91 PRODUCTION OF A CLONED BOER GOAT (CAPRA HIRCUS) BY SOMATIC CELL NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 163
Author(s):  
Y. Tao ◽  
W. Han ◽  
M. Zhang ◽  
J. Ding ◽  
X. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Capra Hircus ◽  
Blastocyst Development ◽  
Tissue Slices ◽  
Boer Goat ◽  
Reconstructed Embryos ◽  
Significant Difference

We reported the birth of a goat clone produced by somatic cell nuclear transfer. The fusion and activation protocols of reconstructed oocytes and embryo transfer procedure were optimized. The donors of somatic cells were fibroblasts derived from ear skin of a Boer goat while the recipient ooplasm was in vitro-matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were activated by ionomycin, 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB) singly or simultaneously (termed as Ionomycin, Ionomycin+6-DMAP, and Ionomycin+6-DMAP+CB). The result showed that the cleavage rate in single ionomycin was significantly lower than that in Ionomycin+6-DMAP and 6-DMAP+CB (34.38% vs. 69.85% and 72.02%; P < 0.05). However, the cleavage rates and blastocyst rates had no significant difference after in vitro culture (P > 0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development rate increased. The reconstructed embryos were equilibrated 1–3 h, 3–6 h, and 6–9 h after fusion, and then activation was undertaken by ionomycin+6-DMAP. We found that the cleavage rates had no significant difference during 1–3 h and 3–6 h (72.58% vs. 72.97%; P > 0.05), but both were significantly higher than during 6–9 h (64.40%) (P < 0.05). A total of 491 reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural estrus. One of those who were treated with hCG after transfer was pregnant and gave birth to a live kid on 153 days. The lamb died accidentally 8 h after birth. The cloned offspring was then dissected and proved well in all organs. Staining of paraffin tissue slices of the viscera suggested that the organs developed well. Microsatellite analysis indicated that the lamb was derived from the somatic cell donor doe genetically.

scholarly journals Production of Boer goat (Capra hircus) by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes

2009 ◽  
Vol 54 (No. 10) ◽  
pp. 448-460 ◽  
Author(s):  
Y. Tao ◽  
W. Han ◽  
M. Zhang ◽  
Y. Zhang ◽  
J. Fang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Microsatellite Analysis ◽  
Capra Hircus ◽  
Fibroblast Cells ◽  
Tissue Slices ◽  
Normal Paraffin ◽  
Boer Goat ◽  
Reconstructed Embryos ◽  
Paraffin Tissue

The present study reports a birth of goat clone produced by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes. The donors of somatic cells were fibroblasts derived from the ear skin of a Boer goat while the recipient ooplasm was <I>in vitro</I> matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were cultured <I>in vitro</I> and then the morphologically normal embryos were transferred to the surrogates. The reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural oestrus. Five of them were treated with hCG after transfer. Among them, one was pregnant and gave birth to a live kid. Due to the improper delivery aid, the cloned kid died accidentally after birth. The cloned kid was then anatomised to observe the viscera development, and the results showed that the organs were normal. Paraffin tissue slices were prepared and stained to ensure the viscera development further, and the results suggested that the organs also developed well in spite of incipient hydropericardium. The microsatellite analysis identified the cloning. It is suggested that the optimised nuclear transfer protocol and proper hCG treatment lead to the successful birth of a goat clone.

