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genetic manipulation in practice
By juragan | 18 July 2008
july 08
Sperm:
- Strip milt from males,
- dilute sperm in 0.85% NaCl (1/200 dilution and then 1/4000 dilution),
- count all of the sperm in 5 blocks (5×5) of hemacytometer,
- calculate sperm concentration (10^10)=(total count/80)x4000Ax4000Bx1000C (A : inverse of dilution, B : volume of hemacytometer cell = 1/4000 uL, C : to convert from concentration per uL to concentration per ml,
- prepare sperm at a final concentration of 8×10^8 sperm/ml, volume of sperm (uL) = 5000×8x10^8/(sperm concentration)
Egg
- Strip the eggs into a glass container
- Place eggs ~10 ml
Triploid (3n)
- Fertilize eggs in one dish with 5 ml of diluted sperm,
- mix eggs and sperm gently with just enough water to cover the eggs, then slowly keep adding water,
- cold shock eggs by pouring eggs through a fine mesh sieve at about 90 seconds post-fertilization and immersed in a water bath at 2 oC, and kept there for 10 min,
- Empty the eggs into a large container of water at the same temperature as the incubation system and from there transferred to one incubator
Meiotic gynogenesis (2n)
- Prepare one petri dish containing 5 ml of diluted sperm,
- place the petri dish into the UV chamber and irradiate for 60 seconds (agitating continuously during UV-irradiation),
- fertilize eggs in one dish with 5 ml of UV-irradiated sperm, mix eggs and sperm gently with just enough water to cover the eggs, then slowly keep adding water,
- cold shock eggs by pouring eggs through a fine mesh sieve at about 90 seconds post-fertilization and immersed in a water bath at 2 oC, and kept there for 10 min.
- empty the eggs into a large container of water at the same temperature as the incubation system and from there transferred to one incubator
recapped from N. Pongthana
Topics: thai notes |
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