93 EFFECT OF SEASON ON QUALITY OF OOCYTES, RESULTS OF IN VITRO MATURATION, AND SOMATIC CELL NUCLEAR TRANSFER IN SWAMP BUFFALO

2007 ◽  
Vol 19 (1) ◽  
pp. 163
Author(s):  
N. T. Uoc ◽  
F. de Rennis ◽  
N. H. Duc ◽  
L. C. Bui ◽  
N. V. Hanh ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
Reproductive Activity ◽  
Blastocyst Stage ◽  
Swamp Buffalo

Reproductive activity in swamp buffalo is characterized by a clearly demonstrated anestrus season. The aim of the present study was to evaluate season effect on the oocyte collection, in vitro maturation, and somatic cell nuclear transfer. The ovaries collected from a slaughterhouse were divided into 3 groups according to the collection period: (1) G1: from January to April; G2: from May to August, which is characterized by higher climate temperature and low reproductive activity; and G3: from September to December. Cumulus–oocyte complexes (COCs) were aspirated from follicles 2-6 mm in diameter using an 18-gauge needle, washed in HEPES-buffered TCM-199 (Sigma-Aldrich, St Louis, MO, USA), and classified following 3 different quality levels: A (with 4–6 layers of cumulus cells), B (with 2–3 layers of cumulus cells), and C (few or without cumulus cells). The oocytes of A and B categories were used for IVM in maturation media currently used in cattle (TCM-199 medium + 10% fetal bovine serum) with an increase of FSH concentration (30 �g mL-1) and estradiol-17β (3 �g mL-1). Maturation was carried out at 39�C in a water-saturated incubator, under 5% CO2 for 22 h. The oocytes were observed for the cumulus expanding and the presence of polar body (PB). The oocytes with PB were used for further enucleation and cell nuclear transfer using buffalo quiescent fibroblast cells and the technique described previously (Nguyen et al. 2000 Theriogenology 53, 235). The percentages of intact and fused oocytes as well as reconstructed embryos developed to blastocyst stage were compared for the oocytes from G1 and G2. The results indicated that the average number of good quality COCs collected per ovary for the G1, G2, and G3 period were 6.00 � 4.08 (n = 426), 2.93 � 2.55 (n = 346), and 4.78 � 1.05 (n = 445), respectively. The percentages of A and B oocytes were 62.4% (1.58 � 0.51 vs. 2.17 � 1.54), 63.2% (0.90 � 0.32 vs. 0.95 � 0.50), and 54.7% (1.12 � 0.25 vs. 1.49 � 0.53), respectively; the maturation rate was 55.08%, 56.28%, and 52.16%, respectively. There were no significant differences between G1 and G2 in the percentage of intact and fused oocytes (93.7% and 59% for G1; 100% and 60% for G2, respectively), but the rate of embryos developed to blastocyst stage was higher for oocytes from G1 (18.5% vs. 10.2%). In conclusion, in swamp buffalo, the hot season affected significantly the number of oocytes collected per animal and the subsequent results of somatic cell nuclear transfer. The optimal period for working with buffalo oocyte is from January to April. This work was aupported by a grant from the Vietnam-Italy 3AB3 Project.

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.

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 < 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 < 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.

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 24 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Shuang Liang ◽  
Zheng-Wen Nie ◽  
Jing Guo ◽  
Ying-Jie Niu ◽  
Kyung-Tae Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Dna Methyltransferases ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

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

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 112 ◽  
Author(s):  
Y. H. Choi ◽  
I. C. Velez ◽  
B. Macías-García ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Direct Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Donor Cells

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 124 ◽  
Author(s):  
F. Du ◽  
J. Xu ◽  
S. Gao ◽  
L. Y. Sung ◽  
D. Stone ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Fusion ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Full Term ◽  
Fusion Method ◽  
Nuclear Injection

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

62 DIFFERENT EFFECT OF TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF PORCINE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETICALLY ACTIVATED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 112 ◽  
Author(s):  
H. X. Wei ◽  
K. Zhang ◽  
Y. F. Ma ◽  
Y. Li ◽  
Q. Y. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Polar Body ◽  
Cell Number ◽  
Cell Numbers ◽  
First Polar Body ◽  
Electrical Fusion

Accumulating evidence suggests that trichostatin A (TSA), a histone deacetylase inhibitor, can increase the success rate of somatic cloning. The objective of this study was to investigate the effect of 50 nm TSA treatment on the development of porcine somatic cell nuclear transfer (SCNT) and parthenogenically activated (PA) embryos. Cumulus-oocyte complexes were matured in vitro. The oocytes with the first polar body (PB1) were chosen for SCNT, and the rest with PB1 or good morphology were selected for PA by a single 100-μs direct current pulse of 1.6 kV cm–1, the same parameter as for electrical fusion. GFP transgenic fetal fibroblast cells were used as nuclear donors. Data were analyzed using SPSS (13.0; SPSS, Inc., Chicago, IL, USA) with one-way ANOVA. In Experiment 1, immediately after electrical fusion and activation, the reconstructed embryos were randomly cultured in porcine zygote medium 3 (PZM3) with 10 μg mL–1 cytochalasin B (CB) and 10 μg mL–1 cycloheximide (CHX), with either 0 nm (control) or 50 nm TSA for the first 4 h, before being cultured for another 20 h in PZM3 without CB and CHX. After being washed, the embryos were cultured in PZM3 medium without TSA until Day 6 at 39.0°C, 5% CO2, 5%O2, 90% N2, and 100% humidity. The same experimental design was used for PA embryos concurrently. The results showed that there were no significant differences in blastocyst rates for SCNT or PA between control and TSA groups (23.0 ± 6.1% v. 27.9 ± 6.3%; 21.0 ± 1.0% v. 17.5 ± 3.2%, respectively). Neither were there differences in the cell numbers of blastocysts (38.3 ± 5.7 v. 32.2 ± 3.4; 42.2 ± 3.5 v. 39.0 ± 1.9, respectively). In Experiment 2, TSA treatment was prolonged to either 36 or 40 h. The blastocyst rates of SCNT were increased (7.3 ± 1.2% (0 h), 13.3 ± 2.6% (36 h), and 20.0 ± 3.3% (40 h)), whereas those of PA were decreased (46.7 ± 5.0% (0 h), 27.7 ± 6.5% (36 h), and 30.8 ± 6.3% (40 h)). The cell numbers of blastocysts from either SCNT or PA were also decreased (SCNT: 47.5 ± 3.8, 37.5 ± 2.0, and 37.1 ± 3.3; PA: 46.1 ± 1.9, 37.5 ± 1.9, and 39.3 ± 2.2; P < 0.05). In Experiment 3, the cell number and the apoptotic index of Day 5, 6, and 7 PA blastocysts treated with 0 or 50 nm TSA were determined by the terminal deoxynucleotide-mediated nick end labeling (TUNEL) assay (Table 1). The results suggested that TSA treatment probably delayed embryo development, which may be one of the reasons for the lower cell numbers in the TSA-treated group. Table 1. Cell apoptosis of PA blastocyst by TUNEL

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