158 IN VITRO DEVELOPMENT OF WOOD BISON (BISON BISON ATHABASCAE) EMBRYOS BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 178 ◽  
Author(s):  
B. M. Kumar ◽  
E. St. John ◽  
P. M. Mackie ◽  
W. A. King ◽  
G. F. Mastromonaco
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Total Cell ◽  
Bison Bison ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Wood Bison

Wood bison (Bison bison athabascae) are currently classified as threatened in Canada. Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for embryo production in non-domestic species in which access to gametes is limited. Unlike fertilization, SCNT allows preservation of the entire genome, thus avoiding dilution of valuable alleles, an important factor for the preservation of genetic diversity. The present study compared the developmental competence of iSCNT embryos reconstructed from adult female wood bison ear fibroblasts (bison NT) with development of embryos reconstructed from adult female cattle ear fibroblasts (cattle NT). Domestic cattle (Bos taurus) oocytes were used as recipient ooplasm for both donor cell types. In vitro fertilized (IVF) and parthenogenetic (PA) cattle embryos were used as controls. Fibroblast cultures at passages 3 to 5 confluent for 5 days were used for SCNT. Mature oocytes were enucleated, reconstructed by transfer of donor cells, and fused with an electrical stimulus of 1.5 kV cm–1 for 40 μs in 0.28 m mannitol containing 100 μm CaCl2 and MgCl2. Oocytes for parthenogenesis and following reconstruction were activated for 5 min in 5 μm ionomycin followed by 5 h in 10 μg mL–1 cycloheximide. Embryos produced by IVF, PA, and SCNT were cultured in modified synthetic oviductal fluid medium at 38.5°C in 5% CO2, 5% O2, 90% N2. Cleavage, blastocyst development to day 8, apoptosis (TUNEL assay, Roche Diagnostics, IN, USA), and total cell number were evaluated. Statistical analyses were carried out using one-way ANOVA, followed by Tukey post hoc analysis or the equivalent nonparametrical Kruskal-Wallis test. Cleavage rate was significantly (P < 0.05) higher in the IVF group than in all other groups (86.9 ± 2.9% v. 71.6 ± 4.5% to 78.1 ± 5.1%). Blastocyst rates, expressed as a percentage of cleaved embryos, were similar among all treatment groups (33.4 ± 3.3% to 39.8 ± 5.7%) except for bison NT which had significantly (P < 0.05) lower development to blastocyst (19.2 ± 5.5%). The percentages of TUNEL-positive cells among PA embryos (6.6 ± 1.5%) and bison NT embryos (6.7 ± 2.4%) were significantly (P < 0.05) higher than in IVF embryos (4.2 ± 1.0%), but similar to cattle NT embryos (5.4 ± 1.7%), which did not differ from the IVF group. Total cell number was significantly (P < 0.05) higher in the IVF group than in all other groups (133.2 ± 10.2 v. 91.2 ± 7.8 to 100.1 ± 12.9). These results confirm that in vitro-matured domestic cattle oocytes can serve as suitable recipients of wood bison somatic cells and that iSCNT may provide a possible alternative for embryo production and genetic preservation of endangered cattle species. Both the incidence of apoptotic cells and total cell number did not differ between cattle and bison NT embryos; thus other factors must play a role in the significantly decreased blastocyst development observed in bison NT embryos. This work was supported by Endangered Species Reserve Fund, Toronto Zoo, and the Canada Research Chairs program.

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.

