Reproduction in Domestic Animals

Cloning in Horses 335embryo culture system. In our experience, the viabilityof cloned embryos is somewhat lower than their IVFcounterpart and for this reason more than one SCNTembryo is transferred into each recipient in our laboratory.Embryo Transfer and OffspringTo date, a few groups have reported the birth of foalsfollowing ICSI of in vitro matured oocytes, after eithersurgical transfer of early embryos to the oviduct(Cochran et al. 1998) or in vitro embryo culture to theblastocyst stage and transcervical transfer to the uterus(Li et al. 2001; Hinrichs et al. 2005; Galli et al. 2007).This has been true also for SCNT embryos where to datethree labs have reported in the scientific literature, thebirth of a dozen equids following oviduct transfer ofearly cleaving embryos (Woods et al. 2003) or uterinetransfer of blastocyst stage embryos (Galli et al. 2003a;Lagutina et al. 2005; Hinrichs et al. 2006, 2007). Ashorse cloning is entering the commercial arena, additionalanimals are currently being cloned or have beencloned by commercial companies. While one suchcompany has announced the birth of cloned foals tothe popular press, information on the methods used andtheir efficiency is not in the public domain.In our laboratory, recipient mares are examined twoto three times a week by ultrasound to determine the dayof ovulation. A total of 3–7 (mostly 5) days afterovulation, each mare receives up to four nuclear transferembryos at the blastocyst stage on day 7 or day 8 ofdevelopment by non-surgical transfer. On day 17, afterovulation, the animals are examined for pregnancydiagnosis and thereafter, are scanned weekly throughoutthe first trimester of pregnancy and later at monthlyintervals until foaling.Table 7 summarizes the results reported in thepublished studies for the transfer of SCNT equidembryos. The work of Woods and Vanderwall for muleand horse SCNT, respectively, refer to the surgicaltransfer to the recipient mare oviduct of one-cellnuclear transfer embryos. Results indicate a total ofthree mule foals born from 305 zygotes transferred, outof 21 pregnancies diagnosed (Woods et al. 2003) andno live foals from 63 zygotes transferred and 7pregnancies diagnosed (Vanderwall et al. 2004). Inour laboratory, the authors have transferred 118embryos (morulae or blastocysts), fresh or frozen, into46 recipients, obtaining 13 pregnancies with oneembryonic vesicle each and 3 live foals, i.e. an embryosurvival rate of 11% and a foaling rate of 23% (3 ⁄ 13)(Galli et al. 2003a; Lagutina et al. 2005). Hinrichs et al.(2006, 2007) reported the transfer of a total of 42blastocyst-stage embryos for 20 pregnancies and 11 livefoals. In our experience, the viability of clonedembryos is lower than for ICSI embryos. In fact,during the same period, in which the SCNT embryoswere transferred a parallel study was conducted transferringICSI embryos both from abattoir and OPUoocytes cultured under the same conditions (Galli et al.2007). Overall, from 34 embryos produced by ICSItransferred into 21 recipients, the authors obtained 13pregnancies at day 17, indicating a survival rate of38% within this study. The best result involved oocytescollected by OPU, with embryos cultured in vitro andcryopreserved, in which 9 vesicles were observed onday 17 out of 13 embryos transferred into 11 recipients.In this case, the survival rate after transfer was 69%and the subsequent foaling rate was 88% (7 ⁄ 8) ofestablished pregnancies. Therefore, results confirm thelower viability of SCNT embryos when compared withfertilized embryos as already described in other species.Pregnancy losses with equine SCNT embryos occurthroughout gestation (Table 7) as described for ruminantsbut at a much lower rate. No large offspringsyndrome has been described in equids. The femalehorse produced by Galli (Galli et al. 2003a) (Fig. 1) iscurrently 4 years old and its foal was born on the 17 thMarch 2008 and it is normal and in good health. Themale produced by Lagutina (Lagutina et al. 2005) hasproduced semen for artificial insemination and it hasconfirmed to be fertile. Altogether, these results suggestthat cloned horses are reproductively normal, as hasbeen shown in other species.Table 7. Pre- and post-implantation development of SCNT embryosreported in the published studiesAuthorNo. ofoocytesfusedNo. ofoocytescleavedNo. ofblastocystsNo. oftransferredNo. of No. ofpregnancies foalsWoods et al.(2003)Galli et al.(2003)Vanderwall et al.(2004)Lagutina et al.(2005)Hinrichs et al.(2006)Hinrichs et al.(2007)307 NA NA 305 a 21 3841 753 21 17 4 172 NA NA 62 a 7 01508 1339 101 101 9 2567 457 14 10 4 2589 480 35 26 16 7a Transferred immediately after fusion to the oviduct.Fig. 1. Prometea, the world’s first cloned horse, born in 2003, in frontof her nuclear donor who was also her surrogate motherÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag