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  • Applied Development Engineering Practice 5: Chromosome Manipulation

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  • ① Female developmental haploid


    • ① Female developmental haploid

      1. Turn on the ultraviolet lamp.

      2. Prepare a flat petri dish that has been cleaned and coated with Vaseline on the bottom sides.

      3. Spermatozoa are diluted 50-100-fold with cherry trout spermatozoa (salmon) or artificial seminal plasma (carp fish).


      4. Spread 0.5 ml of diluted spermatozoa of salmon trout or 100-200 μl of carp fish spermatozoa on the bottom of a flat petri dish to avoid air bubbles.

      5. Process for 90 seconds with an ultraviolet lamp after 10 minutes or more after lighting.

      6. UV-treated sperm (UV sperm) should be transferred to test tubes or hematocrit tubes and stored in a dry manner. (to avoid contamination with normal sperm)

      7. Ultraviolet spermatozoa are inseminated into unfertilized eggs and fertilized by insemination (or fertilization).


      ② Male developmental haploid

      1. Prepare a Petri dish with the saran wrap pinned and place a circular frame on the Petri dish.

      2. Place the cherry ovarian cavity fluid in a circular frame so that unfertilized zebrafish or goldfish eggs are not attached to the edges.

      3. Suck out the cherry salmon ovarian cavity fluid with a pipette man to the very edge of the eggs.

      4. Place the petri dish in a box of ultraviolet lamps and treat with ultraviolet light (about 3 minutes).

      5. After ultraviolet treatment, the ovarian cavity fluid is sucked out and sperm with normal sperm.

      The fertilized egg is placed in the fertilized liquid and fertilized.

  • (2) Polar release inhibition


    • ① Temperature treatment (goldfish: high-temperature treatment)

      1. Keep a thermostatic bath filled with urea-containing fertilizer at 40℃.

      2. Ultraviolet sperm or eggs fertilized with normal sperm are fertilized in a Petri dish filled with fertilized liquid.

      3. After fertilization, place for about 5 minutes. Fertilized eggs adhere to the bottom of the Petri dish.

      4. Discard the fertilized liquor from the petri dish with the fertilized egg and immerse it in a thermostatic bath at 40℃ for 1 to 1.5 minutes.

      5. After treatment, replace immediately with a room-temperature septic solution (second maturation splitting inhibition: female generation diploid (when sperm with ultraviolet sperm) or triple (when sperm with normal sperm).


      ② High-pressure treatment (salmon)

      1. A French press is prepared.

      2. Eggs mated with UV sperm or normal sperm are fertilized in fresh water at 10°C, emptied into a colander to remove excess sperm, and then immersed in more fresh water at 10°C.

      3. After fertilization, place for about 10-20 minutes.

      4. Place fresh water in the cell of the French press.

      5. Put the fertilized egg in the cell and fit the upper lid to the packing.

      6. Fit the rocket on the top cover and then lightly close the knob.

      7. Gently push the top cover to remove air from the cell.

      8. Close the knob by twisting it firmly

      9. Place the cell on the French press body.

      10. Raise the jack and apply pressure to the cell.

      11. After rising to 700 atm, treat for 7 minutes.

      12. When the pressure drops, jack up as appropriate to keep the pressure at 700 atm.

      13. Immediately before 7 minutes, remove the iron bar for the jack.

      14. After 7 minutes, turn the vacuum cock counterclockwise to release the pressure slowly. At this time, note that if the pressure is suddenly lowered, the egg collapses.

      15. After treatment, it is immediately replaced with room temperature fertilizer (inhibition of second maturation division: female developmental diploid (when spermatozoa are stored with ultraviolet spermatozoa) or triploid induction (when spermatozoa are stored with normal spermatozoa).

  • Contents of the following practical training