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Production of Identical Twins from Bovine Embryos Split Pre- of Post-freezing.

Three methods for producing identical twin calves are described. In treatment 1, day 7 bovine embryos were bisected in 12.5% sucrose solution by lateral incision using a fine microsurgical blade after freezing with 1.4 M glycerol and thawing. Both halves remained in the original zona pellucida. In treatment 2, the frozen-thawed embryos were pinned between the blade and the bottom of a plastic petri dish and bisected vertically in PBS. In treatment 3, zona free demi-embryos were cultured at 38.5°C in Ham's F-10, supplemented with 20% bovine serum for 3 hr prior to freezing. In all the treatments, a pair of half-embryos were transferred nonsurgically to a uterine horn of recipients on day 7 of their estrous cycle. In treatment 1, 26 of 81 recipients became pregnant after transfer of the demi-embryos (Pregnancy rates=32%). Five pair of identical twins were obtained from 5 of 22 recipients which delivered offspring. In treatment 2, the pregnancy and twinning rates were 42% (13/31) and 33% (4/12), respectively. One pair of identical twins was obtained from frozen-thawed zona free demi-embryos transferred without removing 1, 2- propanediol after thawing, in treatment 3. These data showed that bovine identical twins could be produced from frozen embryos bisected after thawing, followed by transfer without zona pellucidae.