Effects of hormone-primed oviduct epithelial cell co-culture system on swine SCNT embryo development

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of cumulus donor cells compared to the >20 cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with 20 donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

An increase in intracellular Ca2+ concentration is essential for oocyte activation. Thus most artificial oocyte-activation methods focus on increasing the Ca2+ concentration in the oocytes. Recently, full-term development was reported in mice when oocytes were activated with no increase in intracellular Ca2+ using the Zn2+ chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). During oocyte maturation, Zn2+ is responsible for Cdc25c recognition. Once attached, the Cdc25c-zinc complex dephosphorylates maturation-promoting factor (MPF)/cyclin-dependent kinase 1 (cdk1), thus activating the Cdc25c-MPF positive feedback loop and keeping the oocyte suspended in metaphase II. The TPEN can inhibit the Cdc25c-MPF positive feedback loop, indirectly giving TPEN the power to degrade MPF, allowing the oocyte to exit metaphase II. First, we tested if incubation of porcine oocytes with TPEN could induce oocyte activation. Second, we examined whether the combination of TPEN with conventional activation methods could increase the developmental potential of activated oocytes. Last, based on the results, somatic cell nuclear transfer (SCNT) embryos were further activated with the optimum condition of TPEN to produce clones to confirm developmental competence. Frequencies of blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell numbers in blastocysts were counted and compared by using the Student's t-test. Differences at P < 0.05 were considered significant. When oocytes were incubated with a high concentration of TPEN (100–250 μM) for 10 to 120 min, blastocyst formation was comparable with conventional activation methods; however, the total cell number in the blastocysts was significantly lower (31.3 ± 3.1 v. 24.8 ± 1.9). When oocytes were activated with conventional methods and then incubated with the high concentration of TPEN, embryo development was drastically decreased; no blastocyst development was achieved from TPEN-treated oocytes. Interestingly, when activated oocytes were incubated with a low concentration of TPEN (5–10 μM), surprisingly, the TPEN-treated group showed higher developmental potential compared with the control group. Specifically, the average percent blastocyst formation of TPEN-treated oocytes (5 μM for 30 min) was 27.2 ± 1.7%, but only 10.6 ± 2.5% developed to blastocyst in the control group. Moreover, the average cell number in blastocysts was significantly higher in TPEN-treated oocytes compared with the control group (33.1 ± 2.6 v. 28.2 ± 2.1, respectively). When 290 chemically activated SCNT embryos were treated with 5 μM TPEN for 30 min and transferred into a surrogate, 2 healthy piglets were born. The results indicate that incubation of oocytes with TPEN alone can activate porcine oocytes. Also, when activated oocytes are incubated with the right concentration of TPEN, it can increase embryo quality in vitro. Embryo transfer results also show that TPEN-incubated SCNT embryos are developmentally competent. Additional studies would guide us to develop more efficient way to use TPEN in the activation of SCNT embryos.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

The study’s objectives were to examine the effects of electrofusion on rabbit somatic cell nuclear transfer (SCNT) embryos, and to test melatonin as a protective agent against electrofusion damage to SCNT embryos. The levels of reactive oxygen species (ROS), the epigenetic state (H3K9me3), and the content of endoplasmic reticulum (ER) stress-associated transcripts (IRE-1 and CHOP) were measured. Melatonin was added during the preimplantation development period. The total blastocyst cell numbers were counted, and the fragmentation rate and apoptotic index were determined and used to assess embryonic development. Electrofusion increased (1) ROS levels at the 1-, 2-, 4-, and 8-cell stages; (2) H3K9me3 levels at the 2-, 4-, and 8-cell stage; and (3) the expression of IRE-1 and CHOP at the 8-cell, 16-cell, morula, and blastocyst stages. The treatment of SCNT embryos with melatonin significantly reduced the level of ROS and H3K9me3, and the expression levels of IRE-1 and CHOP. This treatment also significantly reduced the fragmentation rate and apoptotic index of blastocysts and increased their total cell number. In conclusion, the electrofusion of rabbit SCNT embryos induced oxidative stress, disturbed the epigenetic state, and caused ER stress, while melatonin reduced this damage. Our findings are of signal importance for improving the efficiency of SCNT and for optimizing the application of electrical stimulation in other biomedical areas.

