Production of Cloned Lambs: Transfer of Early Cleavage Stage Embryos to Final Recipients

Abstract

ABSTRACT The aim of this study was to obtain cloned lambs by somatic cell nuclear transfer. In vitro matured oocytes were enucleated and injected with serum-starved cumulus cells. After electrofusion and ionomycin treatments, reconstructed oocytes were activated by 10 m g/ml cycloheximide (CHX) for 5 h or 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h and cultured in vitro in SOF medium at 38.5°C in a humidified atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 . Cleavage rates were 18.1% (26/144) in CHX and 46.5% (140/301) in 6-DMAP groups; 11.4% of embryos developed to the morula stage in the 6-DMAP group. To investigate the developmental capacity of the embryos to become live young, early cleavage stage embryos (n= 69) were transferred to the oviducts of synchronized recipients. Five out of eight ewes (62.5%) were diagnosed as pregnant at Day 18 according to progesterone assays, and two ewes (25.0%) were diagnosed as pregnant after 45 days according to ultrasound examinations. Two healthy lambs were born by Cesarean section. In conclusion, although by using different activation protocols blastocyst stage embryos were not obtained after in vitro culture of the somatic cell nuclear transfer embryos, live young that were born after transfer of early stage embryos showed that these embryos possess developmental capacity. Key Words: sheep, somatic cell nuclear transfer, early transfer