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Effect of cell‐free synchronous uterine flushings and microsurgery on the development of porcine embryos in vitro

Experiment I was designed to determine if cell‐free synchronous uterine flushings contain an embryotoxic substance that is normally screened by the intact zona pellucida. Sixty 4‐cell embryos were allocated to three treatment groups: (1) control embryos (n = 20) were cultured in Modified Kreb's Ringer Bicarbonate medium + 10% bovine calf serum (mKRB‐BCS), (2) UF embryos (n = 20) were cultured in 80% mKRB‐BCS + 20% sterile dialyzed uterine flushings (UF), (3) MicroUF embryos (n = 20) received a microsurgical incision in the zona pellucida and were cultured in 80% mKRB‐BCS + 20% UF. Following 72 h in culture at 37°C under a 90% N2, 5% CO2, and 5% O2 atmosphere, the number of nuclei/embryo and the incidence of protrusion of the trophoblast through the zona pellucida (PTZ) were recorded. Addition of UF had no effect on embryo development. A greater (P > .005) proportion of MicroUF embryos exhibited PTZ as compared to UF and control embryos. Experiment II was devised to further characterize the occurrence of PTZ in Micro porcine embryos. Thirty‐three 4‐ to 10‐cell embryos and 14 morulae were distributed across two treatments: (1) control embryos (n = 16 and 6, respectively) were cultured as described in Experiment I; and (2) micro embryos were treated similarly to MicroUF embryos in Experiment I but were cultured in mKRB‐BCS only. At the onset of PTZ, embryos were immediately fixed and examined. The proportion of embryos exhibiting PTZ was greater (P < .007) for Micro versus control embryos. Both the mean time to PTZ and the number of nuclei present were greater for blastocysts cultured from control as opposed to Micro morulae (P < .0001 and P < .0002, respectively). These results indicate that (a) cell‐free synchronous uterine flushings are not detrimental to embryos with incised zonae and (b) microsurgery increases the incidence of and reduces the time to protrusion of the embryo through the zona pellucida in cultured embryos.