Culture of 5-day horse embryos in microdroplets for 10 to 20 days

Embryos were recovered from the uteri of mares 5 d after ovulation. Six embryos, all morulae, were placed singly in 200-ul droplets of Ham's F-12 with 10% fetal calf serum and cultured at 37 degrees C in a 5% CO(2) atmosphere. The embryos expanded to form blastocysts by the third day of culture. The blastocysts hatched from their zona pellucida, rather than the zona thinning and flaking off, as occurs in vivo. Hatching from the zona pellucida began on the third day of culture and was complete in five of six embryos by the sixth day. The embryonic capsule, normally present in equine embryos after Day 6, was not seen in the cultured embryos. The blastocysts continued to expand until 15 to 17 d of age (10 to 12 d in culture), reaching an average diameter (+/- SD) of 2052 +/- 290 um, after which time they either collapsed or contracted. These results demonstrate that equine embryos can be maintained in long-term culture in vitro, exhibiting continued growth and expansion in the absence of the embryonic capsule.