73 IMPROVEMENT OF CANINE CLONING EFFICIENCY BY OPTIMIZED DONOR CELL PREPARATION

2010 ◽  
Vol 22 (1) ◽  
pp. 195
Author(s):  
S. W. Park ◽  
Y. W. Jeong ◽  
J. J. Kim ◽  
K. H. Ko ◽  
S. H. Jeong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Electrical Stimulation ◽  
Somatic Cell ◽  
Somatic Cells ◽  
Contact Inhibition ◽  
Abnormal Development ◽  
Serum Starvation ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Cloned Dog

The Tibetan Mastiff is the oldest dog breed in the world, and it is at the edge of extinction. Li et al. (2008) believe that protection of and research on the Tibetan Mastiff is extremely urgent, yet few studies have been carried out, particularly at the molecular level. Somatic cell nuclear transfer (SCNT) is an efficient technique for the conservation of endangered animals because it can increase the number of individuals within a population. Considering the virtually unlimited value of cloned canids in critical biotechnology applications, including gene conservation of endangered canids and disease models, the effect of cell-cycle synchronization methods, including the use of cycling canine adult skin fibroblasts (CASF), on the cell-cycle stage and viability of donor nuclei was analyzed. To improve the efficiency of cloned dog production, optimal conditions of donor cells were analyzed by culture duration (Days 1, 2, 3, and 4), passages (2, 4, 7, 10, and 11 passages) and mitotic regulator Plk-1/-4 gene expression. Simerly et al. (2003) reported that the depletion of microtubule motors and centrosomal proteins during enucleation of SCNT procedures caused abnormal development of SCNT embryos. We therefore analyzed Plk-1/-4-induced centriole biogenesis in CASF at different passages of donor cells. In this study, somatic cells were collected from a purebred 9-month-old male Mastiff and an 11-month-old female mastiff. In vivo-matured oocytes were retrieved from outbreed dogs by operation. Cycling cells cultured at Day 4 showed a similar effect to that of cells that were artificially synchronized (contact inhibition or serum starvation). It was also confirmed that fresh and short-term culture (<5 passages) resulted in fewer harmful effects and the same cell viability as control cells, using proliferation assays and expression levels of Plk-1/-4 genes. Therefore, 4 passage-cycling cells at Day 4 were used as donor cells of SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then simultaneously fused/activated with 2 DC pulses of 1.9 kV cm-1 for 30 s of electrical stimulation. Finally, 224 embryos were transferred to 16 naturally synchronized recipients. As a result, we were able to use somatic cells collected from both female and male Tibetan Mastiffs to produce 10 female and 6 male mastiffs. Moreover, one surrogate delivered a quartet of identical cloned female Tibetan Mastiffs puppies; each of 3 surrogates also delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. In conclusion, the present study shows that (1) cell-cycle synchronization of donor cells by serum starvation/contact inhibition is not required, (2) Plk-1/-4 mRNA can be used to select the donor cells, (3) electrical stimulation alone is sufficient for the activation of SCNT embryos for the production of SCNT cloned dogs, and (4) the cloned dog delivery efficiency (7.1%) was threefold higher than in previous reports. SWP and YWJ contributed equally to this work. WSH was corresponding author and SHH was co-corresponding author.

Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Ok Jae Koo ◽  
Mohammad Shamim Hossein ◽  
So Gun Hong ◽  
Jose A. Martinez-Conejero ◽  
Byeong Chun Lee
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Contact Inhibition ◽  
Population Doubling ◽  
Serum Starvation ◽  
Population Doubling Time ◽  
Cell Cycle Synchronization ◽  
Significant Difference

SummaryCycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents – dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) – on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

59 SYNCHRONIZATION OF CELL CYCLE STAGE OF BUFFALO (BUBALUS BUBALIS) FETAL FIBROBLAST CELLS BY DIFFERENT TREATMENTS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
N. L. Selokar ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
M. Muzaffar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bubalus Bubalis ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Cloning Efficiency ◽  
Fetal Fibroblast ◽  
Cell Cycle Stage ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Major Factors

