Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep Irina Lagutina a , Giovanna Lazzari a , Roberto Duchi a , Paola Turini a , Irene Tessaro a , Dario Brunetti a , Silvia Colleoni a , Gabriella Crotti a , Cesare Galli a,b, * a Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy b Dip. Clinico Veterinario, Universita ` di Bologna, Via Tolara di Sopra, 50-40064 Ozzano Emilia , Bologna, Italy Abstract Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NTwith mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system. # 2006 Elsevier Inc. All rights reserved. Keywords: Nuclear transfer; Zona-free; Farm animals; Offspring 1. Introduction Cloning mammals by somatic cell nuclear transfer (SCNT) has become a common technology in recent years following the interest raised by the work of Wilmut et al. [1]. Today, after 10 years, most domestic species have been cloned including both laboratory and farm animals. Initially, in farm animals, the application SCNT was focused on the multiplication of superior genotypes of high genetic value. Very soon, however, it became evident that the technique could offer, mainly because of its peculiar failures, an important biological model for basic research as well as a wealth of opportunities for biomedical research [2]. It is in this latter field that most of the potential applications lay today. They range from the creation of genetically modified large animals to the derivation of genetically matched embryonic stem cells derived from cloned embryos [3]. The oocyte provides to date the only environment where full nuclear reprogram- ming can occur, albeit at a low rate for somatic cells, but with the information generated in these experiments new avenues of research have been opened and it is likely that www.theriojournal.com Theriogenology 67 (2007) 90–98 * Corresponding author at: Laboratorio di Tecnologie della Riproduzione, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy. Tel.: +39 0372 437242; fax: +39 0372 436133. E-mail address: cesare.galli2@unibo.it (C. Galli). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.09.011