38 GENERATION OF INTERSPECIFIC CLONED BLASTOCYSTS BY ZONA PELLUCIDA-FREE NUCLEAR TRANSFER IN WILD FELIDS

2013 ◽  
Vol 25 (1) ◽  
pp. 167 ◽  
Author(s):  
L. N. Moro ◽  
J. Jarazo ◽  
A. Sestelo ◽  
D. Salamone
Keyword(s):  
Zona Pellucida ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Total Cell ◽  
Domestic Cat ◽  
Felis Silvestris ◽  
Total Cell Number ◽  
Reconstructed Embryos ◽  
Wild Felid ◽  
Felid Species

Somatic cell nuclear transfer is an assisted reproductive technique that could help to preserve endangered species. Because it is difficult to obtain wild felid oocytes, interspecific cloning using domestic cat (DC) oocytes is an alternative to produce cloned embryos in these species. The aim of this study was to evaluate different cloning strategies in the DC (Felis silvestris catus) and to use the most efficient strategy to generate wild felid embryos by interspecific cloning. First, we evaluated 3 different cloning strategies: (1) enucleation of DC oocytes with zona pellucida (ZP) followed by fusion of a DC fibroblast that was injected into the perivitelline space (ZP-enclosed group), (2) the same enucleation procedure followed by intracytoplasmic injection of a DC fibroblast (ZPi group), and (3) enucleation of ZP-free oocytes followed by adhesion and fusion of a DC fibroblast (ZP-free group). After 2 h of nuclear reprogramming, the reconstructed embryos were activated with 5 µM ionomycin and 1.9 mM DMAP, and cultured in SOF. The ZP-free embryos were cultured in wells of the well system. Embryo development among treatment groups was compared by the Fisher exact test (P ≤ 0.05). The blastocyst rates were similar among the 3 groups: 11.1% (2/18), 11.1% (5/45), and 12.7% (9/71), for ZP-enclosed, ZPi, and ZP-free, respectively. However, the quantity of reconstructed embryos after the procedure was higher in the ZP-free clones because of a higher fusion rate (82.7 vs. 25.4%) and the use of a less-invasive technique than the injection. Moreover, the percentage of expanded blastocysts was also higher (0, 16.2, and 77.8% for ZP-enclosed, ZPi, and ZP-free, respectively). Parthenogenetic controls, with and without ZP, did not differ in blastocyst rates: 47.7% (42/88) and 49.4% (38/77), respectively. After this, the ZP-free strategy was chosen for the successive experiment. In experiment 2, the wild felid species selected for interspecific cloning were Bengal (a hybrid between Felis silvestris and Prionailurus bengalensis; FP group), cheetah (Acinonyx jubatus; AJ group), and tiger (Panthera tigris; PT group). The morula and blastocyst rates were higher in the FP group: 34.3% (36/105), 16.2% (16/99), and 17.5% (11/63) for morulae, and 33.3% (35/105), 1% (1/99), and 3.2% (2/63) for blastocysts of FP, AJ, and PT, respectively. Additionally, total cell number and the expression pattern of octamer-binding transcription factor 4 (Oct-4) were examined in the blastocysts by immunocytochemistry. The mean total cell number in DC, FP, AJ, and PT blastocysts was 177.9 ± 53, 229 ± 40, 53, and 41, respectively. All blastocysts expressed Oct-4 but in different proportions. The percentage of cells expressing Oct-4 in DC, FP, AJ, and PT blastocysts was 48, 66, 100, and 98%, respectively. In summary, ZP-free cloning was found to be an efficient technique in DC, with potential to be used in wild felid species. We also demonstrated that DC oocytes were able to reprogram cells of other genera. This is the first report of felid ZP-free cloning and also the first time that tiger and cheetah embryos were produced by interspecific cloning.

scholarly journals 26 ASSESSMENT OF CHROMOSOME ABNORMALITIES IN SHEEP PARTHENOGENETIC AND NUCLEAR TRANSFER EMBRYOS: EFFECT OF 6-DMAP AND CYCLOHEXIMIDE ON PLOIDY

