OPU-IVF PROTOCOLS

Oocytes Aspiration Plus

SAR IVF

SAR IVC

 

IVF for Large Scale Embryo Production PROTOCOLS

SAR IVF

SAR IVC

 

IVF For Large Scale Embryo Production PROTOCOL

 

SAR IVM

 

SAR IVM contains Medium 199 (M199) with Earle’s salts, L-glutamine, sodium bicarbonate, and HEPES, supplemented with 10 % (v/v) fetal calf serum, 0.5 µg/mL ovine FSH, 5.0 µg/mL ovine LH and 1.0 µg/mL estradiol and other small molecules that promote oocyte maturation in vitro.
Storage temperature: SAR IVM shall be stored at -20 °C, upon arrival.  The expiration: 2 months after production date.

A. Making IVM dishes

1. Prepare dishes early morning. Mark the 35 mm Petri-dishes (Falcon 1008) with marker pen (IVM dish # and date).

2. Using pipetman p200 make 7 drops of SAR IVM with 75 µL per drop in each dish.  Cover the drops with 3 ml mineral oil.

3. Prepare several 35 mm Petri-dishes with 3mL 10% FBS M-199 per dish.

4. Place SAR IVM and M-199 dishes in CO2 incubator. The dishes should be balanced in the incubator 2-4 h prior to use.

 

B. Aspirating ovaries and searching oocytes  

1. Set the ovary aspiration stations by placing three paper towels on top of a square Aluminum foil. Place latex gloves, syringes and needles beside each station. Keep glass beakers (for holding the aspirated ovaries) and 50 mL conical tubes (for collecting the aspirated follicular fluid).

2. With gloved hand, scoop a handful of ovaries from the cooler and place them on the paper towel to mop excess water/saline. With an 18 g needle attached to a 10 ml syringe, aspirate all follicles with size from 2 to 8 mm in diameter from ovary surface.

3. After aspirating, remove the needle from syringe, and push the syringe to empty follicular fluid into a 50 mL conical tube.

4. Settle down the oocytes and debris to the bottom of tube. Remove the supernatant and wash the pellet with RLI’s Oocyte Washing Prior to Maturation (OWP). The pellet is washed 2-3 times with OWP and finally the contents are pulled into a square grid search dish. The oocytes are searched under stereo microscope and picked up from dish using the mouth tubing attached to fine pulled and polished glass pipette. The oocytes are collected in a 35 mm Petri-dish (Falcon 1008) containing 3 mL OWP/dish.

5. Search the dish thoroughly and pick up good quality cumulus-oocyte-complexes (COCs) (at least 4 layers of cumulus cells), COCs are washed in OWP twice and in M-199 twice. At various stages of washing, bad oocytes are discarded, so that the final M-199 dish contains a pool of good quality oocytes.

 

C. Maturation oocytes in vitro 

1. Selected oocytes are put in groups of 25 each in each drop of maturation medium. Thus, each maturation dish (with 7 drops) contains 175 immature oocytes. Write the time, oocyte numbers and initials of the technician on the lid of each IVM dish.

2. Move COCs maturation dishes and kept in the CO2 incubator at 5% CO2, 39 oC and under conditions of maximum humidity. The oocytes are kept in this condition for 22 to 24 hours to complete in vitro maturation process ready for IVF.

 

IVM calculation:
SAR IVM 01 contains 1 mL/vial maturation medium with 5 vials per kit, total 5 mL.  SAR IVM 02 contains 4 mL/vial maturation medium with 5 vials per kit, total 20 mL. 

For each IVM trail, two Petri dishes are prepared with 7 drops per dish for IVF.  If 25 oocytes are added into each fertilized drop, a total of 175 oocytes (25 × 7 = 175) can be matured.  As a result, one package of SAR IVM 01 (5 mL) can be used to mature about 1600 COCs (5000/75 x25 =1666), while SAR IVM 02 (20 mL) about 6400 COCs

 

 

IVF For Large Scale Embryo Production Protocol (pdf format)