Rabbit Embryo Thawing: A Warming Procedure to Recover Cryopreserved Rabbit Embryos for Embryo Transfer

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

1. Thaw the embryos in a single step.
   1. Place the ministraw horizontally 10 cm from liquid nitrogen vapour for 20-30 s.
   2. When the crystallization process begins inside the ministraw, immerse the ministraw in a water bath at 25°C for 10-15 s.
   3. Remove the ministraw plug and cut the cotton plug.
   4. 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. 
   5. Move the embryos to a new plate containing BM solution for another 5 min.

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.