90 SUCCESSFUL VITRIFICATION OF PARTHENOGENETIC PORCINE BLASTOCYSTS PRODUCED FROM DELIPATED IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 153 ◽  
Author(s):  
Y. Du ◽  
P. M. Kragh ◽  
X. Zhang ◽  
H. Yang ◽  
G. Vajta ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Survival Rates ◽  
Cumulus Cells ◽  
Cell Number ◽  
Significant Loss ◽  
Developmental Competence ◽  
Average Cell ◽  
Analogue System ◽  
Hoechst Staining

Cryopreservation of cloned porcine embryos may improve the output of somatic cell cloning considerably by alleviating logistic problems. However, the high lipid content of porcine oocytes and embryos compromises their cryotolerance. Recently a noninvasive procedure was published for delipation of porcine embryos with centrifugation but without subsequent micromanipulation (Esaki et al. 2004 Biol. Reprod. 71, 432-436). This method was applied to our present work with few modifications to compare the cryosurvival of porcine blastocysts produced from delipated vs. intact oocytes with parthenogenetic activation. In four replicates, a total of 192 oocytes were used for the experiments. After in vitro maturation for 44 h, cumulus cells were removed and oocytes were randomly distributed into two groups. For delipation, oocytes were digested with 1 mg/mL pronase in the presence of 50% cattle serum (CS) for 3 min, and washed in HEPES-buffered TCM-199 medium supplemented with 20% CS. Subsequently, 40-50 oocytes were centrifuged (12 000g, 20 min) in HEPES-buffered TCM-199 medium supplemented with 2% CS, 3 mg/mL polyvinyl alcohol and 7.5 �g/mL cytochalasin B (CB). Zonae pellucidae of both centrifuged and intact oocytes were removed completely by further digestion in 2 mg/mL pronase solution. For activation, a single direct current of 85 kV/cm for 80 �s was applied to both groups, followed by 4-h treatment with 5 �g/mL CB and 10 �g/mL cycloheximide. All embryos were then cultured in modified NCSU37 medium. Day 7 blastocysts were vitrified and warmed by using the Cryotop technique (Kuwayama et al. 2005 RBM Online 11, 300-308) at 38.5�C. Survival of vitrified blastocysts was determined according to re-expansion rates after 24 h recovery in culture medium supplemented with 10% CS. Cell numbers of reexpanded blastocysts from both groups were determined after Hoechst staining. Results were compared by ANOVA. Partial zona digestion and centrifugation resulted in successful delipation in 173/192 (90%) of oocytes. The development to blastocysts was not different between delipated and intact oocytes (28 � 7% vs. 28 � 5%, respectively; P > 0.05). However, survival rates of blastocysts derived from delipated oocytes were significantly higher than those developed from intact oocytes (85 � 6% vs. 32 � 7%, respectively; P < 0.01). There was no difference in average cell number of re-expanded blastocysts derived from either delipated or intact oocytes (36 � 7 vs. 38 � 9, respectively; P > 0.05). Our results prove that the simple delipation technique does not hamper the in vitro developmental competence of activated porcine oocytes, and improves the cryosurvival of the derived blastocysts without significant loss in cell number. Future investigations are required to prove the value of the method in an analogue system with blastocysts produced by somatic cell nuclear transfer.

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

2008 ◽  
Vol 20 (1) ◽  
pp. 113
Author(s):  
H. M. Zhou ◽  
B. S. Li ◽  
L. J. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Electrical Pulses ◽  
Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

Effect of delayed enucleation on the developmental potential of nuclear-transferred oocytes receiving adult and fetal fibroblast cells

Zygote ◽  
2002 ◽  
Vol 10 (3) ◽  
pp. 217-222 ◽  
Author(s):  
Xi Jun Yin ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Cell Number ◽  
Heart Beat ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Average Cell ◽  
Developmental Potential ◽  
Membrane Protrusions ◽  
Implantation Sites

