47 EFFECT OF THE TYPE OF CYTOPLASTS, SOURCE OF KARYOPLASTS, AND ACTIVATION PROCESS ON IN VITRO DEVELOPMENT OF BOVINE CLONE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 123
Author(s):  
L. U. Ohlweiler ◽  
J. C. Mezzalira ◽  
R. P. C. Gerger ◽  
E. S. Ribeiro ◽  
F. Forell ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Fluorescent Light ◽  
Activation Process ◽  
Chi Square ◽  
In Vitro Development ◽  
Polar Bodies ◽  
M Phase ◽  
Early Zygote ◽  
Vitro Development

As the recipient cytoplast plays a key role in nuclear reprogramming after somatic cell nuclear transfer (SCNT), the aim of this study was to compare the type of cytoplast/karyoplast [metaphase II (MII) oocyte, early zygote, somatic cells] and the chemical (CA) or sperm-mediated/spontaneous activation (SA) on in vitro development of bovine SCNT embryos produced by handmade cloning (HMC). After 17 h of in vitro maturation, a group of cumulus–oocyte complexes (COCs, n = 945) was manually bisected following zona removal and segregated as enucleated (MII hemi-Cyt) or non-enucleated (MII hemi-Kar). Another group of COCs was in vitro-fertilized, and, 4 h after the onset of IVF, zona-free zygotes with 2 polar bodies (n = 490) were manually bisected under fluorescent light to obtain IVF hemi-Cyt and IVF hemi-Kar. A somatic cell (SC) culture from an adult cow was used for HMC procedures (SC Kar). In 5 replications, experimental groups were composed of: zona-intact MII oocytes (parthenote control, PG); zona-intact zygotes (IVF control); MII Cyt + MII Cyt + SC Kar (SCNT control); IVF Cyt + MII Cyt + SC Kar (G1); MII Cyt + IVF Kar (G2); IVF Cyt + IVF Kar (G3); IVF Cyt + IVF Cyt + SC Kar (G4); and MII Cyt + MII Kar (G5). Following reconstruction and electrofusion, groups G1 to G5 were further divided into 2 sub-groups each, 1 being chemically activated (ionomycin/6-DMAP) along with the control groups PG and SCNT, whereas the others were cultured to verify sperm-mediated (G1 to G4) or spontaneous (G5) activation. Embryos were in vitro-cultured in the WOW system for 7 days. Cleavage (Day 2) and blastocyst (Day 7) rates were compared by the chi-square and Fisher tests, respectively. Cleavage rates in G1-SA, G2-SA, and G3-SA were lower than in their CA counterparts, which were similar to controls (Table 1). Such decrease in cleavage in G1-SA and G2-SA may be caused by the manipulation process rather than by sperm-mediation, since the observed rates were very similar to the G5-SA group. Cleavage in G3 and G4 were also similar to controls, most likely due to the fusion of 2 sperm-activated IVF hemi-Cyt. Blastocyst rates were generally higher in CA than in SA sub-groups except for G4, for which SA benefited from 2 sperm-activated cytoplasts. The lower blastocyst yield in SA sub-groups may reflect at least 2 possible mechanisms: an increased level of heteroplasmy (G1 and G2), potentially caused by an insufficient sperm-activated IVF hemi-Cyt or by a blocking effect imposed by the M-phase-derived hemi-Cyt, and/or a disruption in karyokinetic events caused by the manipulation in sperm-activated IVF hemi-Kar (G2 and G3). In G4, both mechanisms were probably attenuated by the use of 2 sperm-activated IVF hemi-Cyt and a SC-kar, analogous to conditions in the SCNT and G5 groups. Table 1.Effect of cytoplast type and activation process on in vitro development of bovine SCNT embryos This study was supported by a grant from CAPES/Brazil.

