OPU-IVF PROTOCOLS

SAR IVM

Oocytes Aspiration Plus

SAR IVF

SAR IVC

 

IVF for Large Scale Embryo Production PROTOCOLS

SAR IVM

SAR IVF

SAR IVC

 

 

 

 

 

 

 

 

 

 

OPU-IVF PROTOCOL

 

 

SAR IVF

Preparation of Media

SAR IVF Stock II and SAR IVF III stored at -20 C, 6 month after production date
SAR IVF Stock I stored at 4 C, 2 month after production date
Once IVF Fertilization Medium and Sperm Washing Medium are made, the shelf life is 1 WEEK MAX!

 

Sperm Washing Media:

1. Add 800 L of Stock II to 40 mL of SAR IVF Stock I to make a total of 40mL of sperm washing medium. Label as SAR-Sperm.

2. Filter medium using a 0.22 m syringe filter.

3. Aliquot (1) 1.5 mL of sperm washing per bull into a tube suitable for the size of media equilibrating, and place tubes of SAR-sperm into CO2 incubator for 2-3 hours prior to fertilization.

During sperm preparation 1 mL will be used to wash sperm from each bull following Isolate procedure. 0.5 mL will be added back to the 0.5 mL remaining sperm suspension following removal of supernatant.

4.Prepare a second centrifuge nipple tube with (0.5 mL) 45% and (0.5 mL) 90% Isolate layer and warm in warming bath just prior to thawing the straw of semen. Prepare one tube per bull.

 

Sperm washing:

1. Take semen straw from liquid nitrogen tank, gently shake in air for 10 sec.

2. Drop the straw into 37 C water bath till straw is completely thawed, it takes about 30 sec to 1 min.

3. Cut the straw and let semen flow out straw and onto the top layer of Isolate layer.

4. Spin the centrifuge tube at 400xg for 15 min (with Eppendorf Centrifuge 5415C, it is set 2214 rpm.

5. Remove the supernatant and Isolate till the end of pellet.

6. Add SAR-Sperm 1 mL and mix by gently pipetting. Spin the tube at 400xg for 8 min.

7. Remove the supernatant and leave 0.5 mL solution in the tube, add 0.5 mL SAR-Sperm to make up 1 mL. Mix the sperm and ready for IVF.

       

    Fertilization Media:

    1.Add 770 L of Stock III to 10 mL of stock I to make the fertilization media.

    2. Filter medium using a 0.22 m syringe filter into a 15 mL conical tube and label as SAR-Fert.

    3. To prepare for fertilization of 2 cows: a. Aliquot 2 mL of SAR-Fert into a 2 mL cryovial. From the cryovial pipette into a 4 well Nunc plate, 50 L into 2 of the four wells. Immediately cover with 700 L of oil. b.

      Place the remaining SAR-Fert in the cryovial into the CO2 incubator along with the Nunc 4 well plate to equilibrate for 2-3 hours. You will need two drops of 200 L per cow for washing matured oocytes, make in a 35 mm dish at the time of oocyte washing, take this from the remaining 2 mL aliquoted.

      4. At 22-24 h of IVM, place a 60 mm dish on the warming plate and make 2 drops/cow with equilibrated SAR-Fert. Wash one cows oocytes through 2 SAR-Fert drops (200 L/ drop) and place into fertilization dish made before (50 L drop of SAR-Fert covered with oil). You need to wash the oocytes very quickly.

      5. At 22-24 h of IVM, place a 60 mm dish on the warming plate and make 2 drops/cow with equilibrated SAR-Fert. Wash one cows oocytes through 2 SAR-Fert drops (200 L/ drop) and place into fertilization dish made before (50 L drop of SAR-Fert covered with oil). You need to wash the oocytes very quickly.

      6. Incubate dishes in CO2 incubator at 5% CO2, 39 C for exact 18 h.

       

       

      OPU-IVF Protocol (pdf format)