Culture of equine trophoblastic vesicles in vitro

Trophoblastic vesicles have been used to study early embryonic development and maternal recognition of pregnancy in domestic animals. The purpose of this study was to characterize the formation of trophoblastic vesicles from Day-12 to Day-16 equine conceptuses. Conceptuses (n = 19) were collected nonsurgically from mares, the capsule was removed, and the conceptus (trophoblast and inner cell mass) was dissected into 2- to 4-mm fragments. Conceptus fragments were cultured in either Ham's F10 (HF10) or Minimum Essential Media (MEM) with 10% fetal bovine serum (FBS) in 24-well plates. Plates were incubated at 37 degrees C in a humidified atmosphere of 90% N(2), 5% O(2), and 5% CO(2) and were examined at 48 and 96 h for the number and diameter of trophoblastic vesicles formed. There was no significant difference (P > 0.1) between HF10 and MEM in the diameter of trophoblastic vesicles at 48 or 96 h of culture. There was, however, a significant increase (P < 0.01) in the number of trophoblastic vesicles per conceptus between 48 and 96 h of culture for HF10 and MEM. The mean diameters of trophoblastic vesicles after 96 h in culture were 396.4 +/- 19.9 mum and 415.0 +/- 12.0 mum for HF10 and MEM, respectively. Histologic sections of trophoblastic vesicles revealed a bilaminar structure consistent with the presence of trophectoderm and endoderm. Squash preparations of trophoblastic vesicles contained cells similar to those recognized in squash preparations of fresh conceptuses. Areas of increased cell density that resembled the inner-cell mass were seen in both fresh trophoblastic vesicles and in sections of trophoblastic vesicles. Equine trophoblastic vesicles may be useful to further the study of early embryonic development and pregnancy recognition in mares.