Overview
This video demonstrates the procedure for thawing vitrified rabbit embryos and the precautionary measurement steps for optimal embryo recovery.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Embryo Warming
- Thaw the embryos in a single step.
- Place the ministraw horizontally 10 cm from liquid nitrogen vapour for 20-30 s.
- When the crystallization process begins inside the ministraw, immerse the ministraw in a water bath at 25°C for 10-15 s.
- Remove the ministraw plug and cut the cotton plug.
- With a coupled microdispenser, expel all the ministraw content into a plate containing 0.33 M sucrose solution at 25°C in BM for 5 minutes.
NOTE: This step must be done quickly in order to reduce embryo exposure to the vitrification solution. - Move the embryos to a new plate containing BM solution for another 5 min.
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Materials
Name | Company | Catalog Number | Comments |
Liquid Nitrogen | Air Liquide | P1505XXX | |
Petri dishes, 35-mm | Sigma Aldrich | CLS430165-500EA | |
Plastic dressing (Nobecutan) | IBOR medica | 7140028 | |
Plastic Straw 0.25 mL | IMV - technologies | 6431 | |
Scissors | ROBOZ | RS-5880 | Any regular surgical grade steel small straight scissors will work. |
Sterile Gloves | Alvet Escartí | 087GL010075 | |
Sterile gown | Alvet Escartí | 12261501 | |
Sterile mask | Alvet Escartí | 058B15924B | |
Straw Plug | IMV - technologies | 6431 | |
Sucrose | Sigma Aldrich | S7903 | |
Waterbath | RAYPA | BAE-4 | |
Dulbecco's phosphate-buffered saline (DPBS) | Sigma Aldrich | D5773 | Without calcium chloride. |