Colato, C., Albornoz, M., Mellano, M. L., Mellano, P. H., Mellano, J. I., Meltsas, A., Mellano, M. A., Mellano, J. C., Bordignon, V., and Baldassarre, H.
Somatic cell nuclear transfer (SCNT) has been proposed as an outstanding tool for expanding the dissemination capacity of animals of extreme genetic value, as well as for the genetic resurrection of elite animals affected by incurable disease or that died suddenly. Numbers of outstanding males of meat-specialised breeds of goats (Boer) and sheep (Dorper) recently imported into Argentina were expanded using SCNT technology. Oocytes were collected by laparoscopic ovum pickup (LOPU) from 40 Raza Criolla goats and 38 crossbreed sheep that were hormonally stimulated, as described previously (Baldassarre et al.2002 Theriogenology 57, 275). Oocyte maturation, cell transfer, fusion and activation, culture, and transfer to recipients were conducted following procedures previously described (Baldassarre et al.2003 Cloning Stem Cells 5, 279). Briefly, oocytes were matured in vitrofor 24h in TCM 199 supplemented with hormones and 10% serum, at 38.5°C and 5% CO2. 2 caprine fetal fibroblast cell lines (FF1 and FF2) were established from purebred Boer fetuses generated by selective breeding of elite animals, while a fibroblast ovine line was established from a skin biopsy from an elite Dorper ram. Cells were transferred into previously enucleated oocytes, followed by electric fusion using a single DC pulse of 1.6kVcm-1for 70μs. Finally, the reconstructed embryos were activated using ionomycin (5μM/5min) followed by cycloheximide (10μgmL-1) and cytochalasin B (7.5μgmL-1) for 4 to 5h, followed by in vitroculture in mSOF media before transfer into the oviducts of synchronized recipients within 24h after fusion. An average of 10.4 and 12.8 reconstructed embryos were transferred to each of 21 and 12 recipient goats and sheep, respectively. Pregnancy was detected and monitored for the first 3 months by transrectal ultrasound scanning. Initial pregnancy (4 recipients, 33%) was maintained from gestation Day 30 to term in sheep, while goats exhibited a dramatic drop from 9 recipients pregnant (41%) on Day 30 to only 2 (9%) giving birth. Deliveries were by elective C-section. The number of normal offspring with good postpartum survival was 2/2 in goats (100%) and 3/5 (60%) in sheep. Substantial differences were observed between the 2 cell lines used in goats, where pregnancy was 4/11 (36%) for FF1 and 5/10 (50%) for FF2 at Day 30; however, only 2 goats carrying FF2 pregnancies carried. These results are in agreement with previous reports suggesting that cell line may be the largest source of result variation in SCNT. At the time of writing this abstract these clones are ~4 months of age, healthy and growing normally (>40kg weight). To the best of our knowledge, these are the first cloned goats produced by SCNT technology in Latin America, and the second group to produce cloned sheep in the region. [ABSTRACT FROM AUTHOR]