Cell cycle stage of donor cells significantly influences the cloning efficiency during SCNT. Donor cells in G1/G0 stage have better capability to undergo nuclear reprogramming following transfer to an unfertilized oocyte. The lack of availability of cells synchronized at G1/G0 stage is one of the major factors limiting cloning efficiency in buffalo. The aim of this study was to compare the efficacy of various methods for cell cycle synchronization of buffalo fetal fibroblast cells for SCNT. Cells isolated from fetus, 2 to 3 months old, were cultured in DMEM + 10% FBS. The primary culture was sub-cultured 8 to 10 times. For cell cycle synchronization, the cells were cultured to 1) 60 to 70% confluence (controls), 2) 60 to 70% confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (serum starved), 3), full confluence followed by culture for additional 3 to 5 days (full confluent), 4) full confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (full confluent+serum starved) and 5) 60 to 70% confluence followed by treatment with roscovitine (10, 20, or 30 μM) for 24 h. The synchronization efficiency was examined by propidium iodide staining followed by analysis of DNA content using flow cytometry and the data were analysed by 1-way ANOVA followed by Fisher’s l.s.d. test after arcsine transformation. The percentage of cells in G0/G1 phase of cell cycle was significantly higher (P < 0.05) in the full confluent+serum starved and roscovitine treated (20 or 30 μM) groups than that in the full confluent group and that treated with 10 μM roscovitine which, in turn, was higher (P < 0.05) than that in the serum starved and control groups. These results suggest that buffalo fetal fibroblast cells can be synchronized by roscovitine treatment or by serum starvation of fully confluent cell cultures to obtain a high proportion of cells in G0/G1 stage for SCNT. Table 1.Buffalo skin fibroblast cells at various stages following different treatments for cell cycle synchronization Supported by grant No. 1(5)/2007-NAIP from ICAR, India.

Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 111-116 ◽  
Author(s):  
XiuZhu Sun ◽  
ShuHui Wang ◽  
YunHai Zhang ◽  
HaiPing Wang ◽  
LiLi Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Statistical Difference ◽  
Treatment Time ◽  
Day Treatment ◽  
Contact Inhibition ◽  
Developmental Competence ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Cell Cycle Synchronization

SummaryThe purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 ± 2.70 versus 87.40 ± 5.13; 44.10 ± 8.62 versus 58.38 ± 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods.

scholarly journals Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming

PLoS ONE ◽  
2012 ◽  
Vol 7 (4) ◽  
pp. e28203 ◽  
Author(s):  
Mengfei Chen ◽  
Jingjing Huang ◽  
Xuejiao Yang ◽  
Bingqian Liu ◽  
Weizhong Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cells ◽  
Serum Starvation ◽  
Cell Cycle Synchronization

88 CELL CYCLE SYNCHRONIZATION OF FIBROBLASTS DERIVED FROM TRANSGENIC CLONED CATTLE EAR SKIN: EFFECTS OF SERUM STARVATION, ROSCOVITINE, AND CONTACT INHIBITION

2007 ◽  
Vol 19 (1) ◽  
pp. 161
Author(s):  
X.-Z. Sun ◽  
S.-H. Wang ◽  
Y.-H. Zhang ◽  
Y.-P. Dai ◽  
N. Li
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Contact Inhibition ◽  
Developmental Competence ◽  
G1 Phase ◽  
Serum Starvation ◽  
Becton Dickinson ◽  
Cell Cycle Stage ◽  
Cell Cycle Synchronization