2005 ◽  
Vol 17 (2) ◽  
pp. 163
Author(s):  
B. Alexander ◽  
G. Coppola ◽  
D. Di Berardino ◽  
D.H. Betts ◽  
W.A. King
Keyword(s):  
Nuclear Transfer ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Total Cell Number ◽  
Parthenogenetic Activation ◽  
Treatment Groups ◽  
Reconstructed Embryos ◽  
The Mean ◽  
Painting Probes

In current somatic cell nuclear transfer (NT) protocols, the reconstructed embryos are activated by incorporation of secondary oocyte activation compounds such as 6-DMAP or cycloheximide (CHX). The effects of these compounds on the chromosome complement of sheep NT embryos have not been studied in detail. Therefore, the aim of this study was to assess the chromosome abnormalities using sex chromosome specific probes of Day 6 blastocyst-stage sheep embryos produced from parthenogenetic activation and NT. Following 20–22 h of IVM, the oocytes were activated by electric pulsing followed by 30-min culture in cytochelasin B. They were reactivated using ionomycin (5 min) followed by 2-h culture in 6-DMAP or CHX. In contrast, NT embryos were produced using standard NT procedures using male sheep fetal fibroblasts. Reconstructed embryos were activated using the same methods described earlier. The embryos (compact morulae and blastocysts) were fixed and subjected to FISH analysis using cattle X and Y chromosome painting probes. The data were analyzed using Fisher's exact test. Of the parthenogenetic embryos (6-DMAP, n = 28; CHX, n = 32) analyzed, none of the embryos was totally haploid (X) or totally polyploid. When all of the nuclei per embryo were considered, normal (XX) genotype embryos were 6.2% and 0.0% in CHX and 6-DMAP groups, respectively. The rest of the embryos were abnormal due to mixoploidy (100% vs. 93.8%, P < 0.05) in 6-DMAP and CHX treatment groups, respectively. The abnormal nuclei per embryo ranged from 7.3% to 72.2%. The mean total cell number of parthenogenetic blastocysts was 91.2 ± 4.3 and 81.8 ± 6.2 (mean ± SE) in 6-DMAP and CHX, respectively. Among NT embryos analyzed, (6-DMAP, n = 30; CHX, n = 32) only 40.0% and 43.8% of embryos were completely normal for XY chromosomes in 6-DMAP- and CHX-treated groups, respectively. The rest of the embryos were abnormal due to mixoploidy (60.0% vs. 56.2%, P > 0.05) in 6-DMAP and CHX groups, respectively. Monosomy (XO or OY), trisomy (XXY), and tetrasomy (XXYY) were the common abnormalities detected in mixoploid embryos. The abnormal cells per embryo ranged from 3.8% to 41.8% in both treatment groups. The mean total cell number of NT blastocysts was 71.2 ± 9.8 and 63.8 ± 8.4, in 6-DMAP and CHX treatment groups, respectively. In conclusion, the 6-DMAP-treated embryos derived from parthenogenetic activation had significantly higher chromosomal abnormalities than CHX-treated embryo groups (P < 0.05). In contrast, the NT embryos derived from either 6-DMAP or CHX treatment did not show any significant difference in producing chromosomally abnormal embryos at the blastocyst stage. This study also highlights the feasibility of using bovine chromosome painting probes on ovine embryo spreads. This work was supported by NSERC, OMAFRA, and ICCS.

95 DEVELOPMENT OF PORCINE TWO-CELL EMBRYOS WITH OR WITHOUT ZONA PELLUCIDA AND SINGLE BLASTOMERES

2009 ◽  
Vol 21 (1) ◽  
pp. 148
Author(s):  
D. N. Q. Thanh ◽  
K. Matsukawa ◽  
M. Kaneda ◽  
S. Akagi ◽  
Y. Kanai ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Monozygotic Twins ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
First Polar Body ◽  
Blastocyst Quality ◽  
Single Blastomeres

