The physiological significance of the dominant follicle (> 9 mm in diameter in a growing phase; stable for < 3 days) for the superovulatory response in 117 lactating Holstein Friesian dairy cows was investigated. The presence or absence of a dominant follicle was determined retrospectively by analysing videotapes of follicular growth in all the ovaries. Superovulation was induced by 28 mg Armour units (400 mg NIH-FSH-P1) of FSH (Folltropin™) administered either twice or once a day i.m. over 4 days in a decreasing regimen or as a single injection s.c. Donors were scanned daily from day 3 after the oestrus preceding superovulation until embryo recovery. In Expt 1 donors superovulated (two times a day for 4 days) in the absence of a dominant follicle yielded more corpora lutea (11.7 ± 0.9 versus 4.7 ± 1.1, P < 0.01), ova and embryos (8.2 ± 1.2 versus 2.8 ± 1.0, P < 0.01) and transferable embryos (5.0 ± 1.0 versus 2.1 ± 0.9, P < 0.05) compared with donors treated in the presence of a dominant follicle. In Expts 2 and 3 donors were scanned only on the day of superovulation and donors with < 10 follicles 3–8 mm in diameter were considered to have a dominant follicle, while donors with ≥ 10 small follicles 3–8 mm in diameter were classified as having no dominant follicle. In Expt 2 donors superovulated (once a day for 4 days) in the absence of a dominant follicle yielded more corpora lutea (15.5 ± 2.5 versus 4.5 ± 1.4, P < 0.01), ova and embryos (12.9 ± 2.8 versus 1.2 ± 0.4, P < 0.01) and transferable embryos (7.8 ± 2.5 versus 0.3 ± 0.2, P < 0.01) compared with donors treated in the presence of a dominant follicle. In Expt 3 donors superovulated (single s.c. injection) in the absence of a dominant follicle yielded more corpora lutea (11.2 ± 2.7 versus 1.9 ± 0.8, P < 0.01), ova and embryos (9.5 ± 2.7 versus 1.2 ± 0.6, P < 0.01) and transferable embryos (3.4 ± 1.3 versus 0.3 ± 0.2, P < 0.05) compared with donors treated in the presence of a dominant follicle. In Expt 4 donors were superovulated using one injection of FSH per day for 4 days and scanned four times at intervals of 2 days. In the absence of a dominant follicle donors yielded more corpora lutea (19.3 ± 2.3 versus 7.7 ± 1.6, P < 0.01), ova and embryos (17.4 ± 2.6 versus 5.1 ± 1.4, P < 0.01) and transferable embryos (10.3 ± 2.2 versus 1.0 ± 0.5, P < 0.01) than in the presence of a dominant follicle. In cows in which the dominant follicle had been aspirated under sonographical control 2 days before superovulation, the superovulatory response was similar to that in animals treated in the absence of a dominant follicle, and was significantly enhanced compared with animals superovulated in the presence of a dominant follicle (21.6 ± 2.2 corpora lutea, 18.7 ± 12.7 ova and embryos, 10.1 ± 1.5 transferable embryos). The major conclusions from this investigation are: (1) that the presence or absence of a dominant follicle can be detected by a minimized ultrasound scanning schedule using the number of small follicles as the major criterion; (2) the presence or absence of a dominant follicle significantly affects superovulatory responses in dairy cattle; and (3) ultrasound-guided follicular aspiration of the dominant follicle provides an accurate and reliable procedure to increase ovarian responses in dairy cattle possessing a dominant follicle.