To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer

2015 ◽  
Vol 27 (3) ◽  
pp. 544 ◽  
Author(s):  
H. S. Pedersen ◽  
Y. Liu ◽  
R. Li ◽  
S. Purup ◽  
P. Løvendahl ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Oocyte Growth ◽  
Parthenogenetic Activation ◽  
Selection Of

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer

2003 ◽  
Vol 15 (3) ◽  
pp. 179 ◽  
Author(s):  
Goo Jang ◽  
Byeong Chun Lee ◽  
Sung Keun Kang ◽  
Woo Suk Hwang
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Fertilization ◽  
Chondroitin Sulfate ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
Vitro Fertilization

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.

scholarly journals 301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 270
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Cytochalasin B ◽  
Average Rate ◽  
Polar Body ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Average Cell ◽  
Electric Impulse ◽  
Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

scholarly journals Sphingosine-1-Phosphate (S1P) analogue Phyto-sphingosine-1-Phosphate (P1P) improves the in vitro maturation efficiency of porcine oocytes via regulation of oxidative stress and apoptosis

2018 ◽  
Author(s):  
Kyu-Mi Park ◽  
Jae Woong Wang ◽  
Yeong-Min Yoo ◽  
Ji Eun Jang ◽  
Myeong Jun Choi ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Cell Number ◽  
Developmental Competence ◽  
Gene Expressions ◽  
Preimplantation Embryo Development ◽  
Animal Reproduction ◽  
Sphingosine 1 Phosphate

AbstractPhytosphingosine-1-Phosphate (P1P) is a signaling sphingolipid regulating various physiological activities. Yet, little is known of the effect of P1P in the context of reproduction. As such, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM). Here we report the expression of S1PR1-3 among P1P receptors (S1PR1-4) in cumulus cells and oocytes. When P1P was treated by concentrations 10 nM, 50 nM, 100 nM, and 1000 nM during IVM, Metaphase II rate was significantly increased in 1000 nM (=1 μM) P1P treatment group. Maturation rate improvement by P1P supplementation was only observed in the presence of EGF. Oocytes under the influence of P1P decreased intracellular ROS levels yet did not show significant differences in GSH levels. In our molecular studies, P1P treatment up-regulated gene expressions involved in cumulus expansion (Has2 and EGF), antioxidant enzyme (SOD3 and Cat), and developmental competence (Oct4) while activating ERK1/2 and Akt signaling. P1P treatment also influenced oocyte survival by shifting the ratio of Bcl-2 to Bax, while inactivating JNK signaling. We further demonstrated that oocytes matured with P1P significantly displayed not only higher developmental competence (cleavage and blastocyst formation rate), but also greater blastocyst quality (total cell number and the ratio of apoptotic cells) when activated via parthenogenetic activation (PA) and in vitro fertilization (IVF). Despite low levels of endogenous P1P found in animals, exogenous P1P was able to influence animal reproduction as shown by increased porcine oocyte maturation as well as preimplantation embryo development.

356 IMPROVED ENUCLEATION EFFICIENCY FOR PIG SOMATIC CELL NUCLEAR TRANSFER BY DENUDING OOCYTES AT 30 HOURS OF IN VITRO MATURATION

2007 ◽  
Vol 19 (1) ◽  
pp. 293 ◽  
Author(s):  
K. Song ◽  
J. Park ◽  
E. Lee
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Polar Body ◽  
Digital Camera ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Significant Difference