In the mouse, single blastomeres of the 2-cell embryos can develop into adult mice and occasionally both separated blastomeres can give rise to twin animals (reviewed by Tarkowski AK et al. 2001 Int. J. Dev. Biol. 45, 591–596). As a preliminary study for production of monozygotic twins from porcine 2-cell embryos, we investigated the effects of removal of zona pellucida and blastomere isolation at the 2-cell stage on subsequent development of parthenogenetic embryos. Oocytes with the first polar body were parthenogenetically activated after 44 h of in vitro maturation. Stimulated oocytes were then incubated in IVC-PyrLac (IVC medium with pyruvate and lactose) according to the method reported by Kikuchi K et al. (2002 Biol. Reprod. 66, 1033–1041). After 24 to 30 h of parthenogenetic activation, equally cleaved 2-cell embryos were selected and used for the experiments. Some 2-cell embryos were then treated with pronase to remove the zona pellucida and cultured individually as zona-free 2-cell embryos having 2 blastomeres in pair (ZF group), and single blastomeres were split from ZF group and cultured separately (SB group) in V-shaped microwells. In addition, intact 2-cell embryos were cultured individually without pronase treatment as a control group. After 24 h of in vitro culture, IVC-PyrLac was replaced by IVC-Glu (IVC with glucose). The blastocyst rates on Day 6 (Day 0 was defined as the day of electrical stimulation) in control, ZF, and SB groups did not differ (47.6, 50.0, and 42.1%, respectively). Nevertheless, blastocysts derived from the ZF (28.6 ± 3.0) and SB groups (25.9 ± 1.3) had a significantly lower total cell number than that of the control group (41.7 ± 3.2; P < 0.01 by ANOVA). Although the total cell number of blastocysts originating from single blastomeres was significantly lower than that in the intact embryos, the blastocyst formation rates were not different between them. This indicated the possibility of production of monozygotic twins from porcine 2-cell embryos divided into 2 single blastomeres. However, further research is needed to improve blastocyst quality descended from single blastomeres. In conclusion, the removal of the zona pellucida had a negative influence on blastocyst quality but did not affect the development of porcine embryos to the blastocyst stage.

scholarly journals 68 EFFECT OF XENOPUS EGG EXTRACT TREATMENT OF DONOR CELLS ON PORCINE SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 192
Author(s):  
Y. Liu ◽  
O. Østrup ◽  
J. Li ◽  
G. Vajta ◽  
L. Lin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Permeabilization ◽  
Total Cell Number ◽  
Egg Extract ◽  
Donor Cells

Pretreatment of somatic cells to promote subsequent reprogramming during somatic cell nuclear transfer (SCNT) may significantly improve efficiency of the technique. The aim of this study was to evaluate the effect of Xenopus laevis egg extract pretreatment of porcine fetal fibroblast cells using different permeabilization agents prior to SCNT. Fibroblasts were permeabilized using streptolysin O (SLO; 300 ng mL-1, 30 min, 37°C) or digitonin (7 μg mL-1, 2 min, 4°C), and exposed to egg extract for 1 h or 0.5 h, respectively. Cell membranes were resealed in DMEM supplemented with 2 mM CaCl2 for 2 h. After culture for 1, 3, and 5 days (for SLO) or 3 and 5 days (for digitonin), the SLO extract-treated cells (SETC) and digitonin extract-treated cells (DETC) were used as donor karyoplasts for handmade cloning. Controls were SCNT with nontreated cells. Embryos were evaluated for cleavage rate (Day 2), blastocyst rate (Day 6), and total cell numbers of blastocysts. Statistical differences were analyzed by ANOVA. Results are summarized in Table 1. When SETC were used as donors, blastocyst rates were significantly lower compared with the controls, except when the donor cells were cultured for 3 days after treatment. Blastocysts of the latter group also had higher total cell number. With DETC as donors, blastocyst rates and total cell number of embryos at Day 6 reconstructed with cells cultured for 5 days were higher than those in other groups. Results indicate that extract treatment of the donor cells after SLO-permeabilization can give higher number of cells in cloned blastocysts but not improve overall embryo development. However, digitonin treatment for donor cell permeabilization improved both embryo development and cell number of blastocyst. The latter effect was detected only 5 days after the treatment. In conclusion, qualitative efficiency of porcine SCNT could be improved with a combined donor cell permeabilization and extract treatment. Table 1.Effect of different permeabilization agents prior to SCNT

