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Objective—To determine the incidence, ultrasonographic characteristics, and risk factors associated with embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. Design—Prevalence survey. Animals—159 pregnant mares. Procedure—From 1994 to 1998, mares between 11 and 40 days after ovulation with normal and abnormal embryonic development were examined ultrasonographically, and characteristics of each conceptus were recorded. Results—The incidence of abnormal embryonic development in mares characterized by formation of an embryonic vesicle without an embryo was 7/159 (4.4%) during the 5 breeding seasons. Age and breed of mare or type of semen used did not differ for mares with normal and abnormal embryonic development. The percentage of mares in which the conceptus was undersized during ≥ 1 examination was significantly higher for mares with abnormal conceptuses (5/7), compared with mares with normal conceptuses (2/147; 1.4%). The percentage of examinations during which the conceptus was undersized was significantly higher for abnormal conceptuses (12/27; 44.4%), compared with normal conceptuses (4/448; 0.9%). Conclusion and Clinical Relevance—To diagnose an embryonic vesicle without an embryo, mares should be examined by use of transrectal ultrasonography on day 25 after ovulation. When an embryo cannot be identified at that time, mares should be reexamined at intervals of 1 to 3 days until day 30. Because undersized conceptuses are more likely to be abnormal, development of undersized conceptuses should be monitored closely. (J Am Vet Med Assoc 2000;217:58–63)

We recently reported the birth of the first clone of an equine species, a mule, which was produced using a fetal fibroblast cell line (Woods GL et al., 2003 Science 301, 1063). Since the birth of the first foal, two more identical cloned mule foals have been born. All three foals were delivered spontaneously without assistance, and have been healthy and vigorous since birth. Even more recently, the birth of a horse foal cloned from an adult fibroblast cell line was reported (Touchette N, 2003 Nature 424, 635). Despite these successes, the efficiency of equine nuclear transfer (NT) continues to be very low. The objective of this study was to use NT to clone adult horses using cumulus cells. Cumulus-oocyte complexes used for NT were obtained using transvaginal ultrasound-guided follicle aspiration (TVA) 24hrs after hCG treatment; oocytes were used as cytoplasts, while cumulus cells (from one of three different mares) were used as donor cells. Cumulus cells were recovered from TVA fluid, washed two times by suspension in PB1 medium (Whittingham DG, 1974 J. Reprod. Fertil. 37, 159–162), followed by centrifugation (200g) and placement in Glasgow MEM BHK-21 containing 10% FBS. Nuclear transfer procedures were performed as described (Woods GL et al., 2003 Science 301, 1063). Immediately following NT and activation procedures, cloned embryos were surgically transferred to the oviduct of recipient mares (n=2 to 5 embryos/recipient) that had ovulated within 24hrs prior to the transfer. An initial pregnancy examination was performed between Days 14 and 16 (Day 0=surgery); subsequent examinations were then performed at approximately weekly intervals. A total of 136 follicles were aspirated in 96 mares, from which 72 oocytes were recovered (53%). Sixty-two cloned embryos were subsequently transferred to recipient mares, which resulted in 7 (11.3%) ultrasonographically-detectable pregnancies. Cumulus cells from Mare 160 tended (P=0.08) to result in more pregnancies than cumulus cells from Mare 221 (4/17 v. 1/25, respectively). All seven cloned pregnancies underwent spontaneous pregnancy loss between Days 16 and 80. An embryo-proper and heartbeat were detected in three conceptuses. Of four conceptuses in which an embryo-proper was not observed, three did not develop past Day 24; therefore, they were lost before the time at which an embryo-proper generally becomes readily apparent. One conceptus developed to Day 28, yet still failed to form an embryo-proper. There were no premonitory signs of impending embryonic loss in the conceptuses that did not develop an embryo-proper; the conceptus was simply not evident at the subsequent examination. Signs of impending embryonic loss were observed in the three conceptuses in which an embryo-proper was observed, and included: (1) loss of embryonic heartbeat, (2) disorganization of the conceptus membranes, and (3) increased echogenicity of conceptus fluids. One or more of these signs were observed in all three conceptuses prior to pregnancy loss. To our knowledge, this is the first report documenting the establishment of cloned horse pregnancies produced using adult cumulus cells.