Effect of porcine oviductal fluid-derived extracellular vesicle supplementation on in vitro embryonic developmental competence and the production efficiency of cloned pigs.

In this study, we determined the expression of epidermal growth factor (EGF) and its receptor (EGFr) gene, and the effect of exogenous EGF supplementation on preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were fertilized with frozen-thawed semen in vitro or reconstructed with fetal fibroblasts by SCNT. In Experiment 1, total RNA was isolated from oocytes, preimplantation SCNT, or in vitro fertilization (IVF) embryos. The expression of EGF and EGFr mRNA was determined using reverse transcription-polymerase chain reaction (RT-PCR). In SCNT and IVF embryos, the EGF mRNA was detected in oocytes, 2-cell, 4-cell, 8-cell, morulae, and blastocysts, while EGFr mRNA was detected in oocytes, 2-cell, morulae, and blastocysts. In Experiment 2, SCNT embryos at 1-cell stage were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of EGF (0.1, 1, or 10 ng/ml). Supplementing with 10 ng/ml EGF improved cleavage rate (82.8% vs. 76.8%, P<0.05), but not the rate of blastocyst formation compared to the control. At all concentrations, EGF increased (P<0.05) the total cell number in blastocysts (range 50.5-53.7 vs. 43.9). In Experiment 3, EGF (10 ng/ml) was added to NCSU-23 medium at the morula stage. The EGF did not affect blastocyst formation, total cell number in blastocysts or the ratio of inner cell mass (ICM) to total cell number. In conclusion, we demonstrated that EGF and EGFr mRNA are expressed in porcine IVF and SCNT preimplantation embryos, and that EGF increased the quality of blastocysts by increasing total cell numbers in porcine SCNT embryos.

The low full-term developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos is mainly attributed to imperfect epigenetic reprogramming in the early embryos. However, dynamic expression patterns of histone methylation involved in epigenetic reprogramming progression during porcine SCNT embryo early development remain to be unknown. In this study, we characterized and compared the expression patterns of multiple histone methylation markers including transcriptionally repressive (H3K9me2, H3K9me3, H3K27me2, H3K27me3, H4K20me2 and H4K20me3) and active modifications (H3K4me2, H3K4me3, H3K36me2, H3K36me3, H3K79me2 and H3K79me3) in SCNT early embryos from different developmental stages with that from in vitro fertilization (IVF) counterparts. We found that the expression level of H3K9me2, H3K9me3 and H4K20me3 of SCNT embryos from 1-cell to 4-cell stages was significantly higher than that in the IVF embryos. We also detected a symmetric distribution pattern of H3K9me2 between inner cell mass (ICM) and trophectoderm (TE) in SCNT blastocysts. The expression level of H3K9me2 in both lineages from SCNT expanded blastocyst onwards was significantly higher than that in IVF counterparts. The expression level of H4K20me2 was significantly lower in SCNT embryos from morula to blastocyst stage compared with IVF embryos. However, no aberrant dynamic reprogramming of H3K27me2/3 occurred during early developmental stages of SCNT embryos. The expression of H3K4me3 was higher in SCNT embryos at 4-cell stage than that of IVF embryos. H3K4me2 expression in SCNT embryos from 8-cell stage to blastocyst stage was lower than that in the IVF embryos. Dynamic patterns of other active histone methylation markers were similar between SCNT and IVF embryos. Taken together, histone methylation exhibited developmentally stage-specific abnormal expression patterns in porcine SCNT early embryos.