46 EFFECT OF SHORT-TERM TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 123
Author(s):  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
S. G. Hong ◽  
J. T. Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Short Term ◽  
Total Cell Number

Epigenetic reprogramming such as acetylation in somatic cell nuclear transfer (SCNT) has been known as one of problems in cloned embryos. For resolving this acetylation reprogramming, many investigators recently have reported the effect of long-term culture of post-activated SCNT embryos using trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). The objective of this study is to investigate the effect of short-term TSA treatment on in vitro developmental ability and the quality of bovine SCNT embryos. Immature oocytes were aspirated from abattoir-derived ovaries, matured in vitro for 22 h, and enucleated. A bovine fetal fibroblast was placed into the enucleated oocyte and fused by electrical stimulation. The fused couplets were activated by 4-min incubation in 10 μm ionomycin, followed by 4 h of culture in 1.9 mm 6-dimethylaminopurine with or without TSA (0, 50, or 100 nm). The SCNT embryos were subsequently cultured in modified synthetic oviduct fluid medium for 8 days. Developmental competence was assessed by blastocyst formation and total cell number. Total cell numbers were determined by staining with bisbenzimide 33342. As results, developmental competence to blastocysts was higher in 100 nm than control (36.7 v. 27.9%, P < 0.05). In blastocyst hatching rate, TSA 100 nm group (19.5%) at 8 days showed an increased pattern as opposed to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). No significant differences in two cell and morula stage were observed among treatment groups. In terms of development to hatching stage of blastocysts, TSA 100 nm group (19.5%) at 8 days has a significant effect compared to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). Total cell number of blastocysts derived from TSA 100 nm was significantly higher (P < 0.05) than that in TSA 50 nm (116 v. 100), whereas there was not significant difference between control and TSA 100 nm. In conclusion, short-term culture with high concentration of TSA improved the blastocysts formation however total cell number of blastocysts showed contradictory result. The epigenetic modification by TSA treatment on bovine SCNT needs further investigation. This study was financially supported by KOSEF (grant # M10625030005-08N250300510) and the Korean MEST, through the BK21 program for Veterinary Science.

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

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.

185 DEVELOPMENT CAPACITY OF PRE- AND POSTPUBERTAL PIG OOCYTES EVALUATED BY SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETIC ACTIVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 241 ◽  
Author(s):  
H. S. Pedersen ◽  
R. Li ◽  
Y. Liu ◽  
P. Løvendahl ◽  
P. Holm ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Time Interval ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Development Capacity ◽  
Developmental Capacity

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6), and blastocyst cell number (Hoechst 33342) were recorded. For PA embryos in a timelapse incubator (26 oocytes/group; 2 replicates), the first appearance of 2-cell stage was recorded. Between groups, CL% and BL% were analysed by chi-square and cell number by t-test. Results are presented in the table for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences in CL% (90–96) and blastocyst cell number (51–59) between groups. The BL% was higher in medium gilts and sows (41; 45) compared with large gilts (21). The BL% of bad oocytes was 1% from all 4 groups (176 oocytes, 25 replicates). Time interval for appearance of 2-cell stage for embryos developing into blastocysts showed no differences between groups (19–20 h). Within groups, this time interval showed a larger standard deviation for embryos not developing v. embryos developing into blastocysts. It is concluded that (a) sow oocytes have higher developmental capacity compared to gilts, (b) small gilt oocytes are not developmentally competent, (c) measurement of inside-ZP diameter, combined with morphological selection, is useful to remove non-competent oocytes. Further studies are needed to dissect the developmental capacity of medium and large gilt oocytes. Also, further timelapse studies may reveal a time interval in which the first cleavage of embryos with high developmental capacity takes place. Table 1.Rates of cleavage (CL%), blastocyst (BL%), and total no. of cells (mean ± SEM) in blastocysts of PA embryos from gilts and sows1

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer

2015 ◽  
Vol 27 (3) ◽  
pp. 544 ◽  
Author(s):  
H. S. Pedersen ◽  
Y. Liu ◽  
R. Li ◽  
S. Purup ◽  
P. Løvendahl ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Oocyte Growth ◽  
Parthenogenetic Activation ◽  
Selection Of

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer

2003 ◽  
Vol 15 (3) ◽  
pp. 179 ◽  
Author(s):  
Goo Jang ◽  
Byeong Chun Lee ◽  
Sung Keun Kang ◽  
Woo Suk Hwang
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Fertilization ◽  
Chondroitin Sulfate ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
Vitro Fertilization

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.

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.

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