Cell cycle stage plays a critical role in somatic cell nuclear transfer (SCNT), and G0/G1 stage cells are preferred nuclear donors in attempts to produce cloned livestocks. Enhancement of survivability of cloned calves by roscovitine (the cyclin-dependent kinase-2 inhibitor) has been shown (Gibbons et al. 2002 Biol. Reprod. 66, 895–900). The purpose of this study was to evaluate the effects of serum starvation, roscovitine, and contact inhibition on cell cycle synchronization at the G0/G1 stage of transgenic cloned bovine ear skin-derived fibroblasts. The cell line was established from a cloned calf expressing green fluorescent protein (GFP). Data were analyzed by using SAS (8.0) with ANOVA (SAS Institute, Inc., Cary, NC, USA). At passage 2-6, cells were cultured in Dulbecco&apos;s modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) in T-25 culture flasks from immediately after subculture until monolayer cells reached 90% confluence at 39�C, under 5% CO2 in humidified air. Then cells grown in different flasks were randomly distributed to groups: Serum starvation (SS, culture medium changed to DMEM + 0.5% FBS), roscovitine (R, cultured in DMEM + 10% FBS + 15 �M roscovitine), and contact inhibition (CI, DMEM + 10% FBS). From Day 1 to Day 5 after treatment, 3 flasks of cells from each group were subjected to fixation and staining every day, followed by determination of cell cycle stage with Becton Dickinson FACScan (Kues et al. 2000 Biol. Reprod. 62, 412–419). At least 3 replicates were performed for each determination. The results showed that in the cycling fibroblasts (50–60% confluence), 59.29% of the cells were at the G0/G1 phase. For the SS group, the proportion of G0/G1 cells was significantly higher for treatment lasting 3 days, 4 days, and 5 days than for treatment lasting 1 day or 2 days (91.5%c, 91.7%c, and 93.5%c vs. 90.1%b and 88.8%a, respectively; P &lt; 0.05). No statistical differences were observed among 3-day, 4-day, and 5-day treatments. For the R group, one-day treatment was significantly lower in synchronizing cells to the G0/G1 stage than that from Day 2 to Day 5 (86.51%a vs. 91.1%a, 90.1%a, 89.4%a, and 91.3%b, respectively; P &lt; 0.05) during which similar rates of G0/G1 phase were observed. For the CI group, an increase of G0/G1 stage cells was found after 3 days of CI (from 89.4%a for Day 1 and 90.4%ab for Day 2, to 91.4%bc for Day 3; P &lt; 0.05), which was similar to 4 days (91.6%bc) and 5 days (92.1%c) for the CI group. We also found that the efficiency of obtaining G0/G1 phase cells was lower when roscovitine was employed to synchronize the cell cycle than when the SS and CI methods were used (89.7%b vs. 91.1%a and 91.0%a, respectively; P &lt; 0.05), after analysis of the most effective treatment duration of each group. Our data indicate that in attempts to harvest adequate G0/G1-stage cells for re-cloning of transgenic cattle from fibroblasts established from ear skin of a newborn transgenic cloned calf, 3 days of treatment is enough regardless of the method (SS, R, or CI). Further research is needed to evaluate the developmental competence of embryos cloned from cells prepared by the abovementioned treatments. [a-c mean statistical differences; P &lt; 0.05.]

71 DEVELOPMENT OF CLONED DOG EMBRYOS RECONSTRUCTED WITH PRE-ACTIVATED IN VIVO OOCYTES AND DONOR FIBROBLASTS ARRESTED AT G2/M PHASE USING ROSCOVITINE

2010 ◽  
Vol 22 (1) ◽  
pp. 194
Author(s):  
H. Oh ◽  
O. J. Koo ◽  
M. J. Kim ◽  
J. Park ◽  
S. Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cells ◽  
Specific Cell ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
M Phase ◽  
Vitro Development

The coordination between the cell cycle stages of nuclear donor cells and host oocytes has a critical effect on the development of embryos produced by somatic cell nuclear transfer (SCNT). Here, we investigated (1) whether roscovitine, an inhibitor of cyclin-dependent kinases (CDK) could arrest canine somatic cells at S/G2 phase of the cell cycle; (2) whether IVM metaphase II (MII) oocyte could be induced to telophase II (TII) after activation. Last, we investigated embryo development ability of nonactivated oocytes (MII) or activated oocytes (TII) fused with somatic cells at different stages of the cell cycle. Dog fetal fibroblasts were treated with roscovitine (30 or 60 μg mL-1 at 24, 48, or 72 h) and a control group of donor cells was cultured to reach confluency. The cells were then fixed and stained with 1 mg mL-1 propidium iodide for flow cytometric analysis. For SCNT, IVM dog oocytes were obtained by flushing (approximately 72 h after ovulation) from the oviducts of oocyte donor dog (Canis familiaris) and divided into 2 groups; nonactivated oocytes (MII) and activated oocytes (TII) by 10 μg mL-1 calcium ionophore for 4 min. Following preparation of each donor cell arrested in G0 and G2/M phase, cells of G0 stage were placed into enucleated MII oocytes (MII-G0) and cells of G2/M-phase were placed into enucleated TII oocytes (TII-G2/M). After fusion by electric stimulation, the MII-G0 group was chemically activated and cultured in modified SOF medium (mSOF), and the TII-G2/M group was cultured in mSOF without activation. The embryo developmental competence was estimated by assessing in vitro development under the microscope. Data were analyzed using a statistical analysis system program. Based on flow cytometry, the frequency of cells arrested at G2/M-phase in the 30 and 60 μg mL-1 roscovitine groups was significantly higher than that in control (31.95 and 25.99% v. 19.79%, respectively), but differences were not observed between the 30 and 60 μg mL-1 roscovitine groups (P > 0.05). Also, a significant increase in the proportion of cells at G2/M-phase was observed at 48 and 72 h in both roscovitine groups compared with the group not treated with roscovitine. The proportion of cells at G2/M-phase in the 60 μg mL-1 group at 48 h and the 30 μg mL-1 group at 72 h was the highest among all treatments. For the TII-G2/M group, we injected into enucleated TII oocyte and selected a large cell that arrested at G2/M-phase in cells cultured with 60 μg mL-1 roscovitine for 48 h. For the result of in vitro development of cloned embryo from MII-G0 and TII-G2/M, TII-G2/M group (39.4 and 7.8%) showed an increased cleavage rate and development to 8 cells compared with MII-G0 (23.5 and 2.9%). In the present study, we demonstrated that, in combination with nuclear donor cells at specific cell cycle stages, MII and TII dog oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei. This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.

