This article details the process of thawing cryopreserved rabbit embryos, emphasizing the importance of careful temperature management to prevent damage. The procedure involves warming the embryos and rehydrating them in a controlled environment.
Embryo thawing is a process in which cryopreserved frozen embryos are warmed and rehydrated, restoring their biological activity.
To thaw cryopreserved rabbit embryos in a sealed cryogenic straw, remove the straw from liquid nitrogen. The embryos, with minimal volume of cryoprotectant-containing media, are physically isolated by air and media segments within the straw. Briefly position the straw in air, facilitating the release of trapped liquid nitrogen vapors, which otherwise cause embryo damage during exposure to warmer temperatures.
The rapid rise from cryogenic liquid nitrogen temperatures to higher freezing temperatures initiates ice crystal formation within the cryogenic straw, risking embryo damage. As ice crystals begin to form, immediately immerse the straw in a temperature-controlled water bath with gentle agitation for even thawing. This step removes the ice crystals, protecting the embryos from ice crystal damage.
Remove the straw plug. Cut the straw's sealed end; attach a microdispenser to release the contents into a culture plate with sucrose-containing media. The media helps remove cryoprotectants from the embryos; the sucrose avoids excessive swelling of the embryos, restoring their physiological environment during rehydration.
Transfer the embryos into a fresh media-containing plate to completely remove cryoprotectants, ensuring complete rehydration. The thawed embryos are ready for embryo transfer procedures.
To thaw the embryos for a transfer, place the ministraw horizontally, 10 centimeters from the liquid nitrogen vapor for 20 to 30 seconds. When the crystallization process begins inside the ministraw, immerse the straw in a 25-degree Celsius water bath for 10 to 15 seconds. Immediately remove the straw and cotton plugs, and use a coupled microdispenser to expel the ministraw contents into a plate containing 25 degrees Celsius base medium supplemented with 0.33 molar sucrose solution in which embryos must remain during 5 minutes. Then, transfer the embryos to a plate of fresh base medium for another 5-minute incubation.
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