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Publication Number
US-11297831-B2
Publication Date
2022-04-12
Expiration Date
Abstract
Technologies for cryopreserving ungulate embryos for implantation into recipient females are described.
Core Innovation
The invention provides a method for cryopreserving an in vitro produced ungulate embryo. The method includes culturing an immature oocyte in vitro under a first culture condition to obtain a mature oocyte, fertilizing the mature oocyte to generate a zygote, denuding the zygote, and culturing the zygote in a second culture condition. The in vitro produced ungulate embryo is prepared by culturing under an atmosphere of 5% oxygen (O2) and 5% carbon dioxide (CO2).
The cryopreservation comprises freezing the in vitro produced ungulate embryo using ethylene glycol. The freezing includes incubating the embryo in a freezing solution comprising 1.0 to 4 molar (M) ethylene glycol for 5 to 30 minutes at a first temperature between 10°C and 38°C. The method further includes loading the embryo in a receptacle using a sequence of thawing solutions and multiple air bubbles, where the air bubbles separate the thawing solutions and separate the embryo in a freezing solution from neighboring solutions.
After loading, the freezing process includes exposing the receptacle to a temperature of −2 to −10°C for 1 min to 60 minutes. The method then lowers the temperature at a rate of −0.2 to −0.8°C per minute until reaching a second temperature of −30 to −36°C. The receptacle is then immersed in liquid nitrogen for storage, and the cryopreserved embryo is handled as an in vitro produced ungulate embryo.
Claims Coverage
The document includes one independent claim directed to cryopreserving an in vitro produced ungulate embryo, with multiple dependent claims refining culture conditions, thawing, and post-thaw handling. The inventive features center on in vitro preparation under a 5% O2/5% CO2 atmosphere and ethylene glycol freezing using staged receptacle loading separated by air bubbles, followed by controlled temperature exposure, cooling to a target freezing temperature, and liquid nitrogen storage.
In vitro culture under 5% O2 and 5% CO2 atmosphere
Culturing the denuded zygote in a second culture condition comprising an atmosphere of 5% oxygen (O2) and 5% carbon dioxide (CO2) to prepare the in vitro produced ungulate embryo.
Ethylene glycol freezing solution incubation
Freezing the in vitro produced ungulate embryo by incubating the embryo in a freezing solution comprising 1.0 to 4 molar (M) ethylene glycol for 5 to 30 minutes at a first temperature between 10°C and 38°C.
Multi air-bubble receptacle loading with ethylene glycol thawing solutions
Loading the embryo in a receptacle by adding a first thawing solution, a first air bubble separating first and second thawing solutions, a second air bubble separating the second thawing solution and the embryo in a freezing solution, a third air bubble separating the embryo in freezing solution and a third thawing solution, a fourth air bubble separating the third and fourth thawing solutions, and wherein the first, second, third, and fourth thawing solutions comprise ethylene glycol in an isotonic diluent medium.
Staged temperature exposure and controlled cooling to −30 to −36°C
Exposing the receptacle prepared for freezing to a temperature of −2 to −10°C for 1 min to 60 minutes, lowering the temperature at a rate of −0.2 to −0.8°C per minute until reaching a second temperature of −30 to −36°C, and immersing the receptacle in liquid nitrogen for storage.
Across the independent claim, the coverage is anchored in forming an in vitro produced ungulate embryo under a 5% O2/5% CO2 atmosphere and freezing it with ethylene glycol using a receptacle-loading scheme that employs multiple air bubbles to separate staged thawing environments. The independent claim further requires staged temperature exposure, controlled cooling to −30 to −36°C, and liquid nitrogen storage.
Stated Advantages
Improved conception rates, including a reported conception rate of ≥10% and reported ~40% in specific embodiments.
Documented Applications
Cryopreservation of in vitro produced ungulate embryos for transfer to recipients, including transfer of developmental stages such as morula, early blastocyst, blastocyst, and expanded blastocyst into synchronized or natural estrus recipients.
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