Epidermal growth factor (EGF) has been shown to have a positive effect during in vitro maturation (IVM) and has been reported in follicular fluid at levels capable of stimulating meiosis in a variety of species. The aim of the present work was to study the effect on subsequent development of EGF present in defined medium during bovine 1) oocyte maturation or 2) embryo culture. The presence of EGF during IVM, irrespective of concentration (1, 10, 100 ng/mg), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphase II, the rate of cleavage, and the proportion of embryos reaching the 5- to 8-cell stage at 72 h postinsemination. Blastocyst rates on Days 7 and 9 were also significantly improved for oocytes matured in the presence of EGF (10% vs. 18-24% on Day 7 and 21% vs. 31-32% on Day 9, for Tissue Culture Medium 199 [M199] and M199 + EGF, respectively). The presence of fetal calf serum (FCS) during IVM resulted in similarly elevated rates of development. There was no cumulative effect when EGF and FCS were present together during IVM. The presence of EGF also altered the pattern of proteins neosynthesized during maturation. The maturation-promoting effect of EGF was evident for denuded oocytes also, suggesting that EGF may act, at least in part, directly on the oocyte. Immunofluorescence studies revealed the EGF receptor on immature cumulus-oocyte complexes. When present during postfertilization culture in defined medium (synthetic oviduct fluid), EGF stimulated development in comparison to that of the control but could not replace serum. The results suggest a physiological role for EGF in the regulation of bovine oocyte maturation and development.

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Porcine embryo selection prior to transfer is mainly influenced by morphological criteria. However, the relationship between embryonic morphology, developmental potential and cell death by apoptosis in porcine embryos is still unclear. The aim of this study was to establish embryo quality parameters for in vivo fertilised porcine embryos based on timing of development in vitro, embryo morphology and the presence of apoptosis. The kinetics of development and morphological parameters were investigated in a time-lapse cinematographic experiment. Possible links between embryo morphology and apoptosis were examined via a confocal laser scanning experiment, analysing nuclear changes, annexin V and terminal dUTP nick-end labelling. The timing of early cleavages was firmly linked to embryo developmental competence in vitro. Attainment of at least the 5-cell stage before 77 h post insemination and attainment of the morula stage before 102 h post insemination significantly increased the odds for reaching the early blastocyst stage. Overall, a negative effect of fragmentation percentage and fragmentation pattern on subsequent embryonic development was observed, but the developmental potential of embryos experiencing slight fragmentation (0 -5%) was not different from embryos without fragmentation. Correlations detected between developmental arrest and fragmentation, and fragmentation and apoptosis were 0.60 and 0.87 (P < 0.05) respectively. Only a minority of the embryos arrested between the 1-and 4-cell stage displayed biochemical characteristics of apoptosis. Consequently, a significant correlation (0.57) between developmental arrest and apoptosis could only be established for embryos arrested after embryonic genome activation.

The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter !6 mm always gave a higher blastocyst rate than oocytes from follicles <4 mm (UCL: 42% versus 14%, DIAS: 50% versus 35%, INRA: 39% versus 22%; P < 0.05). Blastocyst cell number was not affected by follicle size. Several parameters were investigated for these oocytes. The energy metabolism of cumulus-oocyte-complexes and of denuded oocytes was assessed by the oxygen and pyruvate uptake and by lactate release both at the beginning and the end of the maturation. No effect of follicle size could be detected but lactate release increased after maturation. The global profile of transcripts, the pattern of protein neosynthesis and the kinetics of meiosis resumption were not affected by follicle size. The developmental kinetics of derived embryos was also analysed. Whatever the follicle size, viable embryos had a shorter first and third embryonic cell cycle. Among the viable embryos, the size of the follicle interfered with the fourth cell cycle duration. A higher percentage of blastocysts issued from large follicle presented a short fourth cell cycle (9 h) (35% versus 6%; P < 0.05). Beside, blastocysts derived from small follicles had a delayed cavitation and expansion. Thereby, a higher developmental competence for oocytes from follicle !6 mm versus <4 mm was Theriogenology 63 (2005) [841][842][843][844][845][846][847][848][849][850][851][852][853][854][855][856][857][858][859]

A variety of embryo-based technologies used in farm animal reproduction, including embryo culture, nuclear transfer, embryo-somatic cell co-culture and asynchronous embryo transfer can lead to the production of large offspring; the so-called large calf/lamb syndrome. In some cases, abnormalities in the fetus and newborn are apparent. The nature of these associations is explored with emphasis on the biological differences between in-vivo- and in-vitro-produced embryos. A unifying framework and research programme aimed at explaining anomalies in early embryo development is then proposed in terms of the response of somatic cells and embryos to cellular stress. The review concludes with a caution against developments in assisted conception technologies, in man and domestic animals, being determined too much by the needs of commerce at the expense of research on the molecular, biochemical and physiological basis of early mammalian development.