Cryopreservation of in vitro-produced ovine embryos

The purpose of this study was to evaluate different cryopreservation protocols for in vitro-produced ovine embryos and assess the survival rate after cryopreservation. The experiment was also designed to examine whether this technique is feasible to apply in large-scale operations. In a first experiment, ovine embryos cryopreserved in ethylene glycol using 3 steps (E-3S) showed a higher hatching rate and nuclei number than freezing with glycerol in 3 steps (G-3S) (40.0% versus 20.0% and 135.4 ± 20.7 versus 118.5 ± 19.5, respectively). In a second experiment, vitrification with ethylene glycol + Ficoll 70 + sucrose (EFS) recorded a higher hatching rate and nuclei numbers than vitrification with propylene glycol + glycerol (Pg + Gly) (51.1% versus 31.1% and 133.8 ± 36.8 versus 113.5 ± 22.0, respectively). In a third phase, vitrification of embryos with ethylene glycol + glycerol (Eg + Gly) resulted in higher development and hatching rates and nuclei number than EFS (87.3% versus 65.4%; 76.4% versus 54.5% and 135.7 ± 34.5 versus 113.1 ± 14.1, respectively). In a fourth experiment, fresh in vivo embryos produced a higher lambing rate (67.8%) than the other methods, and E-3S in vitro resulted in lower lambing rates (23.0%). E-3S in vivo, Eg + Gly in vivo, fresh in vitro and Eg + Gly in vitro recorded similar lambing rates (42.8, 37.5, 37.5 and 26.6%, respectively). Ewes receiving in vitro-produced ovine embryos resulted in higher assisted births and perinatal losses than those receiving in vivo-produced embryos (3.6% versus 16.9% and 1.2% versus 10.1%, respectively). Results demonstrate that the transfer of in vitro-produced embryos vitrified with Eg + Gly could have wide application, if the negative side effects of the produced offspring can be managed.