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

Embryo losses in Rasa Aragonesa ewes actively immunized against androstenedione or passively immunized against testosterone

Two-day-old embryos from untreated ewes were transferred to the oviducts of ewes actively immunized against androstenedione (n=26, Group A), passively immunized against testosterone (n=19, Group B) or left untreated (n=25, Group C). Donor ewes superovulated after treatment with follicle-stimulating hormone and fluorogestone acetate (FGA). Recipient ewes were treated with FGA and pregnant mare serum gonadotropin (PMSG, 300 I.U.). Group A received two injections of Fecundin at a 4-wk interval. FGA sponges were inserted when the second injection was given. Group B was treated with antitestosterone antiserum (35 ml) at sponge withdrawal. Each recipient received two morphologically viable embryos 52 to 62 h after the onset of estrus. Antibody titre at embryo transfer and progesterone concentration on Days 2, 4, 6, and 12 after estrus were determined. Fertility was lower in Group A when compared to Group C (42.3 vs 84.1%; P<0.01) while that of Group B (63.2%) did not differ from those of Groups A and C. In immunized groups, most of the embryo losses occurring were complete (both embryos were lost), resulting in a decreased fertility, while in the untreated group embryo losses were mainly partial (only one embryo was lost), hence lowering prolificacy. Fertility in immunized groups changed according to the antibody titre reached. Ewes from Groups A and B with higher antibody titres displayed lower fertility than control ewes. On Days 4 and 12 of the cycle, Group A plasma progesterone concentrations positively correlated with antibody titres and were higher with respect to those of Group C (P<0.05). Progesterone levels in Group B were similar to those of Group C. These results indicate that ewes reaching higher antibody levels had more embryo losses, attributable to the adverse influences of the oviductal and/or uterine environment on embryo development. © 1991

Effect of immunization on reproductive performance, embryo quality and progesterone in Rasa Aragonesa ewes actively immunized against androstenedione or passively immunized against testosterone

A total of 217 Rasa Aragonesa ewes were used to test two immunization treatments 1.Active immunization against androstenedione ewes immunized in previous matings (androstenedione, reimmunized; AR groups, n=58) or not (first immunization; AF groups n=64) were boosted either 2 or 4 wk before mating. 2.Passive immunization against testosterone antisera were injected either at sponge withdrawal (zero time; T0 group, n=21) or 1 wk previously (Tl group, n=22). We used 52 ewes as controls (C group). Half of each group was used either to record reproductive performance or to embryo viability assessment. Prolificacy was significantly increased in ewes which reached a moderate antibody level, independently of the treatment. Fertility was lower in AR ewes that attained a high antibody titre (P<0.01). The percentage of viable embryos recovered was lower in AF ewes (P<0.01), and in ewes whose testosterone antibody titre was high (P<0.05), compared to C group. It was proven that similar or lower antibody levels were more harmful for ewes from AF and Tl than for ewes from AR or T0 groups. The proportion of nonfertilized recovered ova was not significant. Progesterone levels were notably increased in AR ewes (P<0.001) independently of ovulation rate and were positively correlated to antibody titre at mating (P<0.01) but these events were not observed in T ewes. These findings indicate that after androgen immunoneutralization, only those ewes having antibody titres within a limited range at mating had improved reproductive performance. Further research is needed in order to understand the role that progesterone plays in immunized ewes. © 1991

Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol

Embryos were collected on Days 5 and 6 after breeding to investigate the effectiveness of ethylene glycol (ETG) and glycerol (GLY) as cryoprotectants of sheep morulae and blastocysts and to determine their optimum stage of development for cryopreservation. Only excellent (grade 1) and good (grade 2) embryos (196 morulae and 188 blastocysts) were incubated in increasing concentrations of GLY or ETG and submitted to a slow-freezing and quick-thawing procedure. Both cryoprotectants were removed using 0.25 M sucrose solution, and then embryos were cultured or transferred to determine their viability. Freezing medium containing ETG yielded higher in vitro survival rates (P < 0.01) than medium containing GLY (64.6% vs 16.0%); the difference between cryoprotectants was greater when morulae were used (57.9% vs 4.2%, P < 0.005) as compared with blastocysts (70.4% vs 21.5%, P < 0.05). There was a strong interaction between type of cryoprotectant and embryo stage (P < 0.005). After transfer of morphologically viable embryos, the in vivo development rate of embryos frozen with ETG was also higher than that of embryos frozen with GLY (45.5% vs 27.7%, P < 0.05). There were no significant differences in the number of lambs born among procedures and embryo stage, though the lowest lambing rate was obtained with morulae frozen with GLY (21.4%). Similar lambing rates were produced when blastocysts were frozen with either GLY or ETG (36.6% vs 43.0%). The best embryo survival after thawing was observed when blastocysts were frozen with ETG as cryoprotectant. © 1996 Academic Press, Inc

