Use of purified FSH and LH for embryo production, cryopreservation by conventional freezing or vitrification and transfer of embryos in dairy ewes

Three experiments were carried out with the aim of evaluating the efficiency of techniques of in vivo production, storage and transfer of embryos in dairy sheep. Experiment I - For embryo production, thirty-one ewes were synchronized with FGA (vaginal sponges, 40 mg, 9 d) and PGF2 alpha (ICI; 50 mu g, 7th d), and subdivided into three groups corresponding to the following superovulatory treatments over 3 days with purified gonadotrophic preparations: A) control, FSH/LH ratio = 1 (250 IU p-FSH : 250 UI p-LH); B) FSH/LH ratio = 2 (250 IU p-FSH : 125 IU p-LH) and daily FSH/LH ratio of 3.4 - 1.7 - 0.8 in the 3 days of treatment, respectively; C) FSH/LH ratio = 2 (250 IU p-FSH : 125 IU p-LH) and daily FSH/LH ratio of 5.0 - 1.0 - 0.3. On the 7th day after oestrus and mating, ovarian response and embryo production were evaluated. Experiment II - Three freezing methods were evaluated based upon post-thaw embryo quality: CF) conventional slow freezing by 1.5 M ethylene glycol (EG); V-1) one-step vitrification based on exposure of the embryos to one solution (EG 7.15 M + ficoll 2.5 mM); V-3) vitrification in three steps, corresponding to three solutions at increasing concentration of glycerol (GLY) and EG (GLY 1.4 M; GLY 3.4 M + EG 1.4 M; GLY 4.6 M + EG 3.4 M). V-1) and V-3) frozen embryos were directly plunged in liquid nitrogen. At thawing, embryo viability was evaluated on the basis of morphological features. Experiment III - For embryo transfer, a total of 26 recipient ewes were synchronized with donors. On the 7th d from oestrus, 11 recipient ewes received fresh embryos (Group FE - control) and 15 recipients received vitrified-thawed embryos (Group VTE). Each recipient received 2 embryos. Superovulatory treatment B) significantly advanced the onset of oestrus compared to the control (27.3 vs 34.7 h; P0.05) and embryo survival (63.6 vs 56.7%; P>0.05). In conclusion, the results demonstrated that treatments B) and C) did not improve superovulatory response compared to A); for embryo cryopreservation the V3 method can successfully be used for embryo transfer in ewes