62 LONG-TERM STORAGE OF BOVINE EMBRYOS: BIRTH OF A CALF AFTER 22 YEARS OF CRYOPRESERVATION

Abstract

After the pioneering work of Rostand (1946 CR Acad. Sci. III 222, 1524–1525) and Polge et al. (1949 Nature 164, 666–669) on semen cryopreservation and the reports on the first live young born after storage of mouse embryos for up to 8 days (Whittingham et al. 1972 Science 178, 411–414) or 8 months (Whittingham and Whitten 1974 J. Reprod. Fertil. 36, 433–435) in liquid nitrogen, the world’s first calf was obtained in Cambridge nearly 40 years ago, after freezing and thawing of a bovine embryo (Wilmut and Rowson 1973 Vet. Rec. 92, 686–690). Since then, especially in humans (case reports), the birth of healthy offspring after long-term storage of semen, zygotes, and embryos has been repeatedly reported (e.g. Dowling-Lacey et al. 2011 Fertil. Steril. 95, 1120.e1–3). But practical experiences in different other species such as mice or cattle show contradictory results on the fertility after transfer of long-term-stored gametes or embryos. Here, we report the birth of normal Holstein calves after long-term embryo preservation. After storage for 15 to 22 years, 4 embryos were thawed (air-thaw for 10 s and 25°C water bath for 20 s), which had been originally frozen in 10% glycerol (3 embryos) or in 1.5 M ethylenglycol (1 embryo) following a slow freezing protocol. Removal of glycerol was accomplished with a 4-step dilution procedure and 10.3% sucrose (EMCARETM Thawing System, ICPbio Reproduction, Glenfield, New Zealand), whereas ethylenglycol was removed with a washing step in EMCARETM (Holding Solution, ICPbio Reproduction). The embryos were transferred into the uterine horn ipsilateral to the corpus luteum of recipient cows at 7 days post-oestrus. One embryo was classified as expanded blastocyst grade 1 (7-1), 2 embryos were classified as morulae grade 1 (4-1), and the embryo frozen with ethyleneglycol was classified as a morula grade 2 (4-2) (IETS classification; 40× magnification). Three pregnancies were established (1 × 7-1; 1 × 4-1, and 1 × 4-2), and 3 healthy bull calves were born (18.1.2011, stored 15 years; 6.3.2012, stored 22 years; and 29.3.2012, stored 15 years). These case reports document that long-term-stored bovine embryos maintained developmental competence for up to 22 years of cryopreservation in liquid nitrogen. To our knowledge these cases represent the “oldest” cryopreserved bovine embryos resulting in healthy calves in Germany.