Effects of LH administration at the end of an FSH superovulatory regimen on ovulation rate and embryo production in three breeds of sheep

In 3 experiments, 168 ewes of Manchega (n = 72), Churra (n = 62), and Merina (n = 34) breeds were used to test the hypothesis that administration of pure LH, coincident with progestogen removal during superovulation with FSH, causes an increase in the ovulation rate and number of embryos. This administration of LH can further interact with genotype, resulting in breed differential response. In each experiment, the animals were randomly assigned to 1 of 3 treatments. Estrus in all sheep was synchronized with intravaginal sponges of 30 mg of FGA for 12 d, then 270 μg of FSH were administered in 6 injections at 12-h intervals in decreasing doses, starting 48 h before sponge removal. The FSH/LH ratio of the original preparation was 3, and remained constant throughout the treatment in the control group (C). In Treatment 1, (T1) and Treatment 2, (T2), pure LH was administered coincident with progestogen removal-5th FSH injection, and with the 6th FSH injection, at 2 dose levels 60 and 120 μg, (T1), and 120 and 240 μg (T2). Mating occurred 36 and 48 h after the progestogen removal, and the embryos were surgically collected and morphologically evaluated on Days 7 and 8 after sponge withdrawal. Overall, the results showed that LH administration at the end of the FSH treatment did not increase the ovulation rate and number of embryos in Merino (5.9 ± 1.4 and 5.6 ± 1.4 , respectively, T1; 7.0 ± 1.0 and 5.7 ± 1.2, T 2; 4.9 ± 1.1 and 2.6 ± 0.7, C), Churra (6.8 ± 1.4 and 5.2 ± 1.4, T1; 8.1 ± 1.5 and 6.3 ± 1.4 , T2; 6.1 ± 1.5 and 5.4 ± 1.3, C) and Manchega (6.0 ± 1.0 and 4.4 ± 1.0, T1; 5.0 ± 0.8 and 4.2 ± 0.8, T2; 4.8 ± 1.5 and 3.8 ± 1.0, C). Administration of LH induced a significant (P<0.05) increase in the frequency of multiple ovulations (72.3 ± 4.3 %, T1; 74.1 ± 11.5 %, T2; 55.6 ± 5.9 %, C) paralleled to a decrease in the occurrence of ewes with no ovulations (8.7 ± 2.6 %, T1; 7.6 ± 4.6 % T2; 17.3 ± 3.2 %, C) or 1 to 2 ovulations (18.7 ± 4.6 %, T1; 18.1± 7.5 %, T2; 26.8 ± 5.8 % C), regardless of breed or dose of LH. No increase in the mean number of viable embryos was observed, probably due to both the high individual variability and the lower fertilization rates observed in sheep showing multiple ovulations

Effects of FSH commercial preparation and follicular status on follicular growth and superovulatory response in Spanish Merino ewes

Ovarian follicular development was characterized in 24 Spanish Merino ewes to study effects of the follicular status and the FSH commercial product used on follicular growth and subsequent superovulatory response. Estrus was synchronized using 40 mg fluorogestone acetate sponges. The superovulatory treatment consisted in 2 daily im injections of FSH from 48 h before to 12 h after sponge removal. Sheep were assigned randomly to 2 groups treated with 6 decreasing doses (4, 4, 3, 3, 2, 2 mg) of FSH-P(TM) or with 6 doses of 1.25 mL of OVAGE(TM) Growth and regression of all follicles ≥2 mm were observed by transrectal ultrasonography, and recorded daily from Day 6 before sponge insertion to the first FSH injection, and then twice daily until estrus was detected with vasectomized rams. Differences were detected in follicular development from the first FSH injection to detection of estrus (-48 to 36 h from sponge removal) between groups. Administration of FSH-P increased the appearance of new follicles with respect to OVAGEN (6.3 ± 0.7 vs 4.8 ± 0.4; P < 0.05), and the mean number of medium (4 to 5 mm) follicles (8.9 ± 1.2 vs 6.6 ± 0.9; P < 0.05). However, the mean number of follicles that regressed in size after sponge removal (5.9 ± 0.4 vs 3.3 ± 0.4) and the number of preovulatory sized follicles that did not ovulate (60 vs 42.4%) were also higher in FSH-P treated ewes (P < 0.05). So, finally, there were no differences in ovulation rate, as determined by laparoscopy on Day 7 after sponge removal, between ewes treated with FSH-P or OVAGEN (6.3 ± 1.9 vs 7.0 ± 1.7 CL). In all the ewes, the ovulatory response was related (P < 0.05) both to the number of small follicles (2 to 3 mm in diameter) present in the ovaries at the start of treatment with exogenous FSH and to the number of follicles that reached ≥4 mm in size at estrus, despite differences in the pattern of follicular development when using different commercial products. (C) 2000 by Elsevier Science Inc

