OPU-IVF PROTOCOLS

Oocytes Aspiration Plus

SAR IVF

SAR IVC

 

IVF for Large Scale Embryo Production PROTOCOLS

SAR IVF

SAR IVC

 

OPU-IVF PROTOCOL

 

SAR IVC

 

Preparation: for 2 cows

 

1. Aliquot 7 mL of SAR IVC-washing medium (SAR IVC-W) into 15 mL conical tube, label IVC-W and place into CO2 incubator at 39 C for 2-3 h.

2. Aliquot 2 mL of SAR IVC into 2 mL cryovial, label SAR IVC, from the cryovial make a 50 L droplet of SAR IVC in a 4 well Nunc plate for each cow and place cryovial and Nunc plate into CO2 incubator for 2-3 h.

3. Place a 60 mm and 35 mm dish on the warming plate.

 

IVC Procedure:

1. After 18 h of IVF, remove oocytes from SAR-Fert drops to a microcentrifuge nipple tube at volume of 200-300 L and vortex for 1.5 min at the scale 7 (Fisher Scientific Vortex). Spin the vortexed oocyte tube quickly in 3-4 sec in the spinner to let oocytes spun to the bottom of tube. You need make sure that vortexing does not destroy oocytes while stripping off cumulus cells. Test your machine prior to OPU-IVF with slaughterhouse oocytes. Vortexing may lose the oocytes, spinning is important to get fertilized oocytes down to the bottom of tube.

2. After 18 h of IVF, remove oocytes from SAR-Fert drops to a microcentrifuge nipple tube at volume of 200-300 L and vortex for 1.5 min at the scale 7 (Fisher Scientific Vortex). Spin the vortexed oocyte tube quickly in 3-4 sec in the spinner to let oocytes spun to the bottom of tube. You need make sure that vortexing does not destroy oocytes while stripping off cumulus cells. Test your machine prior to OPU-IVF with slaughterhouse oocytes. Vortexing may lose the oocytes, spinning is important to get fertilized oocytes down to the bottom of tube.

3. If not, using the equilibrated IVC-W, rinse the nipple tube with 500 L of IVC-Wash and quickly vortex 3 to 4 times, and spin the tube quickly 3-4 sec, and remove the media from the bottom of the nipple tube and adding it to the oocyte/medium in the lid of the 35 mm dish. Continue rinsing until all oocytes are found. The alternative way is to denude oocytes by pipetting oocytes with glass pipets of different pore sizes. See Addendum below.

4. Using the 60 mm dish and a transfer pipette make 9 drops of IVC- Wash (at least 200 L/drop) and 3 drops of IVC medium (at least 200 L/drop). Wash collected oocytes through 9 drops of IVC-Wash and 3 drops of IVC medium. Then place oocytes in 50 L drop of IVC medium. You need to wash the fertilized oocytes very quickly.

5. Culture embryos in 5% CO2, 5% O2, 90% N2, 39 C for 7 days.

6. On Day 2, check cleavage rate, must remove uncleaved oocytes from the drop. On Day 7, check blastocyst development and its rate. Uncleaved oocytes may generate toxic chemicals during the culture, and they interrupt your judgment of morulae on Day 6.

 

Addendum: Cumulus removal post IVF is the most important step toward to the success of IVF, for high blastocyst development of embryos.

1. You may make several glass pipets with different pore sizes, saying three-four pipets from large size to small size. The last one is just same diameter as that of oocytes.

2. Remove cumulus cells one-by-one pipet, at last time with the smallest pipet. With the largest pore size pipet, you can take several oocytes (5-6 oocytes) at a time into the pipet, and then release the oocytes. In this step, you can remove large portion of cumulus cells from embryos very quickly.

3. Change the smaller size pipets to continue removing cumulus cells, but only take 2-3 oocytes at a time since the pore size is smaller.

4. At last step, just pipet those oocytes attached with small amount cumulus cells. Do not suck whole embryo into the pipet, maybe only 1/3 to 1 of oocyte, then release oocyte immediately. By this process for two-three times, you will remove the cumulus cells completely, but not destroy the oocytes.

5. If you find the one pipet, especially the smallest pipet is fit for your removing cumulus cells easily; you can wash it in 100% ethanol several times, and save for next time to remove CC cells. This glass pipet can be your model for making next pipet, and it also can be used cumulus removal for many times.

 

 

OPU-IVF Protocol (pdf format)