Gather females of interest. One should have at least 2-3 females to ensure that the females lay eggs.
Prime females with HCG using a 1-cc syringe with a 28½ gauge needle in the afternoon- 10 I.U. HCG (Chorulon, Intervet Inc.) diluted with the accompanying diluent for a total volume of 100 uL. It is best to remove the hormone and diluent via syringe and place them in separate eppendorf tubes. Then measure the appropriate volumes of each with a pipet and mix the solution in a third eppendorf.
The females are kept at 25o C overnight.
Day 2 (day of IVF)
Boost females with HCG in the morning-100 I.U. HCG in a total volume of 100uL. It generally takes approximately 4 to 4.5 hours for the females to start producing eggs. The females may remain at room temperature during this time or they may be returned to 25o C.
Shortly before the females should begin to lay eggs, coat 15mm x 60 mm petri dishes with
1x MBS + 0.1% BSA to perform the IVF. Use one dish per IVF to be performed.
The BSA will prevent the sperm from sticking to one place in the dish later.
When the females begin to ovulate, squeeze eggs into a dry petri dish. Aliquot ~100-300 eggs per IVF dish for testing.
Flash thaw the sperm in room temperature water (one aliquot/IVF dish). IMMEDIATELY place the sperm onto the eggs using a cut pipet tip.
Various protocols recommend diluting the sperm with buffer or water before IVF. However, in our tests of this method we found undiluted sperm gave higher rates of fertilization.
Swirl the eggs and sperm gently in the dish to adequately mix the sperm and eggs together.
Wait 2-5 minutes, then flood the eggs with 0.1X MBS or distilled water. Let sit at room temperature until cleavage.
At cleavage, dejelly with 2% cysteine (pH 7.9 + 0.1) for 6 minutes while swirling the dish. Remove cysteine and rinse 4-5 times with 0.1X MBS or 0.05X MBS + 0.1% BSA.
Count cleavers, non-cleavers, and dead embryos; calculate % fertilization.
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Last update: Feb. 13, 2008