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Use of purified FSH and LH for embryo production, cryopreservation by conventional freezing or vitrification and transfer of embryos in dairy ewes
Abstract
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 PGF2α (ICI; 50 μg, 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; P<0.05). Ovulation rate did not differ among the groups (6.5 to
10.8). Transferable embryos in Group B) (7.2) resulted similar to Group A) (5.3) and significantly (P<0.05) different when
compared to Group C) (3.2). V3-method resulted in the highest (P<0.01) transferable embryos (74.5%) compared to
CF- and V1-methods. After transfer, in FE and VTE recipient ewes were comparable in fertility rates (72.7 vs 73.3%;
P>0.05) 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.
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 PGF2α (ICI; 50 μg, 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; P<0.05). Ovulation rate did not differ among the groups (6.5 to
10.8). Transferable embryos in Group B) (7.2) resulted similar to Group A) (5.3) and significantly (P<0.05) different when
compared to Group C) (3.2). V3-method resulted in the highest (P<0.01) transferable embryos (74.5%) compared to
CF- and V1-methods. After transfer, in FE and VTE recipient ewes were comparable in fertility rates (72.7 vs 73.3%;
P>0.05) 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.
