• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.53-59
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• 2003

For somatic cell nuclear transfer in Hanwoo, fetal fibroblast cell lines were established from 35, 50, 70 and 90-day fetuses of Korean native cattle. The sex of these fetal fibroblast cells were analyzed by PCR using Y-specific primers and confirmed that two cell lines were female and the other two cell lines were male. Karyotyping of these cell lines indicates that the chromosome numbers of fetal fibroblast cells were not affected by passage number and more than 80% of fetal fibroblast cells have normal chromosome number. To evaluate Go stage in cell cycle of fetal fibroblast cells, Western blotting was performed to detect the expression level of PCNA which is known to be expressed in all cell cycle stages except G$_{0}$ stage. Following serum starvation or confluent culture for 7 days, fetal fibroblast cells were effectively reached to G$_{0}$ stage. The cell cycle was resumed after culture of these Go stage-fetal fibroblast cells with normal medium. These results indicates that fetal fibroblast cells originated from Hanwoo were successfully isolated and culture system and induction of cell cycle of these cells were established for somatic cell nuclear transfer in Hanwoo.woo.

• Food Science of Animal Resources
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• v.43 no.6
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• pp.1017-1030
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• 2023

Fetal bovine serum (FBS), which contains various nutrients, comprises 20% of the growth medium for cell-cultivated meat. However, ethical, cost, and scientific issues, necesitates identification of alternatives. In this study, we investigated commercially manufactured serum-free media capable of culturing Hanwoo satellite cells (HWSCs) to identify constituent proliferation enhancing factors. Six different serum-free media were selected, and the HWSC proliferation rates in these serum-free media were compared with that of control medium supplemented with 20% FBS. Among the six media, cell proliferation rates were higher only in StemFlex^TM Medium (SF) and Mesenchymal Stem Cell Growth Medium DXF (MS) than in the control medium. SF and MS contain high fibroblast growth factor 2 (FGF2) concentrations, and we found upregulated FGF2 protein expression in cells cultured in SF or MS. Activation of the fibroblast growth factor receptor 1 (FGFR1)-mediated signaling pathway and stimulation of muscle satellite cell proliferation-related factors were confirmed by the presence of related biomarkers (FGFR1, FRS2, Raf1, ERK, p38, Pax7, and MyoD) as indicated by quantitative polymerase chain reaction, western blotting, and immunocytochemistry. Moreover, PD173074, an FGFR1 inhibitor suppressed cell proliferation in SF and MS and downregulated related biomarkers (FGFR1, FRS2, Raf1, and ERK). The promotion of cell proliferation in SF and MS was therefore attributed to FGF2, which indicates that FGFR1 activation in muscle satellite cells may be a target for improving the efficiency of cell-cultivated meat production.

• 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.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.365-373
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• 2000

The purpose of this study was to investigate the effects of the fusion pulses and fusion media on fusion rate and the development of embryos produced by somatic cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus and fetal fibroblast cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 38.5$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. The cumulus cell and cytoplast were fused using one pulse of 70 volts for 40$mutextrm{s}$, two pulses of 70 volts for 40$mutextrm{s}$ and one pulse of 180 volts for 15$mutextrm{s}$. The fetal fibroblast cell and cytoplast were fused using one pulse of 180 volts for 15$mutextrm{s}$ or 30$mutextrm{s}$. The cumulus cell and cytoplast were fused using mannitol and Zimmerman cell fusion medium (ZCFM) as a fusion medium. The fused embryos were activated after the fusion with 10 $\mu$M calcium ionophore for 5 min and 2 mM 6-dimethyl- aminopurine for 3 h. The nuclear transfer embryos were cultured in 500 ${mu}ell$ well of modified CR1aa supplemented with 3 mg/$m\ell$ BSA in th $\varepsilon$ four well dish cove red with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/$m\ell$ BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at 38.5$^{\circ}C$. When the cumulus cells were fused with enucleated oocytes by three different fusion pulses, one pulse of 180 volts for 15 $mutextrm{s}$ yielded the highest fusion rate and developmental rate to blastocyst among the pulses (P＜0.05). When the fetal fibroblast cells were fused with enucleated oocytes, one pulse of 180 volts for 30$mutextrm{s}$ yielded significantly higher fusion rate compared with that for 15 $mutextrm{s}$(P＜0.05). The present result indicates that the fusion rate between karyoplast and cytoplast was affected by the cell type and the optimal fusion condition was different according to cell type or size. When the fusion was conducted by the use of mannitol and ZCFM, the fusion rate was 71.2% and 65.8%, respectively. The developmental rates to blastocyst were 37.8% and 39.8%, respectively. There was no significant difference between two fusion media in the developmental rate of cumulus cell nuclear transfer embryos. These results indicate that optimal electric current should be selected according to cell type.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.87-93
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• 2003