The purpose of this study was to improve the efficiency of somatic cell nuclear transfer (SCNT) in pigs by assessing the development, cell numbers and apoptosis when using different cell types as nuclear donors and different numbers of passages. Primary cultures of the donor cells, porcine fetal fibroblasts (pFFF) from a female fetus at ∼30 days of gestation and adult female ear skin cells (pAESC), were established in DMEM + 15% FCS. For nuclear donor, cells at different passages were cultured for 5 days until confluent. Cumulus-oocyte complexes were matured and fertilized in vitro as controls by the following methods (2000 Theriogenology 54, 787–797). Following enucleation, oocytes were reconstructed by transfer of donor cells and fusion with two DC pulses (1.4 kV/cm, 50 μs) in 0.28 M mannitol containing 0.01 mM CaCl2 and MgCl2. Eggs were then cultured in NCSU23 + 1.9 mM 6-dimethylaminopurine for 3 h. SCNT and IVF embryos were cultured in NCSU23 for 54 h and subsequently in the same medium with 5.55 mM glucose for 90 h at 38.5°C in 5% CO2 in air. In Experiment 1, when the rates of development between IVF and SCNT embryos constituted with cells at 5–7 passages were compared, no significant (P &lt; 0.05) differences were observed in the cleavage rates. The rates of blastocyst formation were significantly (P &lt; 0.05) higher in IVF than in SCNT embryos with pFFF and pAESC (21% vs. 15% and 10%), but it did not differ between SCNT embryos. Total cell numbers in IVF blastocysts (35.4 ± 12) were significantly (P &lt; 0.05) higher than in SCNT blastocysts with pFFF and pAESC (28.4 ± 8 and 26.2 ± 10, respectively). The apoptosis signal by TUNEL was initiated at Day 3 in IVF and SCNT embryos. Apoptosis rates in SCNT blastocysts with pFFF and pAESC (13.1 ± 2.5 and 16.6 ± 4.3, respectively) were significantly (P &lt; 0.05) higher than in IVF embryos (3.6 ± 1.4). As the embryos developed, the rates of apoptosis were increased. On Day 6, the rates of apoptosis in IVF (4.8%) were significantly (P &lt; 0.05) lower than those in SCNT embryos with pFFF (13.1%) and pAESC (16.6%). However, both total cell number and apoptosis in SCNT embryos with pFFF and pAESC revealed no significant differences. In Experiment 2, SCNT embryos with pFFF in different cell passages were compared for the development and apoptosis. No significant (P &lt; 0.05) differences were observed in the cleavage rates of SCNT embryos among different cell passages. The rates of blastocyst formation were significantly (P &lt; 0.05) higher in SCNT embryos with 5–7 passages than those with other numbers of passages (14% vs. 6–8%, respectively). Although total cell numbers of SCNT blastocysts did not differ among different cell passages, apoptosis rates were significantly (P &lt; 0.05) higher when the number of cell passages was increased. These results suggest that fetal fibroblasts at 5–7 passages are ideal nuclear donor cells for obtaining high-quality porcine SCNT embryos. This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.

Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.

Suppressor of variegation 3-9 homolog (Suv39h)1 and 2, Histone H3 lysine 9 trimethylation (H3K9me3)-specific methyltransferases, are mainly involved in regulating the dynamic changes of H3K9me3. Regulating Suv39h expression influences the early development of mice somatic cell nuclear transfer (SCNT) embryos, there are few reports concerning their features in domestic animals. The aim of the present study was to characterize the Suv39h function in early development of Debao porcine SCNT embryos. The global level of H3K9me3 and the expression profiles of Suv39h1/2 in porcine early embryos were analysed by immunohistochemistry and qRT-PCR methods, respectively. Their roles in cell proliferation and histone modification of Debao porcine foetal fibroblast cells (PFFs), and developmental competence of porcine SCNT embryos were investigated by shRNA technology. The methylation levels of H3K9me3 and the expression patterns of Suv39h1 and Suv39h2 were similar (p<.05), and both of them displayed higher levels in Debao porcine SCNT embryos compared with that in PA embryos. The global levels of H3K9me3 and the expressions of G9a, HDAC1 and DNMT1 were decreased by combined inhibition of Suv39h1 and Suv39h2 (p<.05), while the expression of HAT1 was increased (p<.05). Downregulation of Suv39h1/2 also promoted cell proliferation and resulted in a significant increase in the expression of CyclinA2, CyclinB and PCNA in PFFs (p<.05). Furthermore, the use of donor somatic nuclei which depleted H3K9me3 by inhibiting Suv39h1/2 expression markedly increased the cleavage rate, the blastocyst rate and the total cell number of blastocysts of Debao porcine SCNT embryos (p<.05). Altogether, the above results indicate that H3K9me3 levels and Suv39h1/2 expressions display similar patterns in porcine early embryo, and low levels of them are critical to cell proliferation of PFFs and early development of SCNT embryos.