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

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

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

scholarly journals In Vitro Development of Reconstructed Porcine Oocytes after Somatic Cell Nuclear Transfer1

2000 ◽  
Vol 63 (4) ◽  
pp. 986-992 ◽  
Author(s):  
Deog-Bon Koo ◽  
Yong-Kook Kang ◽  
Young-Hee Choi ◽  
Jung Sun Park ◽  
Sun-Kyung Han ◽  
...  
Keyword(s):  
Somatic Cell ◽  
In Vitro Development ◽  
Vitro Development

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.

159 THE EFFECT OF DIFFERENT ZWITTERIONIC BUFFERS AND PBS ON IN VITRO DEVELOPMENT AND MORPHOLOGY OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
J. De la Fuente ◽  
A. Gutiérrez-Adán ◽  
P. Beltrán Breña ◽  
S. S. Pérez-Garnelo ◽  
A. T. Palasz
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Apoptotic Cells ◽  
Bovine Embryos ◽  
Chi Square ◽  
Oh Groups ◽  
In Vitro Development ◽  
Chi Square Analysis ◽  
Vitro Development

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.

50 EFFECT OF AGGREGATION OR FUSION ON DEVELOPMENT AND CELL NUMBER OF BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING AND PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 105
Author(s):  
E. S. Ribeiro ◽  
R. P. C. Gerger ◽  
L. U. Ohlweiler ◽  
I. Ortigari Jr ◽  
F. Forell ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Chemical Activation ◽  
Primary Cultures ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development ◽  
Handmade Cloning

Cloning by somatic cell nuclear transfer has been associated with developmental abnormalities, with the level of heteroplasmy imposed by cell fusion being one of many potential determining factors. As the cytoplast exerts a key role in nuclear reprogramming, embryo aggregation is an alternative to minimize such negative effects during cloning. The aim of this study was to determine the effect of fusion of hemi-cytoplasts or aggregation of hemi-embryos on in vitro development and cell number of clone and parthenote embryos. Bovine cumulus–oocyte complexes (COCs) from slaughterhouse ovaries, after 17 h of IVM, were used for the production of parthenotes by chemical activation, and clone embryos by handmade cloning (HMC) (Vajta et al. 2003 Biol. Reprod. 68, 571–578). Following cumulus and zona removal, oocytes were manually bisected, followed by segregation of nucleated and enucleated hemi-cytoplasts by fluorescence using Hoechst stain. One or two enucleated hemi-cytoplasts were paired with an adult skin somatic cell from primary cultures (>90% confluence) and fused using a 25V AC pre-pulse, followed by a single 1.2 kV cm–1 DC pulse for 10 μs. Reconstructed clone structures and groups of zona-intact oocytes and nucleated hemi-cytoplasts were chemically activated in ionomycin and 6-DMAP. Clone and parthenote structures were in vitro-cultured in the WOW system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) for 7 days, as follows: (G1) clone embryos reconstructed by aggregation of two hemi-embryos per WOW; or (G2) one embryo (two hemi-cytoplasts + cell) perWOW; and parthenote embryos composed of (G3) zona-intact oocytes cultured in wells; or aggregation of one (G4), two (G5), three (G6), or four (G7) nucleated hemi-cytoplasts per WOW. Fusion, cleavage (Day 2), and blastocyst (Day 7) rates, evaluated on a per WOW basis, were compared by the chi-square test (8 replications). Total cell number estimated by fluorescence (Hoechst stain) in blastocysts was analyzed by the Student t-test. Fusion rates of one hemi-cytoplast + cell (G1; 275/592, 46.5%) were lower than for two hemi-cytoplasts + cell (G2; 264/337, 78.3%). Cleavage rates were lower in G1 and G4 and higher in G6 and G7 than G2 and G3. A significant linear increase in blastocyst rates was observed in G5, G6, and G7. Total cell numbers were lower in parthenotes than in clones, except in G6 and G7. The lower fusion and cleavage rates after the aggregation of two clone hemi-embryos (G1) caused nearly a 50% reduction in the overall cloning efficiency. In addition, the aggregation of parthenogenetic hemi-embryos increased cleavage and blastocyst rates and cell number. However, aggregation of hemi structures did not improve blastocyst yield or cell number on a hemi-cytoplast basis. Table 1. In vitro development of parthenote or clone bovine embryos This work was supported by funding from CAPES/Brazil.