120 SUPPLEMENTATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR DURING IN VITRO MATURATION OF PORCINE IMMATURE CUMULUS - OOCYTE COMPLEXES AND SUBSEQUENT DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 165 ◽  
Author(s):  
D. Biswas ◽  
Y.-B. Jeon ◽  
G.-H. Kim ◽  
E.-B. Jeung ◽  
S. H. Hyun
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Total Cell ◽  
Vascular Endothelial ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Endothelial Growth Factor

In the present study, pig cumulus–oocyte complexes were cultured in medium supplemented with different concentrations (0, 5, 50, and 500 ng mL–1) of vascular endothelial growth factor (VEGF), and then the maturation and intracellular glutathione (GSH) concentration of oocytes were examined. In addition, the development of oocytes matured with different concentrations of VEGF after parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT) was observed. Although the maturation rate of oocytes was not affected by VEGF concentrations (81.13 ± 2.61%, 83.93 ± 1.97%, 82.14 ± 4.03%, 75.24 ± 2.68%, respectively), the intracellular GSH concentrations of oocytes matured with 5 and 50 ng mL–1 VEGF were significantly higher (12.68 ± 0.08, 12.33 ± 0.53 pMol/oocyte, respectively) than those of oocytes matured with 0 or 500 ng mL–1 VEGF (10.19 ± 0.66, 10.54 ± 0.54 pMol/oocyte, respectively). The blastocyst formation rates after PA of oocytes matured with 5 and 50 ng mL–1 VEGF were significantly higher (58.99 ± 4.70% and 54.00 ± 1.09%, respectively) than that of oocytes matured with 0 or 500 ng mL–1 VEGF (30.15 ± 4.52%, 34.79 ± 4.01%, respectively). Total cell number of PA blastocyst after oocytes matured with 5 and 50 ng mL–1 VEGF was significantly higher (83.21 ± 4.89, 78.16 ± 6.15, respectively) than that of control and 500 ng mL–1 VEGF (56.91 ± 4.78, 55.93 ± 3.89, respectively). Similarly, the blastocyst formation rate after SCNT of oocytes matured with 5 ng mL–1 VEGF was significantly higher (14.54 ± 1.42%) than that of oocytes matured without VEGF (7.95 ± 1.44%). Total cell number of SCNT blastocyst after oocytes matured with 5 ng mL–1 VEGF was significantly higher (67.83 ± 6.56) than control (48.09 ± 5.36). Fully cumulus cell expansion was significantly higher in the 5 ng mL–1 VEGF treated group (85.37 ± 0.73%) compared with the control (58.89 ± 0.88%). In conclusion, adding 5 ng mL–1 VEGF during IVM improved the developmental potential of PA and SCNT in porcine embryos by increasing the intracellular GSH level. This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.

37 EFFECTS OF TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 99 ◽  
Author(s):  
A. E. Iager ◽  
Z. Beyhan ◽  
P. J. Ross ◽  
N. P. Ragina ◽  
K. Cunniff ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Preimplantation Embryos ◽  
Total Cell Number ◽  
Treatment Groups

