scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P<0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P<0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P<0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

48 TREATMENT OF CLONED BOVINE EMBRYOS WITH HISTONE DEACETYLASE INHIBITORS INCREASES IN VITRO DEVELOPMENT BUT NOT IN VIVO CLONING EFFICIENCY

2009 ◽  
Vol 21 (1) ◽  
pp. 124
Author(s):  
J. E. Oliver ◽  
T. Delaney ◽  
J. N. Oswald ◽  
M. C. Berg ◽  
B. Oback ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Cloning Efficiency ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Previous studies in the mouse have shown treatment of somatic cell nuclear transfer (SCNT) embryos with histone deacetylase inhibitors (HDACi) to significantly increase cloning efficiency (Kishigami S et al. 2006 BBRC 340, 183–189; van Thuan N 2007 Asian Reproductive Biology Society 4, 9 abst). Increasing histone acetylation may open donor chromatin allowing better access for oocyte cytoplasmic factors to facilitate reprogramming. Here, we determined the effect of two HDACi, Trichostatin A (TSA), and scriptaid (Sigma-Aldrich, Castle Hill, NSW, Australia), on bovine cloning efficiency. Zona-free SCNT was performed with serum starved fibroblasts fused to enucleated MII-arrested IVM oocytes. After 4 h, reconstructs were activated with 5 μm ionomycin and 2 mm 6-dimethylaminopurine (DMAP) and cultured individually in 5 μL drops of AgResearch synthetic oviduct fluid (SOF) medium. Treatment with HDACi commenced concomitant with the 4 h DMAP incubation and continued in SOF for the remainder of the treatment period; totalling either 18 or 48 h post activation (hpa). TSA concentrations examined were: 0, 5, 50, and 500 nm, with all treatments containing 0.5% DMSO (n = 1121). Following TSA treatment, increased histone (H) acetylation at lysine (K) of H4K5 was confirmed by semi-quantitative immunofluorescence at the eight-cell stage. Scriptaid concentrations examined were: 0, 5, 50, 250, and 1000 nm, with all treatments containing 0.5% DMSO during DMAP and 0.1% DMSO during IVC (n = 1059). In vitro development on Day 7 was expressed in terms of transferable quality embryos as a percentage of reconstructs cultured. Data were analyzed using a generalized linear model with binomial variation and logit link. Embryos from selected treatments were transferred singularly to recipient cows on Day 7 with pregnancy data analyzed using Fisher’s exact test. Day 7 in vitro development was significantly greater with 5 nm TSA treatment for 18 hpa compared to controls (47.1% v. 34.5%; P < 0.02). Treatment of embryos with TSA for 48 hpa had no effect at any concentration tested. In contrast, scriptaid treatment for 18 hpa had no effect in vitro, while exposure for 48 hpa at 1000 nm significantly increased the development of transferable quality embryos compared to 0 nm (44.0% v. 32.4%; P < 0.005). There was no significant difference in embryo survival rates at D150 of gestation between embryos treated with 0 or 5 nm TSA for 18 hpa (8/48 v. 10/48; 16.7% v. 20.8%). However, in vivo development at Day 150 of gestation following treatment of embryos with 1000 nm scriptaid for 48 hpa was significantly lower compared to controls (1/37 v. 6/31; 2.7% v. 19.4%; P < 0.05). Contrary to the mouse, TSA or scriptaid treatment as used in this study did not increase cloning efficiency in cattle. The use of various HDACi either alone or in combination with DNA demethylating agents may still prove beneficial for reprogramming following nuclear transfer. Supported by FRST C10X0303.

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

scholarly journals 43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
G.-S. IM ◽  
L. Lai ◽  
Z. Liu ◽  
Y. Hao ◽  
C.M. Murphy ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Electric Pulses ◽  
Developmental Rates ◽  
Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P&lt;0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P&lt;0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

136 INVESTIGATION OF LEPTIN ON DEVELOPMENT OF MOUSE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 160
Author(s):  
A. C. Taskin ◽  
A. Kocabay ◽  
M. Yucel
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Development Rate ◽  
Mouse Embryos ◽  
In Vitro Development ◽  
Development Rates ◽  
Significant Difference ◽  
Vitro Development