Porcine morulae and blastocysts were microsurgically bisected and the resulting zona pellucida-free demi-embryos were either cultured in vitro for 48 h or transferred after 24 h of culture into – 24 h asynchronous recipients. All demi-embryos were evaluated according to morphological criteria and classified into three categories (excellent, fair or degenerated). The average diameter and the number of cells were determined. Of 1162 bisected embryos, 764 pairs (66%) were evaluated as transferable after 24 h of culture in vitro. The average diameter after 48 h of culture in vitro was different (P < 0.01) among demi-embryos of the three morphological categories as was the number of cells. The greatest diameter and the greatest number of cells were found in demi-embryos classified as morphologically excellent. A total of 22 of 27 recipients (81.5%) remained pregnant and 21 recipients delivered 126 piglets of which six were stillborn. The survival rate of demi-embryos in farrowing recipients was 21.2% (126 of 594). Litter size was significantly reduced in recipients after transfer of demi-embryos compared with that of mated controls (6.0 ± 2.5 versus 10.8 ± 2.1 piglets). Similarly, the postpartum losses of piglets were higher in the experimental than in the control gilts (26.7% versus 11.6%). Duration of gestation, average birth weight and daily weight gain were not affected. Among the 126 piglets, seven pairs of identical twins (2.3% of 311 transferred pairs) were identified using several genetic markers in blood (blood groups, polymorphic enzymes and plasma proteins) in a total of 25 gene loci. DNA fingerprinting revealed an identical banding pattern between the two partners of each of the seven pairs. Birth and weaning weight as well as daily weight gain varied considerably between monozygotic partners.

Summary. Oestrone accumulation of Day-5 pig blastocysts and the potential physiological significance of oestrone and oestradiol-17β for blastocyst development were investigated in vitro. After 6 h of in-vitro culture in medium supplemented with 10 nm-[³H]oestrone, the accumulation amounted to 550 ± 49 d.p.m. (s.e.m.) per 10 blastocysts. The accumulation of [³H]oestrone (or its metabolite(s)) was reduced (P < 0·001) in the presence of a 100-fold excess of unlabelled oestrone or oestradiol-17β to 135 ± 14 d.p.m. or 148 ± 28 d.p.m. per 10 blastocysts, respectively. The accumulation of [³H]oestrone was not affected in the presence of a 100-fold excess of unlabelled progesterone, testosterone or oestrone sulphate. When blastocysts were post-incubated for 30 or 60 min in [³H]oestrone-free medium, blastocysts retained 74·1 ± 16·8% and 66·0 ± 10·4%, respectively of their initial radioactivity. In parallel experiments with [³H]progesterone the respective values were 23·8 ± 3·0% and 21·7 ± 2·1%. The presence of the antioestrogen nafoxidine (15 μg/ml) in basic culture medium impaired (P < 0·001) the transformation of morulae to blastocysts (21·5 ± 8·9%) compared to controls (98·3 ± 1·7%). The inhibitory effects could be overcome (P < 0·001) by a supplementation with 1 nm- or 100 nm-oestradiol-17β (62·5 ± 12·8% and 80·0 ± 6·2% development to blastocysts) but not with 1 nm- or 100 nm-oestrone (30·3 ± 9·6% and 45·2 ± 10·5%). Blastocyst expansion was also decreased P < 0·01) to 61·0 ± 11·4% of control values in the presence of 15 μg nafoxidine ml. Oestradiol-17β (100 nm) reversed the nafoxidine-induced inhibition of blastocyst expansion to 85·5 ± 5·2% of control values (P > 0·05). It is concluded (1) that early pig embryos possess specific binding sites for oestrogens, (2) that oestradiol-17β is a potentially important factor in morula–blastocyst transformation, and (3) that blastocyst expansion is more independent of oestrogen.

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups. The loss of cells attributed to bisection was 25–30% in category 1 demi-embryos, and increased to 65% in category 3 demi-embryos. Demi-embryos in category 1 derived from early blastocysts and blastocysts experienced no loss of inner cell mass cells. It is concluded that (i) bisection of pig embryos can lead to a substantial proportion of demi-embryos lacking a functional inner cell mass, (ii) the proportion of total cells to inner cell mass cells is similar in demi-embryos and intact embryos, and (iii) the percentage of cell losses attributed to bisection increases with decreasing quality of demi-embryos. Collectively, these results indicate that the blastocyst is the optimal stage to obtain a maximum yield to viable pig demi-embryos.