Faulty epigenetic reprogramming is a likely major cause of the low success rate observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been reported that treatment of reconstructed mouse embryos with the potent histone deacetylase inhibitor, trichostatin A (TSA), results in significantly increased developmental capacity of SCNT preimplantation embryos and live offspring (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 240, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089; Kishigami et al. 2006 J. Reprod. Dev. 53, 165–170). Studies investigating similar reprogramming capabilities of TSA in bovine SCNT embryos report conflicting results (Akagi et al. 2007 Reprod. Fertil. Dev. 19, 24 abst; Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 48 abst). In this study, the effects of TSA treatment on in vitro development of bovine SCNT embryos were examined. Bovine fetal fibroblasts were cultured under contact inhibition for 2 to 5 days and used as donor cells for SCNT. Oocytes were aspirated from abattoir-derived ovaries, and matured in vitro for 18 h prior to enucleation. Reconstructed SCNT couplets were electrofused, and then activated 24 h post-maturation using 5 µm ionomycin followed by 2 mm dimethylaminopurine (DMAP) for 4 h. SCNT embryos were subjected to 0 (control; C-NT) or 50 nm TSA for 13 h post-ionomycin (hpi) TSAa-NT) or 13 hpi + 6 h starting from 40 hpi (TSAb-NT). IVF embryos were produced as an additional control. All embryos were cultured in KSOM supplemented with 3 mg mL–1 BSA for 7.5 days, with 5% FBS added on Day 3. Experiments were repeated 3 or 7 times, and data were analyzed a -way ANOVA procedure. Developmental rates to the blastocyst stage and total cell number of blastocysts were determined. Total cell numbers were determined by fixing blastocysts in 4% paraformaldehyde, and staining with bisbenzimide 33342, followed by microslide mounting and visualization using an epifluorescence microscope. No difference was observed in cleavage rates among the four treatment groups, C-NT, TSAa-NT, TSAb-NT, and IVF, with the rates being 66%, 75%, 73.1%, and 82.3%, respectively (P = 0.33); nor was any improvement seen in the rate of blastocyst development of TSAa-NT or TSAb-NT over C-NT embryos: 36%, 40.2%, and 30.2%, respectively (P = 0.22). Furthermore, there was no significant difference in mean total cell number of blastocysts among treatment groups: C-NT, 120.2; TSAa-NT, 124.2; TSAb-NT, 129.3; and IVF, 141.1 (P = 0.29). These results suggest that 50 nm TSA treatment immediately following activation does not affect the development of bovine SCNT preimplantation embryos.

25 PORCINE OOCYTES SELECTION USING BRILLIANT CRESYL BLUE AND EMBRYO DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER

2014 ◽  
Vol 26 (1) ◽  
pp. 127
Author(s):  
E. J. Park ◽  
K. Y. Song ◽  
J. H. Moon ◽  
B. C. Lee
Keyword(s):  
Nuclear Transfer ◽  
Formation Rate ◽  
Oocyte Quality ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Morphological Criteria ◽  
Selection Of

The efficiency of animal cloning through somatic cell nuclear transfer (SCNT) technology is affected by numerous factors such as oocyte quality and donor cell type. Among the factors, oocyte quality can be enhanced by identification and selection of developmentally competent oocytes before in vitro maturation (IVM). Morphological criteria following homogeneous ooplasm, more than 3 layers of cumulus cells, and oocyte diameter have been used. However, the criteria vary between examiners and even within individual. In contrast, use of Brilliant cresyl blue (BCB), a marker of oocyte growing status related to activity of glucose-6-phosphate dehydrogenase (G6PD), can be a more objective selection method for the detection of fully grown oocytes in bovine, equine and porcine. To our knowledge, BCB has been used to select oocytes in parthenogenesis, intracytoplasmic sperm injection (ICSI), and IVF, excluding SCNT in porcine. The aim of this study was to investigate whether oocytes selected by BCB have better ability to develop into blastocysts than oocytes selected by morphological criteria. After aspirated cumulus–oocyte complexes (COC) from porcine ovaries were washed in HEPES-buffered TCM-199 2 times, the COC were selected through morphological criteria and divided randomly into 2 groups. Group 1 (control; 304 COC) was directly transferred into IVM medium and Group 2 was incubated in TALP supplemented with 26 μM BCB for 90 min at 38.5°C in air. Then, COC of the second group were washed twice in TALP, and COC displaying a blue-coloured ooplasm (BCB+) were selected. The 342 BCB+ COC also were transferred into IVM medium and cultured with 5% CO2 in air at 38.5°C, with hormonal supplementation for 22 h and without the hormones for another 22 h. After denudation, the rate of degenerated oocytes and maturation rate were determined. The matured oocytes were used for SCNT and the development and total cell number of blastocysts were observed. Each experiment was repeated at least 3 times. Data were analysed by unpaired Student's t-test using Graphpad Prism (GraphPad, San Diego, CA, USA). No difference was observed between Groups 1 and 2 in the rate of degenerated oocytes (9.13 ± 0.47% and 10.68 ± 2.73%, respectively), metaphase II rate (94.60 ± 2.30% and 89.26 ± 3.76%, respectively), cleavage rate (77.02 ± 1.56% and 80.05 ± 2.31%, respectively), or blastocyst formation rate (9.74 ± 1.91% and 10.74 ± 1.30%, respectively). However, total cell number of blastocyst showed significant difference between Groups 1 and 2 (57.67 ± 1.76 and 77.50 ± 1.50, respectively; P < 0.01). In conclusion, selection of oocytes through BCB staining does not improve their developmental ability with respect to cleavage and blastocyst formation rate, but enhances embryo quality by means of increased total cell number per blastocyst in porcine SCNT. This study was supported by IPET (#311011-05-2-SB010), MKE (#10033839-2012-21) and TS Corporation.

