In vitro culture of ovine embryos up to early gastrulating stages.

Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes - trophoblast and hypoblast - and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated 'The people behind the papers' interview.

Influence of maternal environment on the number of transferable embryos obtained in response to superovulatory FSH treatments in ewes.

In a first experiment, embryo viability was estimated after recovery in the uterus or the oviduct of 70 Manchega ewes following a treatment of superovulation with decreasing doses of OVAGEN. Fewer viable embryos (5.6 +/- 0.9 vs. 8.3 +/- 0.8, P < 0.05) and more degenerative embryos (31.3% vs. 6.8%, P < 0.005) were obtained from the uterus than from the oviduct respectively. In a second experiment performed on 14 ewes, embryo viability was analyzed in relation to the follicular population estimated by ultrasonography (follicles > or = 2 mm) at the first FSH administration. Progesterone (P4) and oestradiol 17beta (E2) concentrations were also determined from the beginning of the superovulation treatment to the recovery of the embryos. The number of viable embryos (4.3 +/- 1.4) was positively correlated (r = 0.824) with of 2-4 mm diameter follicles (P < 0.05), and with E2 concentrations at -12 h (r = 0.891, P < 0.01) , 0 h (r = 0.943, P < 0.0001) and +24 h (r = 0.948, P < 0.05) from estrus detection. Prolonged high levels of E2 up to 72 h with low levels of P4 on days 3 and 4 after estrus had a negative (P < 0.05) effect on embryo viability. These results indicate that ovarian response to superovulatory protocols is related to the individual variations in the number of follicles of 2-4 mm at the start of FSH treatment, and that embryo viability is conditioned by the steroid patterns during the time spent in the genital tract of the super-ovulated ewes.

New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos.

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.

Transferencia de embriones en ovejas receptoras tratadas con hormona de crecimiento, efectos sobre la viabilidad de los embriones / Embryo transfer in recipient ewes treated with growth hormones. Efects on embryo viability

Para estudiar la supervivencia de embriones transferidos a receptoras sincronizadas con FGA y PMSG, tratadas con hormonas de crecimiento porcina (GHp) al retiro de las esponjas, se realizaron dos experimentos sobre ovejas de la raza Rasa Aragonesa. El experimento 1 utilizó 12 ovejas donantes para transferir un embrión viable a receptoras tratadas (R1; n=29) o no tratadas (R2; n=28) con GHp. La tasa de ovulación (TO) y los niveles de progesterona (P4) resultaron más altos (P <0.01) que las R2. Este último se relacionó directamente con la aplicación directamente con la aplicación exógena de la GHp, mostrando una alta correlación (P < 0.01) entre la concentración de P4 del día 4 y la del día 17 posfecundación, sin diferencias de fertilidad entre tratamientos. En el experimento 2, con 14 ovejas donantes se obtuvieron 95 embriones viables de 2 días, de edad transfiriendo únicamente 68 (8-10 embriones por grupo) a 4 receptoras tratadas con GHp (R1) y a 4 no tratadas (R2). Estos mismos embriones recuperados tres día después a nivel del útero, fueron valorados morfológicamente. Se observó un aumento de la TO (P < 0.05) de las ovejas receptoras tratadas con GHp (R1). La recuperación de embriones fue mayor en las ovejas tratadas con GHp, con una tendencia a una mayor calidad de los embriones (P < 0.2). En conclusión: a) En ovejas receptoras tratadas con FGA y PMSG, la aplicación de GH produce un aumento de la TO y concentración plasmática de P4, cuatro días después de retirar el progestágeno, b) la fertilidad de embriones transferidos no se modifica si se trata con GH a las ovejas receptoras