The relationship between culture duration and embryo morphology is summarized in Figure 1C. Most 2-cell- (67%) and 3-cell- (50%) stage embryos examined at 24 h were normal, but only 14% of 4-cell- and none of the 5- to 8-cell-stage embryos were normal. The proportion of normal embryos at the 4-cell stage increased as culture duration increased to 30-48 h. However, only 17% of 5- to 8-cell-stage embryos at 48 h showed normal morphology. ampicillin antibiotic
Nucleus-Blastomere Relationship and Actin Filament Distribution of Embryos Produced In Vivo and In Vitro (Experiment 2)
As shown in Figure 2, there were great differences in the morphology of embryos produced in vivo and in vitro. In vivo-derived 2- and 4-cell embryos had clear blasto-meres, especially at the 4-cell stage, and all blastomeres were separated. A wide perivitelline space (PVS) was observed in in vivo 2- to 4-cell embryos. After the 8-cell stage, the PVS was fully filled with blastomeres. The inner cell mass was clearly seen in in vivo blastocysts. However, the cytoplasm of in vitro-produced embryos was dark, and it was difficult to see clear blastomeres beyond the 4-cell stage. The PVS was fully filled with blastomeres even at the 2-cell stage. No obvious inner cell mass was observed in in vitro-produced blastocysts.
FIG. 2. Morphology of pig embryos produced in vivo (A, C, and E) and in vitro (B, D, and F). In vivo-produced 2- to 4-cell-stage embryos had clear cytoplasm, distinct blastomeres, and large PVS (A and C), and the blastocyst had a clear inner cell mass (E). In vitro-produced 2- to 4-cell-stage embryos had dark cytoplasm and small PVS (B and D), and the blastocyst did not have an obvious inner cell mass (F). Bars represent 20 ^m in A-D and 40 ^m in E and F.
FIG. 3. Confocal photographs of embryos produced in vivo (left panels) and in vitro (right panels). Normal morphology and actin filament distribution were observed in these embryos. The embryos were stained by rhodamine-phalloidin for actin filaments (red) and by YO-Pro-1 iodide for nuclei (green). Yellow images are an overlay of both red and green. Actin filaments were distributed in the cortex and at the junctions of blastomeres, and in the perinuclear cytoplasm of in vivo- and in vitro-produced 2- to 4-cell-stage embryos. In vivo-produced blastocysts had more cells in the inner cell mass (ICM; E) than in vitro-produced blastocysts (F). Blastocysts had a clear actin filament net (G and H). Pb: polar body. Arrow: sperm at the zona pellucida.
FIG. 4. Confocal photographs of pig embryos produced in vitro. These embryos have either abnormal morphology (D, F, G, and H), abnormal actin filament distribution (A, B, and E), or both (C and I).