27 THE HISTONE DEACETYLASE INHIBITOR 4-IODO-SUBEROYLANILIDE HYDROXAMIC ACID IMPROVES TOTAL CELL NUMBER IN PIG NUCLEAR TRANSFER BLASTOCYSTS

2012 ◽  
Vol 24 (1) ◽  
pp. 125
Author(s):  
K. M. Whitworth ◽  
J. M. Teson ◽  
K. Lee ◽  
J. Mao ◽  
K. J. Tessanne ◽  
...  
Keyword(s):  
Histone Deacetylase Inhibitor ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Suberoylanilide Hydroxamic Acid ◽  
Total Cell Number ◽  
Deacetylase Inhibitor ◽  
Significant Difference ◽  
Hdaci Treatment

Treatment of reconstructed pig clones with the histone deacetylase inhibitor (HDACi) Scriptaid immediately after nuclear transfer (NT) and activation results in increased cloning efficiency. Aberrant gene expression examined in NT blastocyst stage embryos is only partially corrected by Scriptaid use; therefore, 2 other HDACi were examined in this study including the class I and II HDACi, suberoylanilide hydroxamic acid (SAHA) and its hydrophobic derivative 4-iodo-SAHA (I-SAHA). Blastocyst rates and total cell numbers were examined across 6 treatment groups (1 μM SAHA, 10 μM SAHA, 1 μM I-SAHA, 10 μM I-SAHA, 0.5 μM Scriptaid and no HDACi treatment). Nuclear transfer was performed on enucleated MII oocytes using 3 different cell lines. Clones were electrically fused and activated, treated with HDACi for 14 to 16 h and cultured to the blastocyst stage in PZM3 under low oxygen tension for 7 days. Blastocyst number was calculated from the total number of fused oocytes. Blastocysts were then fixed in 4% paraformaldehyde and total cell number was determined by Hoechst staining of nuclei. The results from all 3 cell lines were pooled and 782 embryos were examined for blastocyst development from 7 replicates. All statistical analysis was performed by SAS 9.1 and means were separated by least significant difference (P < 0.05). The treatment group 10 μM SAHA had the highest blastocyst rate of 41.9% (n = 124) and was significantly different than no HDACi treatment (29.2%, n = 161; P < 0.003). There was no significant difference in blastocyst rates between 1 μM SAHA, 10 μM SAHA, 1 μM I-SAHA and 0.5 μM Scriptaid with blastocyst rates of 31.6% (n = 168), 41.9% (n = 124) 34.2% (n = 76) and 40.2% (n = 179), respectively (P < 0.05). Treatment with 10 μM I-SAHA significantly decreased development when compared with the other HDACi treatments (17.6%, n = 74, P < 0.05). There was no interaction between treatment and cell line for blastocyst rates (P > 0.45). Total cell number was significantly higher in blastocysts from the 1 μM I-SAHA (37.9, n = 20) treatment group when compared with Scriptaid (29.9, n = 50) and no HDACi treatment (29.4, n = 42; P < 0.04). There were no significant improvements in total cell number between the other concentrations (P > 0.05). Additionally, there was also a significant interaction between cell line used for nuclear transfer and the total cell number (P < 0.002). Two treatments were selected to determine if 10 μM SAHA and 1 μM I-SAHA treatment postnuclear transfer was compatible with term development. Six embryo transfers were performed and 5 recipient pigs became pregnant and developed to term. The results of this study show that treatment with the HDACi, SAHA and I-SAHA postnuclear transfer has the same blastocyst rates as the commonly used HDACi, Scriptaid. Additionally, treatment with 1 μM I-SAHA improves total cell number when compared with Scriptaid or no HDACi treatment. Funding was provided by Food for the 21st Century.