scholarly journals 69 PRODUCTION OF CLONED PIGS BY NUCLEAR TRANSFER OF PREADIPOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 184
Author(s):  
R. Tomii ◽  
M. Kurome ◽  
H. Ueda ◽  
S. Ueno ◽  
K. Hiruma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Annexin V ◽  
The Other ◽  
Serum Starvation ◽  
Male Adult ◽  
Differentiation Induction ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Fetal Fibroblasts

Since the first success in producing cloned pigs, donor cells have been limited to fetal fibroblasts and a few other cell types. The aim of the present study was to determine if porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (NT) in pigs. Preadipocytes established from subcutaneous adipose tissue of a male adult pig were used as nuclear donor cells. Cell cycle synchronization was carried out by serum starvation (5 days), confluency (5 days), roscovitine treatment (15 μM, 2 days), or differentiation induction by 0.5 mM 3-Isobutyl-1-methylxanthine, 0.25 μM dexamethasone, and 5 μg/mL insulin (5 days). Cell cycle synchronization and apoptosis of the donor cells were examined by flow cytometry and Annexin V staining and TUNEL. IVM oocytes were obtained from abattoir ovaries and matured in NCSU23. Donor cells were fused with the enucleated recipient oocytes by a single DC pulse of 200 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO4. Reconstructed embryos were electrically activated at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed by fixation/staining at 3 h after NT, culture for 7 days in NCSU23, and transfer to the oviducts of estrus-synchronized recipient gilts. The cells immediately entered the G0 phase by differentiation induction (92.5 ± 0.4%), with higher efficiency of synchronization than for the other methods (roscovitine: 80.3 ± 0.2%; confluency: 79.9 ± 0.3%, P < 0.05) except for serum starvation (89.8 ± 0.6%). The proportion of apoptotic cells in the differentiation group was significantly lower than the other groups (Annexin V: 7.7% vs. 15.7 to 19.3%, TUNEL: 8.3% vs. 12.8 to 14.0%, P < 0.05). Incidence of premature chromosome condensation following NT (88.0%) was as high as that observed after NT with fetal fibroblasts previously (data not shown). In vitro developmental rates of the NT embryos did not differ significantly among the cell cycle synchronization methods of the donor cells (7.2 to 10.8%). Cell number of the blastocysts was highest in the differentiation group (49.0 vs. 30.2 to 41.9, P < 0.05). Transfer of 1004 cloned embryos of the serum starvation group to 5 recipients resulted in the production of 4 live and 1 stillborn piglets from 1 recipient. Transfer of cloned embryos reconstructed of donor cells treated by differentiation induction is currently underway. These data demonstrate that preadipocytes collected from an adult pig are promising nuclear donor cells for pig cloning. This study was supported by PROBRAIN.