29 BIRTH OF A FOAL CLONED BY ADULT SOMATIC CELL NUCLEAR TRANSFER WITH ROSCOVITINE-TREATED DONOR CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
Y. H. Choi ◽  
Y. G. Chung ◽  
D. D. Varner ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Direct Injection ◽  
Donor Cell ◽  
Mixed Gas ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Significant Difference

Only one horse foal produced from adult somatic cell nuclear transfer has been reported in the scientific literature (Galli et al. 2003 Nature 425, 680); a second foal from the same laboratory was reported in the popular press in 2005. In these reports, the blastocyst rates were 3 and 17%, and efficiency to birth of a live foal from total reconstructed oocytes was 0.1 and 0.5%, respectively. In cattle, roscovitine treatment of donor cells has been associated with a decrease in blastocyst development, but an increase in live births (Gibbons et al. 2002 Biol. Reprod. 66, 895-900). The present study was performed to determine the effect of roscovitine treatment of donor cells on blastocyst production after equine nuclear transfer and to evaluate the viability of pregnancies established via this treatment. In Experiment 1, fibroblasts were either grown to confluence or treated with 15 �g/mL roscovitine, for 24 h. Enucleated in vitro-matured oocytes were reconstructed by direct injection of fibroblasts using a piezo drill. Recombined oocytes were activated by injection of stallion sperm extract, followed by culture in the presence of 2 mM 6-DMAP for 4 h. They were then placed in culture in DMEM/F-12 with 10% fetal bovine serum (FBS) under mixed gas for 8 days and evaluated for blastocyst development. In Experiment 2, oocytes recombined with either confluent or roscovitine-treated donor cells were activated as above either alone or with the addition of 10 �g/mL cycloheximide at the time of 6-DMAP treatment. Resulting blastocysts from Experiment 2 were transferred transcervically to the uteri of recipient mares. One embryo was transferred per mare. In Experiment 1, there was no difference in rates of cleavage (73-19%) or blastocyst development between confluence and roscovitine treatments (2/55, 3.6% vs. 2/56, 3.6%, respectively). In Experiment 2, there was no significant difference in rates of cleavage (78-18%) or blastocyst development (0-1%; 4/105, 0/104, 0/106, 2/108) among donor cell or activation treatments. Six blastocysts were transferred to mares: two from confluent donor cells and four from roscovitine-treated donor cells. One mare, which received an embryo from the roscovitine donor/6-DMAP treatment, established pregnancy after transfer. The pregnancy continued normally and the mare delivered a colt with minimal assistance on Day 389. Typing for 13 equine microsatellites confirmed that the colt was of the same DNA type as the donor fibroblasts. The colt has grown and developed normally. Results of these studies show that roscovitine treatment of equine donor cells does not negatively affect the proportion of recombined oocytes that progress to the blastocyst stage. A viable colt resulted from an embryo produced with roscovitine-treated donor cells. More work is needed on methods to increase blastocyst rates after nuclear transfer in this species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

2015 ◽  
Vol 27 (1) ◽  
pp. 104
Author(s):  
N. L. Selokar ◽  
M. Saini ◽  
H. Agrawal ◽  
P. Palta ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Frozen Semen ◽  
Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

scholarly journals Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 295-302 ◽  
Author(s):  
Walt Yamazaki ◽  
Christina Ramires Ferreira ◽  
Simone Cristina Méo ◽  
Cláudia Lima Verde Leal ◽  
Flávio Vieira Meirelles ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Standard Treatment ◽  
Oocyte Activation ◽  
Blastocyst Development ◽  
Reconstructed Embryos ◽  
Developmental Capacity ◽  
Bovine Oocytes

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p <0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

33 IN VITRO IMMUNOGENICITY OF SOMATIC CELL NUCLEAR TRANSFER-DERIVED TRANSGENIC CLONED DOGS

2012 ◽  
Vol 24 (1) ◽  
pp. 128
Author(s):  
G. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mononuclear Cells ◽  
Donor Cell ◽  
Foreign Gene ◽  
Beagle Dogs ◽  
Fetal Fibroblasts ◽  
Significant Difference

