap. Store at room temperature over a cover to prevent drying. (end fertilization within 2 hours)
⑥ Transfer the required amount of egg onto the saran wrap pieces using a plastic madra.
⑦ Using a yellow tip, seminate the diluted semen, and mix gently at the back of the toothpick.
⑧ Fill the Petri dish with fertilized semen (0.2%urea, 0.24%NaCl solution), and fertilize the fertilized eggs by inseminating them.
⑨ Agitate and disperse eggs with toothpicks to avoid concentration.
⑩ Discard the fertilized semen, pour the egg membrane removal solution (Ringer's solution containing 0.1% trypsin and 0.4% urea) and allow to stand.
⑪ When the egg membrane becomes clear, move the Petri dish horizontally to collect the egg membrane removal egg in the center of the Petri dish.
