Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2–6 after estrus. In experiment 1, in vitro-derived embryos (≤ 8-cell stage) collected 12–48 h after IVF were separately fixed, stained by orcein, and examined under phase contrast microscopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 74% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blastomere) and/or binucleation (two nuclei in one or more than one blastomere). In experiment 2, actin filament distribution of the embryos at 2-cell to blastocyst stages that were produced in vivo and in vitro were stained by rhodamine-phalloidin and examined by confocal microscopy. Actin filaments were distributed in all in vivo-derived embryos at the cell cortex, and at the joints of cells and perinucleus in 2- to 8-cell-stage embryos and in some cells of morulae and blastocysts. Actin filaments were also distributed in the cortex and at the joints of cells of all in vitro-produced embryos. However, only 20% of in vitro-produced embryos at 2- to 8-cell stages had perinuclear actin filaments in all blastomeres. Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin filaments in some blastomeres. Fragmentation and binucleate blastomeres were not observed in in vivo-derived embryos. In vivo-derived Day 5 (136.5 ± 60.4 nuclei per blastocyst) and Day 6 (164.5 ± 51.9 nuclei per blastocyst) blastocysts had significantly ( p < 0.001) more cells than in vitro-produced Day 6 blastocysts (37.3 ± 11.7 nuclei per blastocyst). In experiment 3, when cytochalasin D, an inhibitor of microfilament polymerization, was included in the culture medium, it prevented 2- to 4-cell-stage embryos from developing to the blastocyst stage. These results indicate that abnormal actin filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture conditions that mediate normal actin filament dis