Improvement of canine somatic cell nuclear transfer procedure G. Jang a , H.J. Oh a , M.K. Kim a , Y.H. Fibrianto a , M.S. Hossein a , H.J. Kim a,b , J.J. Kim a , S.G. Hong a , J.E. Park a , S.K. Kang a , B.C. Lee a, * a Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea b Haemaru Small Animal Referral Hospital, Seohyun-Dong, Bundang-gu, Sung-Nam, Kyung Gi-Do 463-050, Republic of Korea Received 8 December 2006; received in revised form 21 May 2007; accepted 8 August 2007 Abstract The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 ms, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/ cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8–16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6 h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated. # 2008 Elsevier Inc. All rights reserved. Keywords: Canine embryo; SCNT; Parthenogenetic activation; Embryo transfer; Intergeneric 1. Introduction The birth, 10 years ago, of the first cloned sheep by somatic cell nuclear transfer (SCNT) [1] has been the stimulus for further research in a vast array of related areas due to its potential applications across the fields of human health, medicine, agriculture and economics. In addition, there is a growing interest in producing cloned pet animals [2–4]. On the one hand, pet cloning is considered to be motivated by emotional reasons, but it has possible applications for basic research in devel- opmental biology and as a model for human diseases. www.theriojournal.com Available online at www.sciencedirect.com Theriogenology 69 (2008) 146–154 * Corresponding author. Tel.: +822 880 1269; fax: +822 873 1269. E-mail address: bclee@snu.ac.kr (B.C. Lee). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.08.022