• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.64-64
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• 2001

To facilitate the widespread application of somatic cell cloning, improvements in blastocyst production efficiency and subsequent fetal viability are required. Area where technical improvements are needed include donor cell treatments, starvation and passage numbers. This study was carried out to investigate the effect of serum-starvation and passage on the development of ear skin fibroblast cells cloned embryos. A skin biopsy was obtained from the ear of a 2-year-old Korean Hanwoo female. The cells were cultured in 10% FBS＋DMEM up to 2-3 months(up to 10 passages) and then used. In Experiment 1, the Korean bovine Ear Skin Fibroblast cells (KbESF) were either serum starved (culture in 0.05% FBS＋DMEM) or serum fed (10% FBS＋DMEM) for 4-7 days Prior to NT In Experiment 2, the KbESF cells used for nuclear transfer in these experiments were from passages 2 to 10. The development of 208 nuclear transfer (NT) embryos reconstructed from either serum starved or serum fed ear skin fibroblast was assessed. NT embryos reconstructed from serum starved and serum fed cells showed the same developmental rate (cleavage 80.16 vs. 85.37%; blastocyst 20.63 vs. 19,51%). The development of 590 nuclear transfer (NT) embryos reconstructed from passage 2 to 10 was assessed. We observed the same developmental rates for embryos derived from later Passages as compared with those embryos from early passages(blastocyst from 16.69 to 27.91%, average 20.17%). There was no significant difference between serum-fed and serum-starved donor cells. We observed no difference in developmental rates for embryos derived from 2 to 10 passages. These data show that prolonged culture and serum starvation does not affects the cloning competence of adult somatic cells.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.53-58
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• 2006

The study was conducted to evaluate the effects of culture period and fusion method on the development of somatic cell nuclear transfer (SCNT) embryos reconstituted with Korean bovine fetal fibroblast cells (KbFF) and Korean bovine adult ear skin fibroblast cells (KbESF). KbFF were isolated from a day 51 Korean cattle (Hanwoo) fetus, and KbESF were isolated from a 28 month old Hanwoo calf. The cells were cultured up to 15 weeks (passage 15) in vitro for SCNT. Chamber and electrode needles were used for comparing fusion of reconstituted eggs. The doubling times of KbFF and KbESF were 17.3 hr and 24.3 hr, respectively. The fusion and cleavage rates were significantly higher in needle group (76.1 and 81.2% respectively, P<0.05) than those in chamber group. However, the blastocyst development rate was not different between both groups. Fusion and cleavage rates of NT eggs reconstituted with KbESF did not affected by passage number, however, blastocyst rates were lower in passage $1{\sim}4$ group (21.3%) than passage $5{\sim}8$ (39.4%) and $13{\sim}15$ groups (40.4%, P<0.05). Whereas, fusion rate was lower in passage $1{\sim}4$ group (61.5%) than those of passage $5{\sim}8$(75.0%) and $13{\sim}15$ (76.8%) groups, but cleavage and blastocyst rates were similar regardless of passage number in the KbFF. The results suggest that fusion method can affect the development of SCNT embryos, whereas the long term culture up to 15 passages may not affect the development of SCNT embryos.