Day 1: Culture IVC
Below is an introduction provided by Dr. Lotte Stroebech of Stroebech Media, outlining the crucial Day 1 (Culture IVC) steps in bovine IVF. This guide focuses on preparing, warming, and equilibrating media, as well as the proper denudation methods necessary to optimize embryo development. It underscores the importance of precise handling, ensuring minimal cumulus cell debris, and carefully tracking oocytes through each wash and transfer. By following these best practices, laboratories can enhance the consistency and success of their IVF protocols.
Day 1: Culture IVC
Media to be preheated and equilibrated:
WASH Medium, IVC Medium, Stroebech Oil
• Prepare and pre-equilibrate IVC Medium in the desired number of wells for IVC for final culture
• (no more than 30 inseminated oocytes/500 μl)
• Prepare the same number of wells per 4WP or dishes with IVC Medium for rinse
• Add 500 μl IVC Medium to each well of the 4WP and cover with 400 μl oil
• FOR 100 μl micro drops: Add 10 μl IVC Medium, cover with oil, then add 90 μl IVC Medium into the 10 μl drop now covered with oil – this method ensures the drops get round and not flat
• (no more than 10 inseminated oocytes/100 μl)
• Preheat WASH Medium
Denudation - Removal of cumulus cells from the inseminated oocyte
OPU: Denudate in WASH Medium in drops or in 500 μl wells.
Consider to vortex if you have 15 or more inseminated oocytes. Otherwise denudate with pipette.
• Remove the IVF dishes from the incubator – note that cumulus cells have been disrupted from the spermatozoa activity
• Rinse the inseminated oocytes free of oil through several WASH Medium drops
• Use a denudation pipette 125-145 μm - the preferred method
• Or denudate oocytes vigorously with a 200 μl pipette
• Wash the denudated oocytes through the dishes/wells
• Count the denudated inseminated oocytes and record in the IVC worksheet
• Rinse once in the respective equilibrated IVC Medium dish before placement to the 4WP/drop with IVC Medium
Do not use the same wash and/or rinse drops or wells for different donors
SH: Vortex. Use polystyrene plastic for vortexing because it is a harder plastic than the polypropylene centrifuge tubes to ensure the oocytes will be completely denudated.
Be sure that two tubes are hitting hard against each other during vortexing.
For 1-2 min max speed otherwise the cumulus cells remain attached to the zona pellucida.
Prepare two sets of 10 ml tubes and dishes for the SH experiment
If more than two groups are tested prepare wash tubes and dishes corresponding to the number of groups of inseminated oocytes to be denudated.
• Add 1 ml of WASH Medium to two 10 ml vortexing tubes
• Prepare two empty 35 mm Petri dishes both labeled #1
• Add 3 ml WASH Medium to four 35 mm Petri dishes, two labeled #2 and two labeled #3
• Remove the IVF dishes from the incubator – note that cumulus cells have been disrupted from the spermatozoa activity
• Transfer all the inseminated oocytes to the 10 ml tube(s) containing 1 ml WASH Medium
• Vortex for 1-2 min at high speed and pour the content of the 10 ml tube into the empty dish #1
• Add 1-2 ml WASH Medium to the 10 ml tube; pour off again into the same 35 mm dish
• Remove the rest of the medium from the tube with a pipette
• Transfer all the denudated oocytes to dish #2 containing WASH Medium
• Work fast without evaluating individual oocytes
• Wash a second time by transferring the oocytes from dish #2 to dish #3
• For an experiment that requires more groups, randomize the inseminated oocytes at this point
• Count the inseminated oocytes during this last wash step, and finally, rinse once in the equilibrated IVC Medium dish before the final transfer to IVC Medium for culture. Rinse or change pipette tip after each transfer through oil in order not to accumulate too much oil in the IVC Medium rinse dish
• Usually, ~ 30 inseminated oocytes are added to each 500 μl well
Note that cumulus cells will impair embryo development so only few should remain attached to zona pellucida for culture.
Do NOT waste time discarding potential unfertilized oocytes. Give all the benefit of the doubt.
You may have evaluated wrong and unfertilized oocytes do not have a negative effect on the other oocytes!
Write down exactly how many inseminated oocytes that have been placed in each well. Write it on the lid and in the worksheet. Incubate.
Assess the motility of the sperm in the empty wells/drops – there should still be some activity to detect.
If no motile spermatozoa are left, consider sub-optimal conditions in the laboratory
POST IVC
Cleavage can be checked 48 hours after – but embryos are best left alone until after morula compaction.
• Transfer at day 7 – but check day 6 where some embryos have already reached the blastocyst stage
• Use HOLDING Medium for transfer immediately
• If transporting the embryos to recipients preferably use vials and load at the farm do not transport embryos in straws
IVF embryos are NOT in vivo embryos, but more fragile and sensitive to pH and temperature fluctuations