Oocytes for somatic cell nuclear transfer (SCNT) have to be removed from their cumulus cells before enucleation. Denuding oocytes by vortexing or repeated pipetting makes the polar body (PB) deviate from the metaphase (MII) plate, which in turn makes it difficult to remove DNA materials completely during enucleation. We hypothesized that denuding oocytes at 30 h of IVM maintains the MII plate and PB in a closer position and therefore makes it easy to enucleate. To test this hypothesis, oocytes were matured in TCM-199 supplemented follicular fluid, hormones, EGF, cysteine, and insulin for first 22 h, and in a hormone-free medium for 18 h with three modifications: (1) cumulus cells were removed from oocytes just prior to enucleation at 40 h of IVM (control), (2) oocytes were denuded at 30 h of IVM and co-cultured with their detached cumulus cells for 10 h (D+), and (3) oocytes denuded at 30 h of IVM were cultured without cumulus cells (D-). After IVM, some oocytes were stained with Hoechst 33342 and photographed by a digital camera; the distance between the MII plate and the PB were measured using an image analysis program (ImageJ 1.36; http://rsb.info.nih.gov/ij). Also, the enucleation rate after blind enucleation and the in vitro development of SCNT embryos were determined. For SCNT, oocytes were enucleated, and nuclear material from donor cells (skin fibroblasts from a miniature pig) was inserted; oocytes were then electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park et al. 2005 Zygote 13, 269-275) for 6 days. Embryos were examined for their cleavage and blastocyst formation on Days 2 and 6, respectively (the day of SCNT was designated Day 0). Data were analyzed by the GLM procedure and the least significant difference test in SAS (SAS Institute, Cary, NC, USA). The distance between the MII plate and the PB was significantly (P &lt; 0.01) shorter in D+ and D- embryos (19.4 and 18.9 �m, respectively) than in the controls (25.5 �m). Enucleation rates after blind enucleation were significantly (P &lt; 0.01) higher in D+ and D- groups (77% and 72%, respectively) than in the controls (60%). Oocyte maturation (89–91%), SCNT embryo cleavage (71–77%), blastocyst formation (4–5%), and embryo cell number (39-45 cells/embryo) were not altered by different denuding methods. The perivitelline space (PVS) increases with time during maturation and denudation, after PB extrusion markedly enhances PB deviation. It is likely that increased PVS in control oocytes enhanced PB deviation during denudation and then resulted in lower enucleation rate. In conclusion, the results of this study indicated that denuding at 30 h of IVM maintained the MII plate and the PB in a closer position and improved enucleation efficiency without impairing developmental capacity of SCNT embryos. This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

46 EFFECT OF SHORT-TERM TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 123
Author(s):  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
S. G. Hong ◽  
J. T. Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Short Term ◽  
Total Cell Number

Epigenetic reprogramming such as acetylation in somatic cell nuclear transfer (SCNT) has been known as one of problems in cloned embryos. For resolving this acetylation reprogramming, many investigators recently have reported the effect of long-term culture of post-activated SCNT embryos using trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). The objective of this study is to investigate the effect of short-term TSA treatment on in vitro developmental ability and the quality of bovine SCNT embryos. Immature oocytes were aspirated from abattoir-derived ovaries, matured in vitro for 22 h, and enucleated. A bovine fetal fibroblast was placed into the enucleated oocyte and fused by electrical stimulation. The fused couplets were activated by 4-min incubation in 10 μm ionomycin, followed by 4 h of culture in 1.9 mm 6-dimethylaminopurine with or without TSA (0, 50, or 100 nm). The SCNT embryos were subsequently cultured in modified synthetic oviduct fluid medium for 8 days. Developmental competence was assessed by blastocyst formation and total cell number. Total cell numbers were determined by staining with bisbenzimide 33342. As results, developmental competence to blastocysts was higher in 100 nm than control (36.7 v. 27.9%, P < 0.05). In blastocyst hatching rate, TSA 100 nm group (19.5%) at 8 days showed an increased pattern as opposed to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). No significant differences in two cell and morula stage were observed among treatment groups. In terms of development to hatching stage of blastocysts, TSA 100 nm group (19.5%) at 8 days has a significant effect compared to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). Total cell number of blastocysts derived from TSA 100 nm was significantly higher (P < 0.05) than that in TSA 50 nm (116 v. 100), whereas there was not significant difference between control and TSA 100 nm. In conclusion, short-term culture with high concentration of TSA improved the blastocysts formation however total cell number of blastocysts showed contradictory result. The epigenetic modification by TSA treatment on bovine SCNT needs further investigation. This study was financially supported by KOSEF (grant # M10625030005-08N250300510) and the Korean MEST, through the BK21 program for Veterinary Science.

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