Estudio espacial mediante sistemas de información geográfica de los megalitos de la comarca de Tierra de Ledesma (Salamanca, España)

En este trabajo se presenta un inventario de los megalitos documentados en la comarca de la Tierra de Ledesma (Salamanca), a partir del cual se ha realizado un análisis espacial de los mismos utilizando Sistemas de Información Geográfica, con el fin de relacionar estas construcciones con el medio geográfico en el que se encuentran, además de estudiar aspectos como su influencia o dominio sobre una determinada porción del territorio mediante análisis de visibilidad. Queda patente la distribución heterogénea de los megalitos en esta zona, estando su ubicación relacionada con los cursos de agua y con la geología del terreno, además de ubicarse dentro de un determinado rango de altitudes.This paper presents an inventory of the megaliths documented in the region of Tierra de Ledesma (Salamanca), from which a spatial analysis of them has been carried out using Geographic Information Systems, to relate these constructions with the geographical environment in which they are found, in addition to studying aspects such as their influence or dominance over a certain portion of the territory through visibility analysis. The heterogeneous distribution of the megaliths in this area is evident, their location being related to the watercoursesand the geology of the terrain, in addition to being located within a certain range of altitudes.Depto. de Prehistoria, Historia Antigua y ArqueologíaFac. de Geografía e HistoriaTRUEpu

Influence of the FecXR allele in heterozygous ewes on follicular population and outcomes of IVP and ET using LOPU-derived oocytes

Contents Ewes heterozygous for the FecXR allele (R+) in the bone morphogenetic protein 15 (BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the FecXR allele on follicle number and size, oocyte competence and in vitro production (IVP) remains undefined. With these aims, 8 R+ and 8 wild-type (++) ewes were subjected to 2 laparoscopic ovum pick-up (LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles &gt;3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH-treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (-0.2 mm in untreated and -0.8 mm in FSH-treated ewes; p &lt; 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus-oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p &lt; 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture. © 2013 Blackwell Verlag GmbH

Effect of season and duration of FSH treatment on embryo production in sheep

Thirty-six mature Manchega ewes were used in two experiments to determine the effect of season and of 2- or 3-d FSHp treatment on the ovulation rate and number of transferable embryos produced. During the breeding season, estrus was synchronized with FGA (30 mg for 13 d). Begining 48 or 24 h before sponge removal, each ewe received two daily injections of 4-4-3-3-1-1 or 5-5-3-3 mg of FSHp. Concurrently with the two last injections both groups were administered 100 μg of LH. Ewes were tested for estrus and 6 or 7 d later were laparotomized and surgically flushed to recover embryos. The number of corpora lutea (CL), the total number of embryos and of viable embryos were recorded. Six months later (nonbreeding season) the design was repeated, with each ewe receiving the opposite treatment to that received in the fall. Response in ovulation rate and number of viable embryos did not differ between seasons. Mean (SEM) numbers of observed CL and embryos recovered were higher (P<0.001) with the 3-d treatment (8.7±5.8 and 7±4.8) than with the 2-d treatment (5.8±3.2 and 4.4±3) when pooled over the two seasons. The mean number of transferable embryos was higher (P<0.01) with the 3-d (4.2±3.9) than with the 2-d treatment (2.5±2.3). © 1990

Exogenous growth hormone improves the number of transferable embryos in superovulated ewes

The application of pGH (porcine Growth Hormone) to superovulated ewes was studied with the aim of improving the embryo yield. Thirty-seven ewes were superovulated with pFSH for 3 d and 18 of them were cotreated the third day with 0.50 mg of pGH. Embryos were surgically recovered on Day 7 after sponge withdrawal. Then, 102 morphologically healthy embryos were immediately transferred in pairs to 51 synchronized recipient ewes. The GH treatment did not significantly affect the percentage of ewes in estrus, the time of estrus onset or the ovulation rate. However, it improved synchronization by grouping estrus in a narrower range (12 h) in comparison to the control group (24 h); (16 to 28 h after sponge withdrawal vs 12 to 36 h; P < 0.05). The total amount of LH released during the preovulatory surge was lower in the GH than in the control group (P < 0.05). No differences were found between groups for other LH-related parameters such as basal levels, peak values or peak time from sponge removal. The proportions of unfertilized oocytes and degenerate embryos recovered were lower in the GH cotreated group (P < 0.05 and P < 0.01, respectively). This resulted in higher rates of transferable embryos and lambs born per donor ewe in the GH than in the untreated group (3.9 vs 1.7 and 2.28 vs 0.84, respectively; both, P < 0.05). These beneficial effects of GH would likely be due either to a direct action on oocyte maturation or to an indirect action on the oviductal environment. © 2001 by Elsevier Science Inc