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.

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 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.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

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

250 HUMAN EXHALED AIR CAN EFFECTIVELY SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES AND SUBSEQUENT IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 223
Author(s):  
Z. B. Cao ◽  
L. C. Sui ◽  
S. F. Ji ◽  
J. W. Chen ◽  
T. Gui ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Total Cell ◽  
High Oxygen ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Nuclear Maturation ◽  
Air Atmosphere ◽  
Significant Difference

The objective of the present study was to examine the feasibility of culturing porcine oocytes and embryos in vitro using the human exhaled lung air atmosphere. In Experiment 1, the effects of lung air atmosphere on nuclear maturation of prepubertal gilt oocytes and subsequent development in vitro of parthenogenetic-activated and somatic-cell-cloned embryos were explored. Abattoir-derived prepubertal gilt cumulus–oocyte complexes (COC) were matured in TCM-199 supplemented with 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, 10 ng mL–1 of epidermal growth factor, and 10% porcine follicular fluid (pFF) for 40 to 44 h at 38.5°C, 100% humidity, and 5% CO2+20% O2 (high oxygen tension) or human exhaled air encapsulated in plastic, airtight bags (lung air) or 5% CO2+7% O2 (low oxygen tension) in the incubator. Nuclear maturation was evaluated by the presence of the 1st polar body. For parthenogenetic activation, denuded oocytes with the 1st polar body were selected and stimulated with a single 1.6-kV/cm, 100-μs direct current pulse followed by culture in porcine zygote medium-3. For NT, denuded metaphase II oocytes were enucleated, and then the donor cell was directly injected into the perivitelline space. After NT, reconstructed couplets were fused and activated electrically followed by treatment in 7.5 μg mL–1 of cytochalasin B and 10 μg mL–1 of cycloheximide for 4 to 6 h before culture in porcine zygote medium-3. We found no significant difference among groups in terms of nuclear maturation rate (66.5% v. 60.2%, 63.2%), cleavage rate (94.8% v. 94.2%, 85.2%), blastocyst formation rate (39.5% v. 40.3%, 32.5%), and total cell number (37 v. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between the lung-air and high-oxygen (20% O2) groups was observed in the cleavage rate (88.3% v. 80.3%), blastocyst formation rate (7.3% v. 10.7%), and total cell number (34 v. 36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the human exhaled lung air atmosphere. In Experiment 2, in vitro developmental competence of porcine zona-free parthenogenetically activated embryos cultured in a lung air, low oxygen (5% O2), or high oxygen (20% O2) tension gas environment was studied. We found no obvious difference among the 3 groups regarding the rates of cleavage (83.0%, 83.6%, 82.8%), but blastocyst formation rate (26.8% v. 48.6%, 48.2%) and total cell number (23 v. 34, 29) in lung air were lower than those in the rest of the groups (P < 0.05). The results show that lung air could be an alternative for preparing a gas environment for in vitro culture of porcine zona-free parthenotes, although not an ideal alternative. Taken together, porcine oocytes and embryos can be cultured in vitro safely and efficiently using the human exhaled lung air atmosphere. Z. B. Cao and L. C. Sui contributed equally to this work. X. R. Zhang and Y. H. Zhang are the corresponding authors. This work was supported by NSFC (30700574), 863 (2008AA101003).

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.

158 EFFECTS OF AMINO ACIDS IN EMBRYO TRANSPORT MEDIA ON PORCINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 227
Author(s):  
E. J. Park ◽  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
G. A. Kim ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Number ◽  
Total Cell ◽  
Cell Counting ◽  
Buffer System ◽  
Total Cell Number ◽  
Transport Medium ◽  
Significant Difference ◽  
Embryo Transport ◽  
Parthenogenetic Embryos

Due to the distance from the laboratory to the recipient farm, several laboratories, including ours, carry somatic cell nuclear transfer (SCNT)-derived porcine embryos to the farm using a portable incubator for a few hours. If the embryos are nourished well during the transport, viability of embryos might be increased and cloning efficiency can be improved. TALP, which is widely used as a porcine embryo transport medium, lacks amino acids (AA). Proper supply of AA in the uterus is important for the development of pre-implantation embryos because AA have functions as osmolytes, metabolic regulators, or substrates and buffers of intracellular pH. Thus, supplementation of AA could affect the embryonic viability during the transport of SCNT-derived porcine embryos. The aim of this study is to determine whether the transport medium containing AAs affects the in vitro development of parthenogenetic embryos compared to TALP. Porcine zygote medium-5 (PZM-5) was chosen as transport medium containing AA due to its similarity in constituents with TALP except for the AA. Because PZM-5 contains sodium bicarbonate as a buffer system which can not cover wide variation of pH, 10 mM HEPES was added into PZM-5 (PZM+H) as it was normally done with TALP. Porcine cumulus–oocyte complexes (COC) were collected from ovaries of slaughtered pigs and cultured for 44 h using a two-step culture protocol. After denuded, matured oocytes were activated by thimerosal for 10 min followed by dithiothreitol for 30 min. The parthenogenetic embryos were cultured in PZM-5 for 2 days, monitored for cleavage, and loaded in a straw with TALP or PZM+H, respectively. Embryos were stored in a portable incubator (MTG, Bruckberg, Germany; no CO2) at 37°C for three hours and moved to PZM-5 drop for additional 5 days culture. The development was monitored on Day 7 after activation and blastocysts (BL) were collected for total cell number counts and RNA extraction. Ten BL from the TALP group and 11 BL from the PZM+H group were stained with 10 µg mL–1 bisbenzimide (Hoechst 33342) and were visualized for cell counting under fluorescence microscopy. Messenger RNA was extracted from 7 BL of the TALP and PZM+H groups and cDNA were synthesized. Quantitative real-time PCR were done to detect expression levels of apoptosis-related genes using the cDNA. The Bax/Bcl2 ratio was investigated as expression level of apoptosis-related genes and GAPDH was used as control. Each experiment was repeated at least 3 times. Data were analyzed by paired Student’s t-test using Graphpad Prism (version 5, Graphpad Software Inc., La Jolla, CA, USA). No difference was observed between the TALP and PZM+H groups with respect to blastocyst formation rate (22.46 ± 1.47% and 23.17 ± 2.13%, respectively) and total cell number (32.9 ± 2.22 and 37.09 ± 2.18, respectively). There was no significant difference between groups in the Bax/Bcl2 ratio. The use of PZM-5 media, which contains AA, did not affect the development and apoptosis of parthenogenetic embryos. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

Production of somatic cell nuclear transfer embryos using in vitro-grown and in vitro-matured oocytes in rabbits

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 494-500 ◽  
Author(s):  
Hironobu Sugimoto ◽  
Yuta Kida ◽  
Noriyoshi Oh ◽  
Kensaku Kitada ◽  
Kazuya Matsumoto ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase Inhibitor ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Deacetylase Inhibitor ◽  
Number Of Cells

SummaryWe examined growing oocytes collected from follicles remaining in superovulated rabbit ovaries, that were grown (in vitro growth, IVG) and matured (in vitro maturation, IVM) in vitro. We produced somatic cell nuclear transfer (SCNT) embryos using the mature oocytes and examined whether these embryos have the ability to develop to the blastocyst stage. In addition, we examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), on the developmental competence of SCNT embryos derived from IVG–IVM oocytes. After growth for 7 days and maturation for 14–16 h in vitro, the growing oocytes reached the metaphase II stage (51.4%). After SCNT, these reconstructed embryos reached the blastocyst stage (20%). Furthermore, the rate of development to the blastocyst stage and the number of cells in the blastocysts in SCNT embryos derived from IVG–IVM oocytes were significantly higher for TSA-treated embryos compared with TSA-untreated embryos (40.6 versus 21.4% and 353.1 ± 59.1 versus 202.5 ± 54.6, P < 0.05). These results indicate that rabbit SCNT embryos using IVG–IVM oocytes have the developmental competence to reach the blastocyst stage.

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).

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