Histocompatible tissue has been generated by somatic cell nuclear transfer (SCNT) and the resultant tissues were not rejected by the immune system of the nucleus donors. In addition, many transgenic animals combined with SCNT have been produced. However, in vitro immunogenicity of transgenic cloned animals originated from the same donor cell with nontransgenic cloned animals has not been assessed until now. The objective of this study was to evaluate the in vitro immunogenicity of cloned dogs with each other, between cloned dogs and transgenic cloned dogs and between transgenic cloned dogs with each other by mixed lymphocyte reaction. In this study, we used cloned beagles (BG1, 2) derived from SCNT using fetal fibroblasts (BF3). Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, with red fluorescent protein (RFP) gene inserted into their genome. We used 2 age-matched healthy female beagle dogs as control dogs. They have different 3 DLA types with all cloned dogs. Peripheral blood mononuclear cells (PBMC) of 2 cloned beagles and 4 transgenic cloned beagles were isolated from whole bloods using Ficoll gradient solution. PBMC from each dog were mixed to auto PBMC, other transgenic cloned dogs and non-related control dogs under the experimental designs. All the mixtures were incubated at 37°C for 4 days, adding BrdU labeling reagent and re-incubated for 24 h. Results are expressed in absorbance mean value ± standard deviation of 450-nm wavelength read by microplate reader. Each cell combination was assayed in 8 replicates. In Experiment 1, PBMC of cloned beagles were combined with equal concentrations of another cloned beagle's PBMC. In Experiment 2, PBMC suspension of Ruppy 1–3, 5 were mixed with equal concentrations of another transgenic cloned beagle's PBMC suspension. In Experiment 3, PBMC suspensions of cloned beagles were mixed with PBMC suspensions of transgenic cloned beagles and reverse reaction was performed. Statistical analysis was performed by using Mann-Whitney U test. In Experiment 1, whereas the absorbance value of mixture of cloned dogs and control dogs shows apparent proliferation, auto mixture of each dog and allo-mixture of BG1 and BG2 show no proliferation (Table 1), indicating immunological factors exposed to PBMC in 2 cloned dogs were compatible. In Experiment 2 among transgenic cloned dogs, no evidence of proliferations in mixed allo-PBMC was shown (Table 1), suggesting in vitro immunogenicity between transgenic cloned dogs was also not shown. In Experiment 3 among cloned dogs and transgenic cloned dogs, no significant difference was found (Table 1). In conclusion, cloned dogs derived from SCNT shared immunological phenotype. Next, immunogenicity among transgenic cloned beagle dogs was not shown despite random insertion of a foreign gene. Lastly, cloned beagles and transgenic cloned beagles show lymphocyte antigen compatibility irrespective of having a foreign gene or not. Table 1.The absorbance values of mixed lymphocytes of 4 transgenic cloned dogs and 2 cloned dogs This study was supported by RNL BIO (#0468-20110001), IPET, MKE (#10033839-2011-13) and Natural Balance Korea.

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Mohammad Salehi ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Control Group ◽  
Developmental Potential ◽  
Significant Difference ◽  
Mouse Somatic Cell

SummaryThe beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

scholarly journals Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 559-567 ◽  
Author(s):  
Irina Lagutina ◽  
Giovanna Lazzari ◽  
Roberto Duchi ◽  
Silvia Colleoni ◽  
Nunzia Ponderato ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Reconstruction Method ◽  
Blastocyst Development ◽  
Adult Fibroblasts

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

356 IMPROVED ENUCLEATION EFFICIENCY FOR PIG SOMATIC CELL NUCLEAR TRANSFER BY DENUDING OOCYTES AT 30 HOURS OF IN VITRO MATURATION

2007 ◽  
Vol 19 (1) ◽  
pp. 293 ◽  
Author(s):  
K. Song ◽  
J. Park ◽  
E. Lee
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Polar Body ◽  
Digital Camera ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Significant Difference

