58 DEVELOPMENT OF α1,3-GALACTOSYLTRANSFERASE KNOCK-OUT CLONED PIG EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 187
Author(s):  
S. S. Hwang ◽  
M. R. Park ◽  
J. H. Shim ◽  
B. C. Yang ◽  
Y. G. Ko ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cell Number ◽  
Normal Donor ◽  
Blastocyst Development ◽  
Step Method ◽  
Knock Out ◽  
Electric Pulses

This study was performed to increase the developmental rate of cloned embryos with the 1,3-Galactosyltransferase (GalT) gene knocked out (KO). Ovaries were collected from local slaughterhouse and immature oocytes were cultured in TCM-199 + 0.1% PVA + FSH + LH (0.5 μg mL-1) + EGF (10 ng mL-1) + 10% porcine follicular fluid (pFF) at 38.5°C in 5% CO2 humidified chamber for 40 h (1-step) or 20 h (with hormone) +20 h (without hormone; 2-step). After IVM, the oocytes with 1st polar body were enucleated and transferred the GalT KO donor cell originated from miniature pig. The embryos transferred with normal mini-pig ear fibroblast cell were used as control. The reconstructed embryos were fused with 2 electric pulses (DC) of 1.2 kV cm-1 for 30 μs. For the development of cloned embryos, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5°C for 6 days. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle, and pregnancy diagnosis was determined at 28 days after embryo transfer using ultrasonography. Differences among treatment means were determined by a chi-square test. A probability of P < 0.05 was considered statistically significant. The maturation rate was significantly higher in the 2-step method (89.8 ± 2.75) compared with single maturation method (79.6 ± 8.95; P < 0.05). The blastocyst development of cloned embryos reconstructed with GalT KO donor cell (28.4 ± 2.14) was not different from cloned embryos by normal donor cell (27.4 ± 0.01). The cell number of GalT blastocyst (36.1 ± 11.1) was not different statistically from control (26.9 ± 9.3). The apoptosis rate was also not different in both groups (2.9 to 4.9%). Five surrogates were pregnant and the GalT KO fetuses were still ongoing pregnancy at 45 days after embryo transfer. This work received grant support from the Agenda Program (No. 200901FHT010305146 and No. 200901FHT010305535), Rural Development Administration, Republic of Korea.

98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Second Generation ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Blastocyst Development ◽  
First Polar Body

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).

32 PREGNANCY OF EQUINE CLONED EMBRYOS MICROINJECTED WITH PLURIPOTENCY INDUCING GENES (Oct4, Sox2, c-Myc, K1f4)

2014 ◽  
Vol 26 (1) ◽  
pp. 130
Author(s):  
R. Olivera ◽  
R. Jordan ◽  
C. Alvarez ◽  
M. Radrizzani ◽  
G. Vichera
Keyword(s):  
Pregnancy Rate ◽  
Electric Pulse ◽  
Polar Body ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Pregnancy Rates ◽  
Control Group ◽  
Blastocyst Development ◽  
Donor Nucleus ◽  
First Polar Body

Animal cloning is a high impact tool for scientific and economical production, but still with inefficient results. The efficiency of the cloning process depends on the state of differentiation of the donor cell. An adult equine somatic cell can be differentiated to a pluripotent stem cell (iPSC) inducing the expression of certain transcription factors (Oct4, Sox2, c-Myc, and K1f4; Breton et al. 2013). The objective of this work was to assess the effect of the intracytoplasmic injection of pluripotency inducing genes on embryo development and pregnancy rates of equine cloned embryos. Cumulus–oocyte complexes (COC) were obtained from slaughterhouse ovaries. Oocyte collection and maturation procedure were performed as described by Lagutina et al. (2007). After the removal of cumulus cells, oocytes showing first polar body were microinjected with a mixture 1/3 of plasmids/liposomes (Mi group). The plasmid used was the pEP4-E02s-EM2k, which encodes the human genes Oct4, Sox2, Myc, and K1f4. The DNA concentration was adjusted to 0.5 μg mL–1. Microinjected oocytes were enucleated using the zona free method. Adult male skin fibroblasts from the same animal were used as donor nucleus cells. These fibroblasts were attached to the ooplasts with phytohemagglutinin and then fused with an electric pulse. Activation was performed using 8.7 mM ionomycin for 4 min, followed by culture for 4 h in a combination of 1 mM 6-DMAP and 5 mg mL–1 cycloheximide. Zona free reconstructed embryos (ZFRE) were cultured for 7 to 8 days in DMEM-F12 in the well of the well (WOW) system, aggregating 3 embryos per well. A control group (CC group) of not microinjected embryos was included. Cleavage and blastocyst development was assessed at Days 2 and 7, respectively. Transcervical transfer of 49 Day 7 to 8 blastocysts was performed 6 days after ovulation. The mares received 2 blastocysts per transfer. Pregnancy was diagnosed by transrectal ultrasonography 15 days after ovulation. Cleavage and blastocyst rates were analysed by Chi-squared test and pregnancy rate by Fisher test (P < 0.05). Cleavage was 92.1% (n = 58/63) for the Mi group and 90.4% (n = 868/960) for the CC group. Blastocyst rate was statistically higher per well, 28.6% (n = 6/21) v. 13.4% (n = 43/320) but not per oocyte, 9.5% (n = 6/63) v. 4.5% (n = 43/960), for the Mi and CC groups, respectively. Pregnancy rate was 17% (n = 1/6) for the Mi group and 7% (n = 3/43) for the CC group. No twin pregnancies were found and all the pregnancies are still ongoing. The higher blastocyst rates obtained with the embryos microinjected with pluripotency inducing genes compared with the control group showed an improvement in embryo quality. In conclusion, the data presented indicate that the intracytoplasmic microinjection of pluripotency inducing genes in equine zona free cloned embryos improved blastocyst rates on a per well basis and showed a tendency to improve the pregnancy rates. The expression of the Oct4, Sox2, c-Myc, and K1f4 genes could be probably generating better reprogrammed donor nucleus compared with adult differentiated cells used in conventional cloning.