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 229-236 ◽  
Author(s):  
Zubing Cao ◽  
Liucai Sui ◽  
Yunsheng Li ◽  
Suofei Ji ◽  
Xiaorong Zhang ◽  
...  
Keyword(s):  
Early Development ◽  
Nuclear Transfer ◽  
Defined Medium ◽  
Cell Number ◽  
Early Embryo ◽  
Total Cell ◽  
Chemically Defined Medium ◽  
Total Cell Number ◽  
Parthenogenetic Activation ◽  
Blastocyst Rate

SummaryThe present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.

scholarly journals 48EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 SUPPLEMENT TO NCSU-23 MEDIUM ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 146
Author(s):  
S. Kim ◽  
D.H. Nam ◽  
Y.W. Jung ◽  
H.S. Kim ◽  
S.H. Lee ◽  
...  
Keyword(s):  
Growth Factor ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Total Cell ◽  
Insulin Like Growth Factor ◽  
Total Cell Number ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Igf I

The developmental potential of in vitro production of embryos is affected by various factors, including the culture system, oocyte quality, the presence of serum, and embryo paracrine and autocrine growth factors. Insulin-like growth factor is a good stimulator of oocyte maturation and embryo development. The present study investigated the effect of insulin-like growth factor-I (IGF-I) supplement on the preimplantation development of porcine embryos derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. The number of total cells and inner cell mass (ICM) cells in blastocysts were counted after differential staining at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). In Experiment 1, a total of 2,462 in vitro-matured oocytes (527, 458, 498, 481 and 498, respectively) were inseminated with frozen-thawed boar semen and subsequently cultured in North Carolina State University (NCSU)-23 medium supplemented with various concentrations of IGF-1 (0, 1, 10, 50 and 100ngmL−1). As a result, significant model effects on the development to the 2-cell stage (P=0.033) and to the blastocyst stage (P=0.0067) were found, and more blastocysts (16.9, 16.6, 17.5, 21.8 and 14.7 %, respectively) were obtained in medium supplemented with 50ngmL−1 of IGF-I. Moreover, increase in the total cell number (56.5, 53.2, 74.0, 76.4 and 58.4) and ICM (6.6, 5.8, 9.3, 9.4 and 6.1) cells was observed in IVF embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 IGF-1. In Experiment 2, porcine cloned embryos were produced by our standard protocol using fetal fibroblasts as donor cells (Hyun SH et al., 2003 Theriogenology 59, 1641–1649) and cultured in NCSU-23 supplemented with the same concentration of IGF-1 as Experiment 1. As a result, a total of 501 reconstructed oocytes (99, 98, 102, 99 and 96, respectively) were cultured and significant model effects on the development to the 2-cell stage (P=0.0179) were found. More blastocysts (10.5, 11.2, 11.8, 20.8 and 10.1%) were produced when embryos were cultured in NCSU-23 medium supplemented with 50ngmL−1, even though no statistical significance was found (P=0.1182). Increases in the total cell number (42.7, 46.0, 45.9, 51.1 and 38.2) and ICM cells (3.8, 3.8, 5.6, 6.6 and 4.8, respectively) were observed in cloned embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 of IGF-I. In conclusion, the present study demonstrated that IGF-1 at the concentration of 50ngmL−1 improves the development of preimplantion embryos derived from IVF and SCNT. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).

Sign in / Sign up

Export Citation Format

Share Document