Nuclear transfer (NT) is one of the most advanced technologies to increase animal efficiency in livestock production. Re-cloning can be utilized to investigate more effective methods for agricultural, biological, and medical research. However, few studies have been undertaken on re-cloning from cloned animals. The present study was conducted to examine some factors affecting in vitro development of re-cloned embryos and pregnancy by somatic cell nuclear transfer (SCNT). Ear fibroblast cells as karyoplast donors were isolated from a cloned Korean goat, Jinsoonny, 3 weeks after birth and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. Recipient oocytes were surgically collected by flushing the oviducts at 35 h after hCG injection from FSH-stimulated goats. The zonae pellucidae of the oocytes were partially drilled using a laser system and each somatic cell was individually transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at 39�C in an atmosphere of 5% CO2, 5% O2, 90% N2 for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients. Estrous synchronization was induced by 10 days of treatment with a CIDR and a single injection of PGF2�. Pregnancy was diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion and cleavage rates of re-cloned oocytes were 87.5% (182/208) and 56.0% (102/182), respectively. A total of 175 re-cloned embryos were transferred into 28 recipients. Eleven (39.3%) and 4 recipients (14.3%) were confirmed pregnant on Days 21 and 60, respectively. In comparison of pregnany rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were confirmed pregnant on Days 21 and 63, while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. There were no differences in pregnancy rate when the recipients were in estrus within 12 h of the donors (40 to 60%). However, the pregnancy rate was significantly decreased when the difference was greater than 24 h (0 to 35%; P << 0.05). Re-cloning can be used for many purposes, and synchronization between donors and recipients may be an important factor for further success of nuclear transfer.
Somatic cell nuclear transfer: failures, successes and the challenges ahead