To elucidate the developmental differences occurring after in vitro fertilization (IVF) of pig oocytes matured either in vitro (n = 1934) or in vivo (n = 1128), the present experiment investigated the morphological changes from penetration to the two-cell stage. Oocytes were examined every 2–4 h from 2 to 32 h after in vitro insemination to study sperm penetration, male and female pronucleus formation, synkaryosis and first cleavage. The penetration rate was significantly higher (P < 0.05) for in vivo matured oocytes (69.8%) than for in vitro matured oocytes (35.0%). Penetration of spermatozoa into the ooplasm was first recorded 6 h (in vitro matured oocytes) and 4 h (in vivo matured oocytes) after addition of the spermatozoa to the oocytes. For both in vivo and in vitro matured oocytes, 2 h were required for sperm head decondensation. However, maximum sperm head decondensation occurred 2 h later in in vitro matured oocytes. Within 6 h, 41.7 ± 5.6% of the in vivo matured oocytes had completed second meiotic division, whereas only 20.8 ± 6.5% of the in vitro matured oocytes reached this developmental stage (P < 0.01). For in vitro matured oocytes, male pronucleus formation was retarded 2–4 h after onset of insemination and development of the female pronucleus was enhanced compared with in vivo matured oocytes. Synchronized opposing pronuclei were observed 14 h after insemination in in vitro matured oocytes and after 8 h in in vivo matured oocytes. Synkaryosis was first observed at 16 and 18 h in in vivo and in vitro matured oocytes, respectively. First cleavage was observed 32 h (in vitro matured oocytes) and 28 h (in vivo matured oocytes) after insemination. It is concluded that under our IVF conditions, oocytes matured in vitro display lower penetration and cleavage rates and asynchronous pronucleus development, as well as delayed cleavage, compared with oocytes matured in vivo.

The hypothesis that high concentrations of IGF1 can impair embryo development was investigated in a bovine in vitro model to reflect conditions in polycystic ovary syndrome (PCOS) patients. Embryos were either cultured in the absence or presence of a physiological (100 ng/ml) or supraphysiological (1000 ng/ml) IGF1 concentration. Cell allocation, apoptosis, transcript and protein expression of selected genes involved in apoptosis, glucose metabolism and the IGF system were analysed. Supraphysiological IGF1 concentration did not improve blastocyst formation over controls, but induced higher levels of apoptosis, decreased TP53 protein expression in the trophectoderm and increased the number of cells in the inner cell mass (ICM). The increase in ICM cells corresponded with an increase in IGF1 receptor (IGF1R) protein in the ICM. A small, but significant, percentage of blastocysts displayed a hypertrophic ICM, not observed in controls and virtually absent in embryos treated with physiological concentrations of IGF1. Physiological IGF1 concentrations increased total IGF1R protein expression and upregulated IGFBP3 transcripts leading to an increase in blastocyst formation with no effects on cell number or apoptosis. In conclusion, the results support the hypothesis of detrimental effects of supraphysiological IGF1 concentrations on early pregnancy. However, our results do not support the premise that increased apoptosis associated with high levels of IGF1 is mediated via downregulation of the IGF1R as previously found in preimplantation mouse embryos. This in vitro system with the bovine preimplantation embryo reflects critical features of fertility in PCOS patients and could thus serve as a useful model for in-depth mechanistic studies.