Platelet activating factor (PAF) is known as an autocrine growth/survival factor in mammalian preimplantation embryos. This study investigated the expression of porcine PAF receptor (PAFr) mRNA and its role in porcine in vitro fertilized (IVF) or somatic cell nuclear transfer (SCNT) embryo development. The expression of PAFr mRNA in IVF or SCNT blastocysts was shown by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Semiquantitative RT-PCR and Southern blot analysis demonstrated that PAFr mRNA was expressed during preimplantation embryo development, it was highly expressed through the 2-cell to 8-cell embryo stage, and it decreased at the morula stage. PAFr mRNA expression was detected steadily in IVF embryos, whereas it was varied at the 2-cell, 4-cell, and blastocyst stages in SCNT embryos. To determine the role of PAF in IVF and SCNT embryo development, embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of PAF (0, 0.037, 0.37, 3.72, or 37.2 nM). The PAF supplement significantly increased the rate of blastocyst formation in SCNT embryos, but not in IVF embryos. The PAF supplement for the entire 168 h of culture showed significantly higher blastocyst formation in SCNT embryos. Upregulation of PAFr mRNA by PAF in SCNT embryos indicated that the embryotrophic effect of PAF was mediated through its functional receptors in SCNT embryos. In conclusion, the present study demonstrated that PAFr mRNA was expressed in porcine IVF and SCNT embryos, and that PAF supplement improved the developmental competence of SCNT embryos through its specific receptors.

Somatic cell nuclear transfer (SCNT) embryos can develop at relatively high rates during the preimplantation period; however, most of these fail after implantation. Development of extraembryonic tissue is indispensable for normal embryonic development. Hence, an abnormality of trophoblast development might be a significant factor in post-implantation lethality of SCNT embryos. A transcription factor, caudal-related homeobox 2 (Cdx2), appears to be involved in the segregation of ICM and trophectoderm (TE) in preimplantation embryos (Niwa et al. 2005 Cell 123, 917–929). Both Cdx2 and Oct3/4 are expressed in all cells at the morula stage, and then Cdx2 expression becomes restricted to the TE and Oct3/4 to the ICM as the blastocyst develops. Mouse embryos deficient in Cdx2 are able to develop to normal blastocysts but die soon after implantation, probably because of defects in the TE lineage. Moreover, dysplasia of the spongiotrophoblast layer might attribute to an abnormality of Tpbpa expression in mouse SCNT embryos (Wakisaka-Saito et al. 2006 Biochem. Biophys. Res. Commun. 349, 106–114). In this study, we examined the expression profiles of transcription factors implicated in trophoblast development in mouse SCNT embryos and intracytoplasmic sperm injection (ICSI) embryos by immunohistochemistry and real-time PCR analysis. SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998 Nature 394, 369–374). In brief, B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. After nuclear transfer, the oocytes were activated and cultured in KSOM to the morula and blastocyst stages. Immunohistochemical analysis demonstrated that in ICSI embryos Cdx2 was only partially expressed at the 8-cell stage but completely in early morulae. In contrast, in SCNT embryos, it was absent at the 8-cell stage and appeared partially at the early morula stage. Thereafter, Cdx2 expression became restricted to the TE cells in both the ICSI and the SCNT blastocysts. However, ectopic expression of Oct3/4 was observed in the TE cells of SCNT, but not in ICSI blastocysts. Real-time PCR analysis showed that at the 8-cell stage, Cdx2 was expressed in ICSI but not in SCNT embryos. In addition, the expression level of Cdx2 in SCNT embryos at the blastocyst stage was only half that in ICSI embryos (P &lt; 0.05). However, there was no significant difference in expression level of Oct3/4 between ICSI and SCNT embryos. Eomesodermin (Eomes) is also implicated in trophoblast development and its expression depends on Cdx2, BMP4, and FGF4. In SCNT embryos, the expression level of Eomes was also only half that in ICSI embryos. These results indicate that the delayed expression of Cdx2 in SCNT embryos may lead to the ectopic expression of Oct3/4 in blastocysts and, along with the limited expression of Cdx2 and Eomes, may contribute to disorders in the function of the trophoblast lineage for normal placental development. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

