Reagents and supplies
| Reagent | Source | Catalog Number |
|---|---|---|
| EmbryoMax mineral oil | Sigma | ES-0005-C |
| FERTIUP (from CARD media kit) | Cosmo Bio | KYD-004-EX-X5 |
| Paper clip for plunger | ||
| Scissors | Fisher | 08950 |
| Long forceps | Fisher | 10316C |
| Beaker with 37 degree water | ||
| 35mm tissue culture dish | Fisher | 0877230 |
| 60mm tissue culture dish | Fisher | 0877231 |
Prepare culture dishes (one set per straw thawed)
- Prepare either a 35 mm or a 60 mm dish with 90 μl FERTIUP and overlay drop with mineral oil.
- Incubate for 30 minutes before use.
Thawing sperm in straws
- Remove straw from LN2 storage; hold in air for 5 seconds.
- Immerse straw in 37°C water for 10 minutes.
- Remove straw, wipe with 70% ethanol.
- Cut off the seal and cut through the PVA plug, leaving half of the cotton plug.
(Created in https://BioRender.com) - Using a metal rod as a plunger, push against the remaining portion of the cotton plug and expel the contents into FERTIUP drop; avoid dropping the plug.
- Incubate sperm dish for 30 minutes.