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 viab ility. The present study was conducted to examine the morphological charact eristics and the relationship between actin filament organization and morph ology of porcine embryos produced in vitro and in vivo, In vitro-derived em bryos were produced by in vitro maturation, in vitro fertilization (IVF), a nd in vitro development, In vivo-derived embryos were collected from insemi nated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived emb ryos (less than or equal to 8-cell stage)collected 12-48 h after IVF were s eparately fixed, stained by orcein, and examined under phase contrast micro scopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 7 4% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blast omere) 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 b lastocyst stages that were produced in vivo and in vitro were stained by rh odamine-phalloidin and examined by confocal microscopy. Actin filaments wer e 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. How ever, only 20% of in vitro-produced embryos at 2- to 8-cell stages had peri nuclear actin filaments in all blastomeres, Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin fil aments in some blastomeres. Fragmentation and binucleate blastomeres were n ot 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 incl uded in the culture medium, it prevented 2- to 4-cell-stage embryos from de veloping to the blastocyst stage. These results indicate that abnormal acti n filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture condition s that mediate normal actin filament distribution may result in an improvem ent in embryo quality.