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 &lt; 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 &lt; 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 &lt; 0.05) compared to MII oocytes. During in vitro culture, the H2O2 and ·OH radical levels of SCNT embryos were significantly higher (P &lt; 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).

Activation treatment is one of the important factors that affect the development of somatic cell nuclear transfer (SCNT) embryos. We examined the effect of post-activation (PA) treatment on the change in donor nucleus and SCNT embryo development in pig. Cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and in fresh hormone-free medium for 18 h. After 40 h of IVM, oocytes with a polar body were enucleated, injected with a donor cell (ear skin fibroblasts bearing the human decay accelerating factor gene), electrically fused, and activated 1 h after fusion. Then, SCNT embryos were cultured in a modified NCSU-23 medium (Park et al. 2005 Zygote 13, 269–275) containing no additives (control), 5 �g mL-1 cytochalasin B (CB), 0.4 �g mL-1 demecolcine (D), or CB+D for 4 h. CB and D were prepared from stock solutions of 5 mg mL-1 CB in DMSO and 10 �g mL-1 D in Hank&amp;apos;s balanced salt solution (HBSS), respectively. After PA treatment, SCNT embryos were cultured in a modified NCSU-23 medium for 6 days. The embryos (n &amp;equals; 188, 189, 187, and 186 for control, CB, D, and CB&amp;plus;D, respectively) were examined for cleavage and blastocyst (BL) formation on Days 2 and 6, respectively (Day 0 &amp;equals; the day of SCNT). Cell number of BL was examined by counting the number of nuclei stained with Hoechst 33342 under fluorescence. To assess the nuclear structure, some of the fused oocytes were fixed at 12 h after PA and stained with aceto-orcein (n &amp;equals; 42, 44, 43, and 45 for control, CB, D, and CB&amp;plus;D, respectively). Nuclear state was classified as 1 pseudopronucleus (PPN), multi-PPN, and others. Data were analyzed by ANOVA (GLM procedure) in SAS (SAS Institute, Inc., Cary, NC, USA). PA treatment with D and CB&amp;plus;D significantly (P &amp;lt; 0.05) increased 1 PPN formation (84 and 80&amp;percnt;, respectively) compared to control and CB (62 and 64&amp;percnt;, respectively). Conversely, a higher (P &amp;lt; 0.001) rate of multi-PPN was observed in control and CB (31 and 36&amp;percnt;, respectively) than in D and CB&amp;plus;D (9 and 7&amp;percnt;, respectively). This result was in contrast with the finding in mouse that nocodazole, another microtubule depolymerizing agent, induced multi-PPN in reconstructed zygotes. Pig meiotic spindles differ at their poles from those in mice by lacking &amp;gamma;-tubulin. Absence of &amp;gamma;-tubulin in pig oocytes would make spindle dynamics more sensitive to depolymerization, which might lead to a different result in this study. Embryo cleavage (77&amp;ndash;85&amp;percnt;) was not altered by PA treatments, but BL formation was significantly (P &amp;lt; 0.05) increased by CB, D, or CB&amp;plus;D (26, 28, and 28&amp;percnt;, respectively) compared to control (16&amp;percnt;). Total cell number of BL (36&amp;ndash;40 cells/BL) was not different among groups. These results indicate that PA treatment with CB and/or D improved in vitro development of SCNT pig embryos and that D treatment effectively prevented the formation of multi-PPN. This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.