Effects of donor sex and different cell treatments on development of yak–bovine interspecies nuclear transfer embryos

2008 ◽  
Vol 5 (1) ◽  
pp. 55-60
Author(s):  
Liu Ying ◽  
Zhu Shi-En ◽  
Li Rong ◽  
Wang Li-Li ◽  
Wang Hai-Ping ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Cell Group ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Significant Difference ◽  
Interspecies Nuclear Transfer

AbstractThe purpose of this study was to evaluate the effects of donor sex, treatments of cell cycle synchronization and donor nuclei obtained from fresh or frozen–thawed conditions on developmental competence of yak–bovine interspecies nuclear transfer embryos. Bovine (Bos taurus) oocytes were used as recipients and yak (Bos grunniens) ear fibroblast cells were used as donors. Results indicated that the development rate of male blastocysts was higher than that of female (56.6% versus 39.5%, P<0.05), whereas cleavage and total cell number showed no difference between the two groups. No significant difference was observed in the development and quality of blastocysts with donor cells treated by serum starvation or contact inhibition, and there was no significant difference in embryo development with fresh or frozen–thawed donor cells, whereas the cleavage rate in the group of frozen–thawed cells was significantly lower than that of the fresh cell group (54.5% versus 78.2%, P<0.05). The results demonstrated that donor sex could impact the developmental competence of yak–bovine interspecies nuclear transfer embryos, whereas different treatments of cell cycle synchronization and freezing had little influence.

28 GENERATION OF REACTIVE OXYGEN SPECIES IN BOVINE CULTURED SOMATIC CELLS AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DURING MICROMANIPULATION PROCEDURES AND EARLY IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 120 ◽  
Author(s):  
H. K. Bae ◽  
J. Y. Kim ◽  
I. S. Hwang ◽  
C. K. Park ◽  
B. K. Yang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Serum Starvation ◽  
Ros Generation ◽  
Oh Radical ◽  
Oxygen Species ◽  
Donor Cells

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in the donor cells, recipient oocytes, and somatic cell nuclear transfer (SCNT) embryos during nuclear transfer procedures. Bovine ear skin cells were classified by serum starvation, confluence, and cycling cells. Bovine metaphase II (MII) oocytes matured in vitro for 22 h and denuded by vortexing were enucleated and electrofused with serum-starved donor cells, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture for 4 h. In vitro fertilization (IVF) was performed for controls. SCNT and IVF embryos were cultured in CR1aa supplemented with 3 mg mL–1 BSA for ∼36 h. Donor cells, recipient oocytes, and SCNT embryos were stained in 10 μM dichlorohydrofluorescein diacetate (DCHFDA) or 10 μM HPF dye each for 30 min at 39°C to measure the H2O2 or ·OH radical levels after various micromanipulation steps. SCNT and IVF embryos were also stained at the 1-, 2-, and 4-cell stages after 8, 24, and 42 h of fusion or insemination, respectively. The fluorescent emissions from the samples were recorded as JPEG file using a digital camera (F5.0, 4 s) attached to a fluorescent microscope with filters at 450 to 480 nm for excitation and at 515 nm for emission. The images were analysed using ImageJ software 1.37 (NIH) by the intensity of fluorescence (pixels) in each cell (total 70 to 75 cells in each group), oocyte and embryo (total 50 to 60 eggs or embryos in each group). 4 to 7 replicates were performed for each experiment, and data were analysed by Duncan′s multiple-range tests. H2O2 and ·OH radical levels of cultured somatic cells were high in confluence group and significantly low in serum starvation group (P < 0.05). During micromanipulation, H2O2 levels in recipient oocytes and SCNT embryos were increased by enucleation (37.2 pixels), electrofusion (49.7 pixels), and activation (40.6 pixels) treatments (P < 0.05) compared to that in MII oocytes (33.1 pixels), and the level of H2O2 was extremely increased immediately after electrofusion. ·OH radical levels were significantly higher during manipulation procedures (51.6 to 55.7 pixels; P < 0.05) compared to MII oocytes. During in vitro culture, the H2O2 and ·OH radical levels of SCNT embryos were significantly higher (P < 0.05) compared to IVF embryos at 1- (32.4 v. 17.3 and 52.0 v. 29.6 pixels, respectively), 2- (27.2 v. 22.0 and 33.4 v. 26.0 pixels, respectively), and 4-cell (25.1 v. 16.5 and 26.9 v. 20.7 pixels, respectively) stages. These results suggest that the culture type of donor cells can affect the ROS generation level and the cellular stress during micromanipulation procedures also can generate the ROS in bovine SCNT embryos, which may lead the cellular damages in bovine SCNT embryos. This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008–313-F00067).

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