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Publication Number
US-11793193-B2
Publication Date
2023-10-24
Expiration Date
Abstract
Technologies for cryopreserving ungulate embryos for implantation into recipient females are described.
Core Innovation
The invention relates to cryopreservation of in vitro produced ungulate embryos by a slow-freezing/direct-transfer approach. It freezes an in vitro produced ungulate embryo by incubating the embryo in an ethylene glycol freezing solution and loading the embryo-containing freezing solution into a straw-like receptacle with multiple air-separated thawing-solution regions.
The receptacle positions the embryo in a freezing solution while the loading arrangement includes air bubbles separating first and second thawing solutions and separating the freezing solution and the embryo from adjacent thawing solutions. The thawing solutions comprise ethylene glycol in an isotonic diluent medium, including a 1:1 dilution of the freezing solution or a 0.75 M ethylene glycol thawing solution.
After loading, the invention exposes the receptacle to subzero temperatures, maintains the embryo at the defined subzero range, and lowers the temperature at a controlled cooling rate until reaching a second subzero temperature. The frozen embryo is then immersed in liquid nitrogen for storage, producing a frozen in vitro produced ungulate embryo, with thawing and direct use described using thawing environments and transfer to synchronized recipient ungulates, including transfer to a uterine horn.
Claims Coverage
Two independent claims are present. Their inventive coverage centers on ethylene glycol exposure of ungulate embryos, loading into a straw-like receptacle with air-separated thawing/freezing columns, controlled subzero temperature handling including a controlled cooling rate to a second subzero temperature, and immersion in liquid nitrogen for storage.
Ethylene glycol incubation in a freezing solution
Incubating the in vitro produced ungulate embryo in a freezing solution comprising ethylene glycol at specified concentration and time at a first temperature.
Straw-like receptacle with air-separated thawing/freezing regions
Loading the in vitro produced ungulate embryo in a receptacle by adding thawing solutions separated by air bubbles and separating the embryo in a freezing solution from adjacent thawing solutions, wherein the thawing solutions comprise ethylene glycol in an isotonic diluent medium.
Subzero temperature exposure and controlled cooling rate
Exposing the receptacle comprising the in vitro produced ungulate embryo to subzero temperature conditions, maintaining it within a subzero range, and lowering the temperature at a controlled cooling rate until reaching a second subzero temperature.
Immersion in liquid nitrogen for storage
Immersing the in vitro produced ungulate embryo in liquid nitrogen for storage, thereby producing a frozen in vitro produced ungulate embryo.
Across the independent claims, the core inventive features are ethylene glycol exposure, straw-like receptacle loading with air-separated solution columns, controlled subzero handling with a controlled cooling rate, and liquid nitrogen storage to produce a frozen embryo.
Stated Advantages
Improved conception/pregnancy rates versus standard vitrification/recovery for direct transfer IVP embryos.
Reduced toxicity via shorter ethylene glycol exposure and controlled rehydration using side/thawing columns.
Higher pregnancy/conception outcomes reported for direct transfer compared with vitrified embryos at 30 days and similar trends at 60 days.
Documented Applications
Cryopreservation and direct transfer of in vitro produced ungulate embryos, including transfer to synchronized recipient ungulates and transfer to a uterine horn.
Use of frozen in vitro produced ungulate embryos across developmental stages including morula to expanded blastocyst as part of freezing and thawing workflows and downstream transfer.
Multi-embryo/device workflows and storage as cryopreserved embryos using described media conditions for in vitro produced embryos.
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