Reproduction in Domestic Animals

Reprod Dom Anim 43 (Suppl. 2), 331–337 (2008); doi: 10.1111/j.1439-0531.2008.01181.xISSN 0936-6768Somatic Cell Nuclear Transfer in HorsesCesare Galli 1,2 , Irina Lagutina 1 , Roberto Duchi 1 , Silvia Colleoni 1 and Giovanna Lazzari 11 Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, Cremona, Italy; 2 Dip. ClinicoVeterinario, Universita` di Bologna, Cremona, ItalyContentsThe cloning of equids was achieved in 2003, several years afterthe birth of Dolly the sheep and also after the cloning ofnumerous other laboratory and farm animal species. The delaywas because of the limited development in the horse of moreclassical-assisted reproductive techniques required for successfulcloning, such as oocyte maturation and in vitro embryoproduction. When these technologies were developed, theapplication of cloning also became possible and cloned horseoffspring were obtained. This review summarizes the maintechnical procedures that are required for cloning equids andthe present status of this technique. The first step is competentoocyte maturation, this is followed by oocyte enucleation andreconstruction, using either zona-enclosed or zona-freeoocytes, by efficient activation to allow high cleavage ratesand finally by a suitable in vitro embryo culture technique.Cloning of the first equid, a mule, was achieved using anin vivo-matured oocytes and immediate transfer of the reconstructedembryo, i.e. at the one cell stage, to the recipientoviduct. In contrast, the first horse offspring was obtainedusing a complete in vitro procedure from oocyte maturation toembryo culture to the blastocyst stage, followed by nonsurgicaltransfer. Later studies on equine cloning report highefficiency relative to that for other species. Cloned equidoffspring reported to date appear to be normal and those thathave reached puberty have been confirmed to be fertile. Insummary, horse cloning is now a reproducible technique thatoffers the opportunity to preserve valuable genetics andnotably to generate copies of castrated champions andtherefore, offspring from those champions that would beimpossible to obtain otherwise.IntroductionCloning mammals by somatic cell nuclear transfer(SCNT) has become a common technology in recentyears, following the work of Wilmut and co-workers(Wilmut et al. 1997). Today, a decade later, mostdomestic and laboratory species have been cloned,including the horse (Campbell et al. 2007). Initially, infarm animals, the application of SCNT was focussed onthe multiplication of superior genotypes of high geneticvalue. Very soon, yet, it became evident that thetechnique could offer, mainly because of its peculiarfailures, an important biological model for basicresearch as well as a wealth of opportunities forbiomedical research (Hochedlinger and Jaenisch 2006).It is in this latter field that most of the potentialapplications lie today. They range from the creation ofgenetically modified large animals to the derivation ofgenetically matched embryonic stem cells derived fromcloned embryos (Rideout et al. 2002).With regard to Equids, the birth of three mule foalscloned from foetal cells (Woods et al. 2003) and onehorse foal cloned from adult somatic cells (Galli et al.2003a) were reported in 2003 and additional cloned foalshave also been documented (Lagutina et al. 2005;Hinrichs et al. 2006, 2007). The relatively late applicationof SCNT to horse reproduction is a consequence ofthe limited information available in the publishedstudies on assisted reproductive techniques in Equids,in particular oocyte maturation, activation and in vitroculture of early pre-implantation embryos. The cloningof the mule by Woods and co-workers (Woods et al.2003) relied on the use of in vivo matured oocytes thatwere transferred to the oviducts of recipient maresimmediately after nuclear transfer and activation. Incontrast, Galli (Galli et al. 2003a), Lagutina (Lagutinaet al. 2005) and Hinrichs (Hinrichs et al. 2006, 2007)have all reported cloning of horse embryos up to theblastocyst stage carried out completely in vitro.In general, few studies are available in the publishedstudies on equine in vitro embryo production and it isonly recently that reports have been published oncomplete in vitro production of equine pre-implantationembryos by means of in vitro oocyte maturation,fertilization by intracytoplasmic sperm injection (ICSI)and in vitro culture (Galli et al. 2002a; Lazzari et al.2002a; Choi et al. 2004; Hinrichs et al. 2005). Theselatter reports demonstrate the feasibility of applying acompletely in vitro procedure to obtain horse ICSIblastocysts able to establish a pregnancy (Galli et al.2002a,b; Lazzari et al. 2002a) and develop into live termoffspring (Galli et al. 2007).One additional reason for the limited development ofassisted reproduction techniques in Equids is the lack ofinterest demonstrated by the horse industry. SCNT, inparticular, has received a sceptical welcome in mostequestrian disciplines. Some of the mayor players in thefield (such as the American Quarter Horse Associationand the Jockey Club) are opposed to the use of cloningand will not register offspring produced by this technique(Church 2006).The refinement of the in vitro culture conditionssuitable for equine oocyte maturation and embryogrowth (Hinrichs et al. 2005; Galli et al. 2007), thedevelopment of adequate horse oocyte activation protocols(Lazzari et al. 2002b) and the application of apiezoelectric device (Westhusin et al. 2003) or zona-freemanipulation for embryo reconstruction (Booth et al.2001; Oback et al. 2003; Lagutina et al. 2007), have allbeen the fundamental steps for defining a successfulin vitro procedure for SCNT in the horse.Although nuclear transfer, in general, and SCNT, inparticular, are routine techniques in several laboratoriesworldwide, both in farm animals and in laboratoryspecies, they are very demanding. The major hurdle inÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag