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.

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.

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.

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.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

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.

83 IN LOW OXYGEN CULTURE, IS HYPOTAURINE NECESSARY FOR IN VITRO DEVELOPMENT OF PORCINE EMBRYOS?

2016 ◽  
Vol 28 (2) ◽  
pp. 171
Author(s):  
J. A. Benne ◽  
L. D. Spate ◽  
B. M. Elliott ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Free Radical Scavengers ◽  
Total Cell Number ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

For decades it has been known that reactive oxidative species (ROS) form during in vitro embryo culture. A buildup of ROS can be detrimental to individual cells in the embryo and lead to a decrease in development and quality. To overcome oxidative stress in culture systems, additives, such as taurine and/or hypotaurine, have been used. In the pig, taurine or hypotaurine addition is deemed necessary for normal in vitro development. Another commonly used technique to reduce ROS is to culture embryos in a lowered oxygen environment (e.g. 5%). Porcine zygote medium 3 (PZM3) base culture medium is used in the following experiments and contains 5 mM hypotaurine, which is one of the most costly additives in the medium. The objective of this experiment was to determine if hypotaurine is still necessary if the embryos were cultured in 5% O2 from the zygote to the Day 6 blastocyst stage. In Experiment 1, oocytes were matured for 44 h and fertilized in vitro. After fertilization, presumptive zygotes were then transferred to 500 µL of MU-1 medium (PZM3 with 1.69 mM arginine) that either contained or did not contain hypotaurine for overnight culture at 20% O2. On Day 1, the same embryo culture plates were moved to 5% O2, 5% CO2, and 90% N2 and cultured to Day 6. The percent blastocyst stage was determined, and total cell number was counted in 3 of the 5 replicates in order to give us an indication of the embryo quality. The percent blastocyst in the controls (+hypotaurine) was 34.4% ± 2.8 and not different from the no hypotaurine (32.9% ± 2.2; N = 830; 5 replications; P > 0.10). Furthermore, total cell number was not different between the two groups (30.8 ± 1.5 v. 33.6 ± 1.8, respectively, N = 146; 3 replications; P > 0.10). In Experiment 2, the same experiment was repeated in somatic cell nuclear transfer derived embryos, which may be more sensitive to ROS due to the micromanipulation procedure. Wild type fetal fibroblast cells were used as donor cells. There was no significant difference in development to the blastocyst stage due to the presence or absence of hypotaurine (17.7% ± 2.5 v. 11.8% ± 2.3, respectively; N = 454; 4 replications; P = 0.07). All blastocyst data were analysed using the GENMOD procedure in SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and cell number data were analysed using the PROC GLM also with SAS 9.4. These data show that porcine embryos can be efficiently cultured to the blastocyst stage without adding any oxygen free radical scavengers to the media when culturing in reduced oxygen atmosphere. Further studies include evaluating term development via embryo transfers and measuring ROS production of these embryos. Funding was provided by Food for the 21st Century and the National Institutes of Health (U42 OD011140).

scholarly journals 141 BENEFICIAL EFFECT OF ETHYLENEDIAMINETETRAACETIC ACID COMBINED WITH HEMOGLOBIN ON PRE-IMPLANTATION DEVELOPMENT OF PORCINE IN VITRO PRODUCED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 221
Author(s):  
J.H. Kim ◽  
G.S. Lee ◽  
H.S. Kim ◽  
S.H. Lee ◽  
D.H. Nam ◽  
...  
Keyword(s):  
Beneficial Effect ◽  
Embryo Development ◽  
Ethylenediaminetetraacetic Acid ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Porcine Embryo

Developing a porcine embryo culture system is important for increasing the rates of implantation and pregnancy of somatic cell nuclear transfer (SCNT) embryos. Ethylenediaminetetraacetic acid (EDTA) was shown to inhibit glycolytic activity of cleavage stage embryos, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for later-stage embryos as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development. On the other hand, addition of a nitric oxide (NO) scavenger, hemoglobin (Hb), to the culture medium is known to promote embryo development to the blastocyst stage. This study was conducted to evaluate the beneficial effect of EDTA combined with Hb on pre-implantation development of porcine embryos in vitro. Porcine embryos produced by in vitro maturation and fertilization were cultured for 6 days in North Carolina State University (NCSU)-23 medium supplemented with EDTA or/and Hb. All data were subjected to one-way ANOVA and protected least significant difference (LSD) test using the general linear models (GLM) procedure of the statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Statistical significance was determined when the P value was less than 0.05. In Exp. 1, culturing porcine zygotes with 100 mM EDTA (n = 537) significantly increased cleavage rates (85.3%) at 48 h post-insemination compared to supplementing with 0, 1, or 10 mM EDTA (78.9, 79.7, or 78.2%, respectively). However, EDTA at these concentrations did not promote blastocyst formation compared to the control. In addition, no difference was observed in total cell numbers in blastocysts among the experimental groups (41.8, 42.6, 45.8, 44.5, respectively). In Exp. 2, in vitro-fertilized oocytes were cultured with 0, 1, or 10 mg/mL Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the total cell number of blastocysts obtained from 1 mg/mL Hb supplementation (n = 566) compared to that of the control (56.8 vs. 41.6). In Exp. 3, culturing embryos (n = 548) with 100 mM EDTA + 1 mg/mL Hb significantly improved rates of cleavage (84.0% vs. 75.2%) and blastocyst formation (19.2% vs. 12.7%), and the total number of cells in blastocysts compared to those of the control (58.4 vs. 42.3). In conclusion, our results demonstrated that EDTA or Hb have different roles in supporting in vitro pre-implantation development of porcine embryos; EDTA mainly stimulated early cleavage up to the 2- to 4-cell stage, and Hb promoted the total cell number of blastocysts. However, combined supplementation with these two chemicals improved cleavage, blastocyst formation, and total cell number in blastocysts. This study was supported by a grant from Korea Ministry of Science and Technology (Biodiscovery).

190 EFFECT OF TRANS-ε-VINIFERIN ON IN VITRO PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENTAL COMPETENCE IN PRE-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 207
Author(s):  
Y. Jeon ◽  
S.-S. Kwak ◽  
S.-A. Jeong ◽  
R. Salehi ◽  
Y. H. Seong ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Vitis Amurensis ◽  
Total Cell Number ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Porcine Oocyte

Trans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoids family. Trans-ε-viniferin is isolated from Vitis amurensis, 1 of the most common wild grapes in Korea, Japan and China. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after IVF or parthenogenesis (PA). At the laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Chungbuk National University, trans-ε-viniferin was purified from the leaves and stems of Vitis amurensis. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. First, in total, 594 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM) with 10% porcine follicular fluid, 10 IU mL–1 of eCG and 10 IU mL–1 of hCG. After 22 h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22 h and then nuclear maturation was evaluated. Second, in total, 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. Lastly, the developmental competence of oocytes matured with different concentrations of trans-ε-viniferin (0, 0.5 and 5.0 μM) was evaluated after IVF or PA. In total, 711 embryos were evaluated. As results, we observed that trans-ε-viniferin treatment during IVM did not improve the nuclear maturation of oocytes in any group (84.2, 86.6, 85.5, 83.3 and 79.2%, respectively), but significantly increased (P < 0.05) intracellular GSH levels in the 0.5 μM group (0 μM vs 0.5 μM; 14.6 vs 16.8 pmol oocyte–1) and reduced ROS levels (0 μM vs 0.5 μM and 50 μM; 174.6 vs 25.7 and 23.8 pixel oocyte–1). Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after IVF, but total cell numbers were significantly higher (P < 0.05) in the 0.5 and 5.0 μM treatment groups (53.6 ± 4.0 and 47.9 ± 3.1) compared to the control group (36.4 ± 2.2). The PA embryos showed similar results; there were no significant differences in cleavage rates and blastocyst formation rates, but the total cell number significantly increased in the 0.5 and 5.0 μM treatment groups (59.6 ± 4.2 and 60.8 ± 4.6) compared to the control group (43.1 ± 2.1). In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased total cell number of blastocysts, possibly through increasing intracellular GSH synthesis and reducing ROS levels. This work was supported by a grant from the Korea institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries, Republic of Korea.

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