This study was conducted to investigate the effects of embryo development by fusion condition on the nuclear transfer with brindle coated cow's ear cells. Ear cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion condition with 1.9kv/cm, 2.0kv/cm, 2.1kv/cm each 10 and 20ug duration Nuclear transfer embryo were activeted with a combination of 5ug/ml and 1.9mM 6-DMAP (4min, 4h). Fusion rate was 51∼68% range among fusion condition (1.9, 2.0, 2.1kv/cm; 10, 20us). But, cytoplasm lysis rate was increased by higher electric condition (0∼51.8% range). Each parameter's cleavage and blastocyst formation rate were 1.9kv/cm for 10us (75.8 and 19.5%), 20us (69.8 and 48.6%), 2.0kv/cm for 10us (76.9 and 20.0%), 20 us (68.5 and 40.9%), 2.1kv/cm for 10us (70.5 and 44.2%), 20 us (68.5 and 27.0%). We compared the effectiveness of cloning for between brindle coated cow's ear cells and Hanwoo fetal fibroblast cells. There was no significant differences in the fusion rate and developmental rate to the blastocyst stage. After transfer of blastocysts derived from nuclear transfer embryos, pregnancy rates of the Hanwoo fetal fibroblast cells and brindle coated cow's ear cells were checked pregnant on day 60 as assessed by ultrasonography, 40% (2/5) and 15.8% (3/19), respectively. This studies conclude that brindle coated cow's ear cells have the developmental potentiality to term by nuclear transfer. These results demonstrate that the increased the field strength was to be profitable for development of blastocyst or reduce of cytoplasm's damage than increasing the pulse duration.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.177-181
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• 2008

This study was performed to investigate the effects of hCG treatment on pregnancy and delivery rates in the Hanwoo recipients. There were significantly higher pregnancy and delivery rates in the recipients treated with hCG at 7 days after artificial insemination (p<0.05), respectively. The SCNT embryos from bovine fetal fibroblast cells were transferred into the synchronized recipients. The recipients were administered saline (n=89) or hCG (1,500 IU) (n=48) at 7 days after heat, respectively. The pregnancy rate was significantly higher in the recipients treated with hCG compared to that of saline treated group (p<0.01), however, the delivery rate was not different in both treated groups. The concentration of plasma progesterone (P4) was not different in both groups before hCG treatment, but the P4 level was increased significantly in hCG treated group after hCG injection (p<0.05). Although the pregnancy rate was very high in early stage of pregnancy, it was decreased dramatically after 50 days of pregnancy and maintained basal level. Taken together, the treatment of hCG in the SCNT recipients after day 7 of heat was effective method to increase the P4 concentration and to increase the pregnancy rate. But it did not affect directly to delivery.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.199-203
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• 2008

This study was performed to analyze the characterization of plasma hormonal levels during pregnancy in the Hanwoo recipients pregnant by artificial insemination (AI) or somatic cell nuclear transfer (SCNT) embryos. The synchronized recipients pregnant by SCNT embryos produced by Hanwoo fetal fibroblast cells (n=8) and by AI (control, n=5) were used. The plasma hormonal levels were measured by RIA (P4 and E2) and ELISA (cortisol), respectively. In control, the increase of E2 and the decrease of P4 were occurred immediately before the initiation of parturition. The expression pattern of plasma P4 was similar in both groups from 50 to 10 days before parturition, however, it did not decrease even at the expected date of labor in the SCNT recipients. The plasma cortisol was expressed a lower level during pregnancy in the SCNT recipients. But, the cortisol was increased in the cow aborted around 100 days of pregnancy (n=1). Based on these results, it can be postulated that the failure of the hormonal changes immediately before parturition in the SCNT recipients may be one of the most important reasons for a delayed parturition in clone calving.