Leptin is a hormone-like protein of 167 amino acids. As an adipocyte-related hormone it has an important role in weight regulation and physical fitness but also has effects on reproductive and other physiological mechanisms. The aim of the present study was to investigate the effects of different concentrations of leptin added to the culture media, the quality, in vitro development rate, and in vivo rate of mouse embryos. Superovulated CB6F1 (C57BL/6XBalb/c) hybrid female mice (5–6 weeks of age) were killed ~18 to 20 h after hCG administration. Single-cell embryos were flushed from the oviducts of the dead mice with human tubal fluid medium supplemented with HEPES and 3 mg mL–1 of BSA. They were cultured in Quinn's cleavage medium supplemented with 4 mg mL–1 of BSA in 5% CO2, 37°C until reaching 2-cell stage. The 2-cell embryos were randomly divided into 4 groups and cultured in Quinn's blastocyst medium supplemented with 4 mg mL–1 BSA + 0, 10, 50, and 100 ng mL–1 leptin (L0, L10, L50, and L100) in 5% CO2, 37°C until the blastocyst stage. Some of the developing blastocysts were used for differential staining for the inner cell mass and trophectoderm (TE) cells [Mallol et al. 2013 Syst. Biol. Reprod. Med. 59,117–122]. Some of them were transferred into pseudopregnant females (CD1) on the 2.5 to 3.5th days and kept until the 13.5th day of pregnancy for the in vivo development rate. The results were evaluated using one-way ANOVA with Bonferroni post-hoc test in SPSS 22.0. The P-values <0.05 were considered statistically significant. Each experiment was repeated at least 4 times. The blastocyst development rates of L0, L10, L50, and L100 were 92.57% (162/175), 97.16% (205/211), 97.80% (178/182), and 97.85% (182/186), respectively. The in vitro development rates were significantly higher in the L10, L50, and L100 compared with L0 (P < 0.05). The inner cell mass cells of L0, L10, L50, and L100 were 13.17, 14, 16, and 15.43. There was no significant difference between the groups in terms of inner cell mass cells (P > 0.05). The TE cells of L0, L10, L50, and L100 were 47, 56.4, 53.7, and 58.57, respectively. The TE numbers were significantly increased in the presence of L10 and L100 compared with L0 (P < 0.05). The in vivo development rates of L0, L10, L50, and L100 were 13.51% (5/37), 48.72% (19/39), 15.38% (6/39), and 41.03% (16/39), respectively. The in vivo development rates of L10 and L100 were significantly higher than for L0 and L50 (P < 0.05). The resorption rates of L0, L10, L50, and L100 were 10.8% (4/37), 30.8% (12/39), 12.8% (5/39), and 20.5% (8/39), respectively. There was no significant difference between the groups in terms of the resorption rates (P > 0.05). This study found that L10, L50, and L100 were supporting the development of the embryos in the in vitro culture. The L10, L50, and L100 significantly increased the total cell numbers. The L10 and L100 were particularly effective on the number of the TE cells. In conclusion, 10 and 100 ng mL–1 leptin have a positive effect on the in vitro, quality and in vivo development of the mouse embryo. Therefore, leptin seems to play an important role on the embryo development and in vivo development. Research supported by TUBITAK-113O223.

Serum starvation of bovine fetal fibroblasts prior to nuclear transfer increases in vitro development rates

1999 ◽  
Vol 51 (1) ◽  
pp. 204
Author(s):  
J.R Hill ◽  
Q.A Winger ◽  
K.L Jones ◽  
J.A Thompson ◽  
R.C Burghardt ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Serum Starvation ◽  
In Vitro Development ◽  
Development Rates ◽  
Fetal Fibroblasts ◽  
Vitro Development

139 TRICHOSTATIN A IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY A NEW ACTIVATION METHOD

2011 ◽  
Vol 23 (1) ◽  
pp. 173
Author(s):  
L. C. Sui ◽  
W. Wang ◽  
Y. S. Li ◽  
Y. L. Zhang ◽  
S. F. Ji ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Trichostatin A ◽  
Formation Rate ◽  
Activation Method ◽  
Electrical Activation ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
Vitro Development ◽  
Equal Contribution

Recently, it has been reported that a new activation method, brief exposure to cycloheximide before electrical activation, could increase development rates and reduce cell death. In our study, we allocated reconstructed SCNT embryos into 3 groups: electrical activation followed by exposure to cycloheximide (10 μg mL–1) for 4 h (ELE+CHX); exposure to cycloheximide (10 μg mL–1) for 10 min followed by electrical activation (CHX+ELE); and electrical pulse treatment alone (ELE). We found the CHX+ELE (10 min) group had a similar blastocyst formation rate and total blastocyst number with the ELE+CHX (4 h) group, and both groups could increase in vitro development compared with the ELE group. Trichostatin A (TSA), an inhibitor of histone deacetylase, has been reported to potentially enhance cloning efficiency. We examine the effect of TSA on nuclear transfer embryos produced by the CHX+ELE activation method. The reconstructed embryos were treated with 50 nM TSA for 0 and 36 h. We found that 50 nM TSA for 36 h after activation had an increased blastocyst rate compared with the control (15.35 v. 8.84%; P < 0.05), but there was no difference in cleavage rate or in total blastocyst numbers. Our data demonstrate that TSA treatment could significantly improve pig nuclear transfer embryos produced by a new activation method. S.L.C., W.W. equal contribution; Corresponding author ZH. X.R., ZH. Y.H.; Supported by NSFC (30700574), 863 (2008AA101003).