Oocytes for somatic cell nuclear transfer (SCNT) have to be removed from their cumulus cells before enucleation. Denuding oocytes by vortexing or repeated pipetting makes the polar body (PB) deviate from the metaphase (MII) plate, which in turn makes it difficult to remove DNA materials completely during enucleation. We hypothesized that denuding oocytes at 30 h of IVM maintains the MII plate and PB in a closer position and therefore makes it easy to enucleate. To test this hypothesis, oocytes were matured in TCM-199 supplemented follicular fluid, hormones, EGF, cysteine, and insulin for first 22 h, and in a hormone-free medium for 18 h with three modifications: (1) cumulus cells were removed from oocytes just prior to enucleation at 40 h of IVM (control), (2) oocytes were denuded at 30 h of IVM and co-cultured with their detached cumulus cells for 10 h (D+), and (3) oocytes denuded at 30 h of IVM were cultured without cumulus cells (D-). After IVM, some oocytes were stained with Hoechst 33342 and photographed by a digital camera; the distance between the MII plate and the PB were measured using an image analysis program (ImageJ 1.36; http://rsb.info.nih.gov/ij). Also, the enucleation rate after blind enucleation and the in vitro development of SCNT embryos were determined. For SCNT, oocytes were enucleated, and nuclear material from donor cells (skin fibroblasts from a miniature pig) was inserted; oocytes were then electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park et al. 2005 Zygote 13, 269-275) for 6 days. Embryos were examined for their cleavage and blastocyst formation on Days 2 and 6, respectively (the day of SCNT was designated Day 0). Data were analyzed by the GLM procedure and the least significant difference test in SAS (SAS Institute, Cary, NC, USA). The distance between the MII plate and the PB was significantly (P &lt; 0.01) shorter in D+ and D- embryos (19.4 and 18.9 �m, respectively) than in the controls (25.5 �m). Enucleation rates after blind enucleation were significantly (P &lt; 0.01) higher in D+ and D- groups (77% and 72%, respectively) than in the controls (60%). Oocyte maturation (89–91%), SCNT embryo cleavage (71–77%), blastocyst formation (4–5%), and embryo cell number (39-45 cells/embryo) were not altered by different denuding methods. The perivitelline space (PVS) increases with time during maturation and denudation, after PB extrusion markedly enhances PB deviation. It is likely that increased PVS in control oocytes enhanced PB deviation during denudation and then resulted in lower enucleation rate. In conclusion, the results of this study indicated that denuding at 30 h of IVM maintained the MII plate and the PB in a closer position and improved enucleation efficiency without impairing developmental capacity of SCNT embryos. This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

67 PIG OOCYTES WITH A LARGE PERIVITELLINE SPACE MATURED IN VITRO HAVE GREATER DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 139
Author(s):  
J. You ◽  
N. Kim ◽  
S. Kang ◽  
E. Lee
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Separation Procedure ◽  
Perivitelline Space ◽  
Blastocyst Formation ◽  
Significant Difference

The size of perivitelline space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P < 0.05. In Experiment 1, oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.0 pixels/oocyte v. 0.6 pixels/oocyte) and blastocyst formation (54% v. 37%) after PA than oocytes with smaller PVS. In Experiment 2, maturation culture of oocytes in PZM-3 with reduced (61.6 mM) NaCl concentration significantly increased (P < 0.05) the size of the PVS (5.2 μM v. 3.3 μM) compared with control oocytes that were matured in PZM-3 containing 108 mM NaCl, although the treatment did not alter the nuclear maturation. Moreover, oocytes with increased PVS expressed more CDK1, PCNA, and ERK2 mRNA and had higher (P < 0.05) intracellular GSH levels (1.6 pixels/oocyte v. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52% v. 41%) and SCNT (32% v. 18%) compared with control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation resulting from the enhanced GSH level and transcription factor expression and that enlargement of PVS by the culture in low-NaCl medium also improves developmental competence of pig oocytes. This work was supported by grants (#20070301034040 and #20080401034072) from the BioGreen 21 Program (Rural Development Administration, Republic of Korea).

Sign in / Sign up

Export Citation Format

Share Document