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Day One: Administer PMS via IP injection to females of
appropriate strain and age for ova donors at about noon.
Day Three: Administer hCG via IP injection to females ~48 hours
post PMS injection.
Prepare dishes for egg collection, sperm collection and IVF.
One dish of 1 ml IVF medium per male for fresh sperm
One dish of 3 ml IVF medium per three
One IVF/wash dish per three females with 400 µl drop for
fertilization and four 150µl drops for wash post fertilization
covered with oil – no lids on these dishes. Keep at 37° C under
mixed gas. (mixed gas is 5%CO2, 5% O2, 90% N2)
Day Four: IVF
Collect sperm – See sperm thawing protocol.
Add 5 – 10 µl of sperm to IVF drop in dishes. (Frozen = 10 µl
and fresh = 5-10 µl depending on motility.) Incubate under mixed
gas for one hour (fresh be can used immediately). Record time sperm
13-14.5 hours post hcg collect oviducts from superovulated
females. Work quickly on this step, trying to take no more than
five minutes per group of three females. Dissect out clutches of
eggs and transfer into IVF+sperm drop with as little extra media as
possible using 10 µl pipetman. Record time of addition to
Repeat until eggs have been added to all IVF dishes. Incubate
for 4-6 hours at 37° C under mixed gas.
Using mouth pipette, wash eggs/zygotes into first 150 µl drop.
Move best looking ones into next drop, leaving behind dead,
fragmented cells. Divide healthy ones between the last two drops.
Record number of live, fragmented, dead, and zona free cells.
Again, try to take less than five minutes for this step and return
promptly to incubator.
Incubate overnight under mixed gas.
Day Five: Score IVF
For each IVF dish, count and record number of two-cell embryos,
one cell, fragmented and dead oocytes. These embryos can be
cryopreserved, transferred to 0.5 dpc pseudopregnant females or
kept in culture up to the blastocyst stage. KSOM medium or other
culture medium is used if culturing beyond the two-cell stage.