53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 174
Author(s):  
R. Olivera ◽  
C. Alvarez ◽  
I. Stumpo ◽  
G. Vichera
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Nuclear Reprogramming ◽  
Chi Square ◽  
In Vitro Development ◽  
First Polar Body ◽  
Group 3 ◽  
Group 2 ◽  
Vitro Development

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.

55 EFFECT OF FUSION-ACTIVATION INTERVAL AND EMBRYO AGGREGATION ON IN VITRO AND IN VIVO DEVELOPMENT OF CLONED BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
F. Forell ◽  
J. C. Mezzalira ◽  
F. Zago ◽  
F. K. Vieira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Rate ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development ◽  
Handmade Cloning ◽  
Activation Intervals

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitro and in vivo development of cloned bovine embryos This study was supported by FAPESP and CAPES, Brazil.

54 IN VITRO DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY HANDMADE CLONING FROM DISTINCT CELL CULTURE CONFLUENCES AND AGGREGATION SCHEMES

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
E. S. Ribeiro ◽  
J. C. Mezzalira ◽  
L. U. Olwheiler ◽  
F. Forell ◽  
...  
Keyword(s):  
Cell Culture ◽  
Somatic Cell ◽  
Nuclear Transfer ◽  
Plateau Phase ◽  
In Vitro Development ◽  
Distinct Cell ◽  
Aggregation Scheme ◽  
Vitro Development ◽  
Handmade Cloning

The coordination of the cell cycle of the donor nucleus and the recipient cytoplasm is thought to be one of the major essential factors needed for successful development of cloned embryos and offspring from somatic cell populations. Cell cycle synchronization protocols used for somatic cell nuclear transfer (SCNT) vary in preference among groups, with the confluence inhibition by contact appearing to be one of the most widely used methods today. However, the relationship between the level of cell confluence in a culture dish at or near the plateau phase of growth and blastocyst yield following cloning by SCNT still needs to be better characterized. The aim of this study was to examine the effect of 3 distinct cell culture confluences before nuclear transfer and embryo aggregation on in vitro development of clone bovine embryos. In vitro-matured bovine COC were used for the production of clone embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells 11). Oocytes were manually bisected following cumulus and zona removal. After selection of hemi-cytoplasts by DNA staining, 1 (50%) or 2 (100%) enucleated hemi-cytoplasts were paired with an adult Nelore skin somatic cell and then electrofused (15 V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs). Cells were selected from 1 out of 3 distinct culture confluences: (1) 70 to 80%; (2) 80 to 90%; and (3) >90%; assessed by morphological evaluation before the SCNT procedure. Reconstructed clone embryos and groups of zona-intact oocytes (parthenote controls) were activated in ionomycin and 6-DMAP. Clone embryos (100%) and hemi-embryos (50%) reconstructed from the 3 groups underwent IVC in the well of the well (WOW) system for 7 days, allocating 1 embryo (1 × 100%) or aggregating two hemi-embryos (2 × 50%) per WOW. After 11 replications, cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, were compared using the chi-square test. Results are summarized in Table 1. Cleavage rates were similar for all groups. The aggregation scheme did not appear to have influenced, either positively or negatively, the developmental outcome to the blastocyst stage. However, blastocyst rates increased nonlinearly (7.0, 17.5, and 29.4%) with the increase in cell confluence. A highly confluent cell culture has already been shown to have a greater proportion of cells in G0/G1 than cycling cells at the log phase (>91% v. 59%; Sun et al. 2008 Zygote 16, 111-116). However, blastocyst development in this study was lower than anticipated for cells in the early plateau phase (70 to 80%), when predicting such development based on the expected G0/G1 proportion in that cell population. Table 1.In vitro development of bovine cloned embryos from distinct cell culture confluences and aggregation scheme This study was supported by FAPESP and CAPES/Brazil.

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