76 CLONED HORSE PREGNANCIES PRODUCED USING ADULT CUMULUS CELLS

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) 24 hrs 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 24 hrs 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.