Cryopreserving ungulate embryos

A method for cryopreserving an in vitro produced ungulate embryo comprising the steps of:

• culturing an immature oocyte in vitro under a first culture condition to obtain a mature oocyte;
• fertilizing said mature oocyte to generate a zygote;
• denuding said zygote;
• culturing said zygote in a second culture condition comprising an atmosphere of 5% oxygen (O2) and 5% carbon dioxide (CO2) to prepare said in vitro produced ungulate embryo;
• freezing said in vitro produced ungulate embryo wherein the freezing comprises the steps of:
• (1) incubating said embryo in a freezing solution comprising of 1.0 to 4 molar (M) ethylene glycol for 5 to 30 minutes at a first temperature between 10° C. and 38° C.;
• (2) loading said embryo in a receptacle comprising the steps of adding:
• i) a first thawing solution to said receptacle;
• ii) a first air bubble and a second thawing solution, wherein said first air bubble separates said first and second thawing solutions;
• iii) a second air bubble and said in vitro produced ungulate embryo in a freezing solution, wherein said second air bubble separates said second thawing solution and said in vitro produced ungulate embryo in a freezing solution;
• iv) a third air bubble;
• v) a third thawing solution, wherein said third air bubble separates said in vitro produced ungulate embryo in a freezing solution and said third thawing solution;
• vi) a fourth air bubble;
• and
• vii) a fourth thawing solution, wherein said fourth air bubble separates said third and fourth thawing solutions and said first, second, third, and fourth thawing solutions comprise ethylene glycol in an isotonic diluent medium;
• (3) exposing the receptacle prepared in step (2) to a temperature of −2 to −10° C. for a time period of 1 min to 60 minutes;
• (4) lowering said temperature at a rate of −0.2 to −0.8° C. per minute until reaching a second temperature of −30 to −36° C.;
• and
• (5) immersing the receptacle in liquid nitrogen for storage.