Cryopreserving ungulate embryos

Abstract

Technologies for cryopreserving ungulate embryos for implantation into recipient females are described.

We claim: 
     
         1 . An ungulate animal produced by a method comprising:
 (1) freezing an in vitro produced ungulate embryo, wherein the freezing comprises the steps of:
 (a) incubating the in vitro produced ungulate 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.; 
 (b) loading the in vitro produced ungulate 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; 
 
 (c) exposing the receptacle comprising the in vitro ungulate produced embryo to a temperature of −2 to −10° C. for a time period of 1 min to 60 minutes; 
 (d) lowering said temperature at a rate of −0.2 to −0.8° C. per minute until reaching a second temperature of about −30 to about −36° C.; and 
 (e) immersing said in vitro produced ungulate embryo in liquid nitrogen for storage, thereby producing a frozen in vitro produced ungulate embryo; 
   (2) thawing said frozen in vitro produced ungulate embryo, wherein the thawing comprises the steps of:
 (a) exposing the receptacle that contains said frozen in vitro produced ungulate embryo to a first thawing environment for a first period of time sufficient to thaw said thawing solutions, wherein the first thawing temperature is from 10° C. to 38° C.; 
 (b) exposing said receptacle to a second thawing environment at a thawing temperature from 10° C. to 38° C. for a second period of time, sufficient to thaw the freezing solution; and 
 (c) mixing the thawing solutions, freezing solutions and said frozen in vitro produced ungulate embryo within the receptacle, thereby generating a thawed in vitro produced ungulate embryo; and 
   (3) transferring the thawed in vitro produced ungulate embryo to a recipient ungulate.   
     
     
         2 . The ungulate animal of  claim 1 , wherein the in vitro produced ungulate embryo is in a developmental stage selected from the group consisting of morula, early blastocyst, blastocyst, and expanded blastocyst. 
     
     
         3 . The ungulate animal of  claim 1 , wherein said in vitro produced ungulate embryo is an in vitro produced ungulate embryo selected from the group consisting of Bos taurus, Bos indicus, and crossed breed Bos indicus-taurus.