Pregnancy Miracle

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1. Two days before the IVF procedure, start the superovulation of the donor females as described in Subheading 4.

2. On the day before the IVF or at least 3 h before, preincubate one 35-mm sterile tissue-culture dish containing 1 mL FM without paraffin and six dishes containing 0.5 mL FM under paraffin oil at 37°C with 5% CO2.

3. Early on the morning of the IVF, prepare three tissue-culture dishes containing 2-3 mL M16 medium and one tissue-culture dish containing M16 microdrop cultures. Incubate at 37°C with 5% CO2.

4. At 0645 h, kill the male donor by cervical dislocation. Place it abdomen side up, and soak with 70% ethanol. Open up the body cavity. Pull out the testes with a pair of watchmaker's forceps. Dissect out the epididymis (a white mass of coils at the base of the testes) with a pair of fine scissors, and immediately transfer to the pregassed tissue culture dish containing 0.5 mL FM under paraffin. Repeat for the other epididymis.

5. Rapidly tease away the fine membrane with a pair of fine forceps, and squeeze the sperm out of the epididymis. The sperm should exit in a continuous stream. Do this fast since the sperm should be at room temperature for the minimum possible time.

6. Incubate the sperm at 37°C with 5% CO2 for 30 min.

7. To each preincubated tissue-culture dish containing 0.5 mL FM under paraffin, add 50 |L of sperm. The final concentration of sperm should be approx 1-2 x 106 sperm/mL.

8. Incubate the diluted sperm mixture for 2 h at 37°C with 5% CO2.

9. At 0940-l000 h, kill the superovulated females by cervical dislocation, and dissect out the oviducts from both sides (Subheading 13.3.). Collect all the oviducts into a pregassed dish containing 1 mL of FM without oil.

10. Tease away the ampulla to release the cumulus mass from the oviducts. Transfer the cumulus masses from four oviducts to each pregassed dish containing 0.5 mL FM plus sperm.

11. Incubate the sperm-egg mixture for 4 h (min 3 h) at 37°C with 5% CO2.

12. At 1500 h, wash the eggs three times in pregassed M16 medium to remove excess sperm. Transfer the eggs to the M16 microdrops and culture overnight.

13. On the next day, surgically transfer the two-celled embryos to the oviducts of 0.5-d pc pseudopregnant recipient females.

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Pregnancy Nutrition

Pregnancy Nutrition

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