scholarly journals 36COMPARISON OF THE DEVELOPMENTAL POTENTIAL OF CAPRINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM IN VITRO AND IN VIVO MATURED OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 140
Author(s):  
Y. Echelard ◽  
E. Memili ◽  
S.L. Ayres ◽  
M. O'Coin ◽  
L.H. Chen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Reproductive Tract ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Student’S T

The objective of this study was to compare the development to the blastocyst stage of reconstructed caprine nuclear transfer (NT) embryos derived from two sources of ova. In vivo oocytes were flushed from the oviduct of superovulated donors by exposing the reproductive tract via a small ventral laparotomy. In vitro oocytes were collected from ovaries supplied by an abattoir located in Purdue, IN. Oocytes were aspirated, cultured in maturation medium (M199 +10% goat serum, 3μgmL−1 LH, 3μgmL−1 FSH and 0.22mM sodium pyruvate), and shipped overnight (38°C, air). Donor cell preparation and NT procedures were as previously reported (Behboodi et al., 2001 Theriogenology 55, 254 abst). Donor cells were transfected female fetal fibroblasts that were synchronized by 4 days of serum starvation, followed by a 10-hour exposure to medium containing 10% FCS. Oocytes were enucleated, karyoplast-cytoplast couplets were reconstructed, fused and then activated simultaneously by a single electrical pulse. Couplets containing in vitro oocytes were incubated in the presence of 5μgmL−1 ionomycin after fusion. Fused couplets were co-cultured in TCM199 with 10% FCS and oviductal epithelial cells for 8–10 days (38°C, 5% CO2). Embryos that developed in vitro to the blastocyst stage were surgically transferred to recipients. Pregnancies were confirmed by ultrasonography. One live kid was delivered on Day 150 of gestation via elective C-section. Southern blotting analysis confirmed that it was derived from the transgenic donor cell line. These experiments show that in vivo matured oocytes not only better support caprine NT embryo development to the blastocyst stage, but also can result in live birth (table). Although fusion and cleavage rates were similar in the two groups, development to the blastocyst stage was significantly higher (Student’s t-test) in the group utilizing in vivo-matured oocytes. In conclusion, this is the first live goat produced from goat NT blastocysts developed in vitro. This suggests that in vivo matured oocytes may be superior to oocytes developed in vitro for generating live animals from NT blastocysts. Table 1

39 BOVINE SOMATIC CELL NUCLEAR TRANSFER USING CUMULUS - OOCYTE COMPLEXES COLLECTED FROM THE IDENTICAL INDIVIDUAL BY OVUM PICKUP

2007 ◽  
Vol 19 (1) ◽  
pp. 138 ◽  
Author(s):  
K. Hasegawa ◽  
S. Takahashi ◽  
S. Akagi ◽  
K. Takeda ◽  
K. Imai ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Single Strand Conformation Polymorphism ◽  
Blastocyst Stage ◽  
Polymorphism Analysis ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Developmental Rates ◽  
Vitro Development

We previously produced a cloned calf by nuclear transfer (NT) using cumulus cells removed from cumulus–oocyte complexes (COCs) after IVM. If both cumulus cells and oocytes are obtained identically and individually, and can be used simultaneously for NT, the production of cloned cows will be more expedient. And the cloned offspring produced from them will not exhibit the heteroplasmic mixed mtDNAs of donor cells and recipient oocytes. In this study, we examined the developmental potential of NT embryos using cumulus–oocyte complexes (COCs) collected from cows individually by ovum pickup (OPU). The cumulus cells were removed from COCs after IVM. The cumulus cells and cumulus-free MII oocytes derived from the same cow were used as donor nuclei and recipient oocytes, respectively. NT was performed as previously described (Akagi et al. 2003 Clon Stem Cells 5, 101–108). In Experiment 1, we examined the in vitro development of NT embryos using COCs collected by OPU. The aspiration of the follicles was performed once a week consecutively for 6 weeks in 6 cows (Cows A, B, C, D, E, and F) without hormone stimulation. In Experiment 2, we examined fetal development after the transfer of NT embryos. A Japanese black cow (Cow G) was used for OPU. On Day 7, 13 NT blastocysts were transferred to 7 recipient cows. The mtDNA genotypes of the donor cow and the cloned calf were analyzed by PCR-mediated single-strand conformation polymorphism analysis as previously described (Takeda et al. 2003 Mol. Reprod. Dev. 64, 429–437). The results of Experiment 1 are summarized in Table 1. Fusion rates did not differ among individual cows. However, the developmental rates of NT embryos at the blastocyst stage varied widely among individual cows, with a range of 19 to 64%. In Experiment 2, 2 of 7 recipient cows became pregnant on Day 30. One pregnant cow aborted on Day 60, and another cow calved a healthy calf. The mtDNA genotype of the cloned calf was confirmed to be identical with that of the donor cow. These results indicate that COCs from an identical individual can be used as donor nuclei and recipient oocytes for NT in order to produce female clones with the same mtDNA as that of the donor cow. Table 1.In vitro development of NT embryos using COCs collected by OPU

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