IVF CARD Protocol

IVF Procedure (using cryopreserved sperm in straws)

1. Day 1 — Superovulation
   1. Superovulate 3 to 5 week old female mice
   2. Inject 100 μl CARD HyperOva intraperitoneally (i.p.) between 2:00 pm and 6:00 pm.

2. Day 3 — hCG and preparation

   1. 48 to 52 hours after HyperOva injection, administer 5 IU hCG i.p.
   2. Place beaker of distilled water in the incubator to warm overnight (for sperm thawing).
3. Day 4 — IVF Day
   1. Prepare CARD fertilization medium (formulation depends on sperm type).
   2. Prepare mHTF washing medium or KSOM Advanced (MR-101D, Sigma) if using alternative.
   3. Prepare culture dishes:
      1. Sperm dish
         1. One 60 mm dish per sperm straw/vial.
         2. Add 90 μl of FERTIUP sperm preincubation medium.
         3. Cover with mineral oil. 
         4. Place in the incubator 30 minutes before thawing sperm.
      2. Fertilization dish
         1. One 60 mm dish per superovulated injected female.
         2. Add 200 μl CARD fertilization medium for fresh sperm or 90 μl CARD fertilization medium for frozen sperm.
         3. Cover with mineral oil.
         4. Place in the incubator 10 minutes before sacrificing the first female.
      3. Washing dishes
         1. One 60 mm dish per fertilization dish. 
         2. Add four 80 μl drops of mHTF or KSOM, cover with mineral oil.
         3. Place in the incubator at least 30 minutes before embryo rinsing.  (These can also be prepared immediately after insemination.)

Collection of oocytes:

1. Sacrifice each female 15 to 17 hours after hCG. Process one female at a time.
2. Expose the ovaries/oviducts and remove oviducts, minimizing fat, blood, and fluid.
3. Place oviducts into the oil layer of the fertilization dish.
4. Tear open the ampulla with #5 forceps to release the cumulus-oocyte complexes (COC).
5. Drag the COCs into the CARD medium drop and return dish to the incubator.
6. After all oocytes are collected, incubate fertilization dishes for 30 to 60 minutes before insemination.

Thaw sperm:

1. Remove a frozen sperm straw from liquid nitrogen; hold in air for 5 seconds.
2. Immerse straw in 37°C water for 10 minutes.
3. Remove straw, wipe with 70% ethanol.
4. Cut off the seal and cut through the PVA plug, leaving half of the cotton plug.(Created in https://BioRender.com)
5. Using a metal rod as a plunger, push against the remaining portion of the cotton plug and expel the contents into the FERTIUP drop; avoid dropping the plug.
6. Incubate sperm dish for 30 minutes.

Insemination:

1. Pipette 10 μl of sperm suspension from the edge of the FERTIUP drop (most fertile sperm).
2. Add sperm directly onto the cumulus masses in the fertilization dishes.
3. Return dishes to the incubator.

Post-Insemination Handling:

3 hours post-insemination:

1. Wash oocytes three times through the mHTF or KSOM drops.
2. Leave oocytes in the third mHTF or KSOM drops overnight.

6 hours post-insemination:

1. Examine oocytes and discard unfertilized ones.
2. Continue overnight incubation.

Day 5 – Embryo Handling

1. The next morning, transfer two-cell embryos to the fourth mHTF drop.
2. Embryos may now be cryopreserved, cultured, or transferred to 0.5 dpc recipients.