32 FIRST SHEEP CLONES BORN IN SOUTH AMERICA

Abstract

Since the birth of the first animal produced by nuclear transfer using adult somatic cells (Wilmut et al. 1997 Nature 385, 810–813), cloning has been used in many species. Although sheep were the first species to be cloned, the limited data available from different research groups indicate sheep to be among the lowest in terms of efficiency (0.5–2%), mainly due to high losses during pregnancy and the postnatal period. Since there were no previous reports on cloning of small ruminants in South America, the aims of the present study were to perform a larger scale sheep cloning program to evaluate the efficiency under local conditions in Argentina and describe the main postmortem findings observed in fetuses and extraembrionary membranes. Male adult fibroblast cell lines from different animals were used. Matured oocytes were recovered by flushing the oviducts of synchronized and superovulated donor ewes. From 437 ewes, 4720 oocytes were recovered (10.8 oocytes/ewe). Enucleation and nuclear transfer were performed as described previously, with modifications. Cell fusion was induced by a double electrical pulse in sorbitol medium (1.20 kV cm^–1, 30 μs). Fused couplets were activated using ionomycin for 5 min and then cycloheximide and cytochalasin B for 5 h. Embryo culture was performed at 38.5°C in a 5% O₂, 5% CO₂, 90% N₂ atmosphere, in KSOM supplemented with 2% FCS and 0.2 mm glucose on Day 3 of culture. After 6 to 7 days in culture, embryos were graded and then surgically transferred into synchronized recipients. Pregnancy rate was checked 23 days after embryo transfer and then weekly until 60 days, using a transrectal 5-MHz probe (Toshiba, Tokyo, Japan). From 70 to 150 days, a 3-MHz convex probe was used to perform monthly transabdominal scans. Most of the losses (70%) occurred within the first 90 days of pregnancy, and 15% of the fetuses were aborted late in gestation. Main ultrasound findings were fetal and placental hydrops and bilateral hydronefrosis. In addition, 6 of the ewes developed hydrallantois. Ewes beyond 148 days of gestation were induced for parturition using four IM injections of betamethasone 12 h apart (3.96 mg each). If labor did not commence, a C-section was performed (9/11). Eleven cloned lambs were born; two of them are still alive (6 months). All newborns were clinically evaluated after birth and their health assessed. Of the newborns, 80% died in the first 24 h after birth; they were depressed, with severe respiratory distress that developed into acidosis. At necropsy, fetuses were found with: collapsed lungs (9/9), fatty liver (6/9), ascitis (7/9), hydronefrosis (6/9), and placental edema with partial degeneration of placentomes (8/9). The final efficiency obtained in this large-scale cloning program (0.6%) was very similar to that reported by others in a smaller trials. Many studies on gene expression of cloned embryos and placental development have partially explained why this technique has a particular low efficiency in the sheep species; therefore further studies should be done in order to improve the outcome.

Table 1. Efficiency of sheep cloning