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  • Applied Development Engineering Practice 2: Egg Collection and Fertilization, Egg Membrane Removal

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  • (1) Zebra fish


    • ① Males and females will be prepared in the evening of the day before the day of egg collection.

      ② 2-3 male animals are transferred to a water tank for egg collection. Distinguish between males and females by color tone (males are more orange)

      ③ Place a stainless steel cage on the water tank for egg collection. Check that the bottom of the car is submerged in water.

      ④ Select two females from the female aquarium and place them in a cage above the aquarium containing males.

      ⑤ Place the lid of the water tank on the basket to prevent males and females from jumping out.

      ⑥ On the morning of the next day, the male under the tank is caught by the net and transferred to the upper basket.

      ⑦ Check if males follow females.

      ⑧ The egg laying is confirmed soon after it begins to catch up. (sometimes not done)

      ⑨ Allow to stand for about 20 minutes and wait for the fertilized egg to accumulate at the bottom of the tank.

      ⑩ The basket containing both sexes is transferred onto another tank.

      ⑪ Prepare a siphon of the hose, a plastic petri dish, and a brown strainer.

      ⑫ Put water into the hose, place one mouth in the tea pepper to prevent water from exiting, and place the pepper on a plastic petri dish.

      ⑬ Collect eggs submerged under the tank in a tea filter using siphons of hoses.

      ⑭ The breeding water is put in the plastic case, and the fertilized egg accumulated in the tea lump is put in it.

      ⑮ Transfer the fertilized egg to a glass Petri dish using a pipette with a large mouth.

      ⑯ Use a large pipette to remove water from the glass petri dish.

      ⑰ Place 0.1% trypsin Ringer's solution (about 120 μl of 0. 1N NaOH solution in 100ml) from the top of the fertilized egg and allow to stand. Observe that the egg membrane separates into two sheets under a stereomicroscope.

      ⑱ When the egg membrane is peeled off, use a large pipette to move the trypsin solution so that the egg is not sucked in, and separate the egg membrane and embryo. Do not foam. When foamed, the embryo explodes in contact with the foam.

      ⑲ Move the Petri dish gently to collect the egg membrane removal egg in the center.

      ⑳ Transfer the membrane-depleted eggs to an agarose petri dish filled with the primary culture.

  • (2) Goldfish


    • 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.

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      Hormonal injection to goldfish File
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      Egg Collection and Artificial Insemination of Kingyo File
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  • (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)

      ④ Place the eggs in a colander, absorb the moisture by placing a kim-towel under the colander, and transfer the eggs to a container filled with 0.2% Pancreatin-Ringer's solution (prepared at pH 11.5 with NaOH). Place the container in an incubator (set at 10°C to 15°C).

      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).

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