For successful fertilization by the male gamete, oocyte cytoplasmic organelles such as the Golgi apparatus have to undergo specific changes: the entire process is known as cytoplasmic maturation. The goal of this study was to unravel the dynamics of the Golgi apparatus in bovine oocytes at critical stages of in vitro maturation, i.e. germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II, and to investigate the role of various molecules critically involved therein. The cytoplasmic distribution of proteins was assessed by immunocytochemistry and laser confocal microscopy. We applied specific inhibitors, including nocodazole to unravel the functional role of the microtubular elements; sodium orthovanadate, which primarily inhibits cytoplasmic dynein ATPase activity; monastrol which inhibits the kinesin EG5; and roscovitine to inhibit the kinase cyclin-dependent kinase 2A (CDC2A). Prior to GVBD, the Golgi apparatus was translocated from the centre of the cytoplasm to the cortical area in the periphery, where it underwent fragmentation. A second translocation was observed between GVBD and MI stages, when the Golgi apparatus was moved from the cortex to the centre of the cytoplasm. Incubation with the specific inhibitors revealed that microtubules played an active role in the final localization at GVBD, while CDC2A was essential for Golgi fragmentation at GVBD stage. This partitioning was a precondition for the second movement. In conclusion, for the first time we show basic mechanisms critically involved in the regulation of the dynamic changes of Golgi apparatus during meiosis of the bovine oocyte.

IGF1 plays an important role in bovine follicular growth, acquisition of oocyte competence and embryo viability. Current data also indicate a critical role for IGF1 in both the ovarian response and the embryo yield following the superovulatory treatments. IGF1 can have either positive or negative effects on embryo viability which is related to the concentration of IGF1 induced by superovulation treatment. These effects impact either on oocyte competence or directly on the embryo. Concentrations in the physiological range appear to result in the production of higher quality embryos, mainly due to the mitogenic and the anti-apoptotic activities of IGF1. However, high superovulatory responses are associated with decreased embryo viability and a concomitant increase in apoptosis. Studies in mice suggest that this increase in apoptosis is related to the downregulation of the IGF1 receptor in the embryo associated with high IGF1 concentrations. Strategies capable of controlling the IGF1 concentrations could be one approach to improve superovulation responses. A range of possible approaches for research within the IGF system in gonadotrophin-stimulated cattle is discussed in this review, including the possible use of superovulated female cattle as an alternative animal experimental model for research on reproductive disorders in humans associated with abnormal IGF1 concentrations.

The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.

Insulin improves development of mammalian preimplantation embryos and, in addition to the regulation of glucose transport, it exerts mitogenic and anti-apoptotic activities. The expression of glucose transporters (Glut) mediating the uptake of this essential energy substrate is critical for embryo survival. An impaired expression of Glut leads to an increase in apoptosis at the blastocyst stage and involves Bax. The various effects of insulin were unravelled by supplementing the in vitro culture medium with insulin (1.7 micromol l(-1)) and (i) the rates of cleavage and blastocyst development were recorded; (ii) mitogenic activity was studied by determining the total number of blastocyst cells and the ratio between trophectoderm and inner cell mass (ICM) cells; (iii) the frequency of apoptosis in blastocysts was determined by the TdT-mediated duTP nick-end labelling (TUNEL) assay and by quantification of the relative amounts of mRNA for Bax and Bcl-XL; and (iv) expression for Glut1, Glut3 and Glut8 transcripts was compared between embryos cultured in the presence or absence of insulin. Insulin increased rates of cleavage (81.2+/-2.2 (control) to 86.0+/-2.5) and blastocyst development (24.7+/-1.9 to 31.3+/-1.2), and number of blastocyst cells (123.7+/-6.0 to 146.3+/-6.6); the increase in the number of blastocyst cells was due to a significantly higher number of trophectoderm cells (82.3+/-5.0 versus 100.3+/-5.5). Blastocysts derived from cultures supplemented with insulin showed a significant decrease in apoptosis as determined by the TUNEL assay (14.8+/-0.9 to 12.2+/-0.7). No effects of insulin on the mRNA expression of Glut isoforms and Bax and Bcl-XL were found. These results demonstrate that the mitogenic and anti-apoptotic effects of insulin on bovine preimplantation embryos did not correlate with changes in the amounts of mRNA for the glucose transporter isoforms Glut1, -3 and -8, or transcripts for Bax and Bcl-XL.