100 EFFECT OF DIFFERENT POST-ACTIVATION TREATMENTS ON THE DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER PIG EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 167
Author(s):  
H. Y. Yong ◽  
K. Song ◽  
E. Lee
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Cell Number ◽  
Donor Nucleus ◽  
Gamma Tubulin ◽  
Stock Solutions

Activation treatment is one of the important factors that affect the development of somatic cell nuclear transfer (SCNT) embryos. We examined the effect of post-activation (PA) treatment on the change in donor nucleus and SCNT embryo development in pig. Cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and in fresh hormone-free medium for 18 h. After 40 h of IVM, oocytes with a polar body were enucleated, injected with a donor cell (ear skin fibroblasts bearing the human decay accelerating factor gene), electrically fused, and activated 1 h after fusion. Then, SCNT embryos were cultured in a modified NCSU-23 medium (Park et al. 2005 Zygote 13, 269–275) containing no additives (control), 5 �g mL-1 cytochalasin B (CB), 0.4 �g mL-1 demecolcine (D), or CB+D for 4 h. CB and D were prepared from stock solutions of 5 mg mL-1 CB in DMSO and 10 �g mL-1 D in Hank&apos;s balanced salt solution (HBSS), respectively. After PA treatment, SCNT embryos were cultured in a modified NCSU-23 medium for 6 days. The embryos (n &equals; 188, 189, 187, and 186 for control, CB, D, and CB&plus;D, respectively) were examined for cleavage and blastocyst (BL) formation on Days 2 and 6, respectively (Day 0 &equals; the day of SCNT). Cell number of BL was examined by counting the number of nuclei stained with Hoechst 33342 under fluorescence. To assess the nuclear structure, some of the fused oocytes were fixed at 12 h after PA and stained with aceto-orcein (n &equals; 42, 44, 43, and 45 for control, CB, D, and CB&plus;D, respectively). Nuclear state was classified as 1 pseudopronucleus (PPN), multi-PPN, and others. Data were analyzed by ANOVA (GLM procedure) in SAS (SAS Institute, Inc., Cary, NC, USA). PA treatment with D and CB&plus;D significantly (P &lt; 0.05) increased 1 PPN formation (84 and 80&percnt;, respectively) compared to control and CB (62 and 64&percnt;, respectively). Conversely, a higher (P &lt; 0.001) rate of multi-PPN was observed in control and CB (31 and 36&percnt;, respectively) than in D and CB&plus;D (9 and 7&percnt;, respectively). This result was in contrast with the finding in mouse that nocodazole, another microtubule depolymerizing agent, induced multi-PPN in reconstructed zygotes. Pig meiotic spindles differ at their poles from those in mice by lacking &gamma;-tubulin. Absence of &gamma;-tubulin in pig oocytes would make spindle dynamics more sensitive to depolymerization, which might lead to a different result in this study. Embryo cleavage (77&ndash;85&percnt;) was not altered by PA treatments, but BL formation was significantly (P &lt; 0.05) increased by CB, D, or CB&plus;D (26, 28, and 28&percnt;, respectively) compared to control (16&percnt;). Total cell number of BL (36&ndash;40 cells/BL) was not different among groups. These results indicate that PA treatment with CB and/or D improved in vitro development of SCNT pig embryos and that D treatment effectively prevented the formation of multi-PPN. This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

scholarly journals Blastocyst formation, embryo transfer and breed comparison in the first reported large scale cloning of camels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. O. Olsson ◽  
A. H. Tinson ◽  
N. Al Shamsi ◽  
K. S. Kuhad ◽  
R. Singh ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
Large Scale ◽  
Donor Cell ◽  
Blastocyst Formation ◽  
Short Term ◽  
Embryo Formation ◽  
Large Expansion ◽  
Breed Differences ◽  
Breed Comparison

AbstractCloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

Comparative study of a new composite biomaterial fluor-hydroxyapatite on fibroblast cell line NIH-3T3 by direct test

Biologia ◽  
2008 ◽  
Vol 63 (2) ◽  
Author(s):  
Marica Theiszová ◽  
Soňa Jantová ◽  
Silvia Letašiová ◽  
Ľuboš Valík ◽  
Martin Palou
Keyword(s):  
Comparative Study ◽  
Cell Line ◽  
Dna Content ◽  
Fibroblast Cell ◽  
Cell Number ◽  
Fibroblast Cell Line ◽  
3T3 Cells ◽  
End Points ◽  
Basal Cytotoxicity ◽  
Nih 3T3

AbstractThe number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH− groups and F− ions. The aim of this experimental investigation was to use the embryonal mouse fibroblast cell line NIH-3T3 for comparative study of basal cytotoxicity of fluoridated biomaterials FHA and FA discs. Hydroxyapatite (HA) disc, high-density polyethylene as negative control and polyvinyl chloride (PVC) containing organotin stabilizer as positive control were used as standard biomaterials. The appropriateness of the use of NIH-3T3 cells and their sensitivity for tested biomaterials were evaluated on the basis of five cytotoxic end points: cell proliferation, cell morphology, lactate dehydrogenase (LDH) released, protein and DNA cell content. The basal cytotoxicity of FHA, FA and HA discs was measured by direct contact method. FHA composite, FA and HA demonstrated in cell line NIH-3T3 nearly similar basal cytotoxicity increasing with the time of treatment. After 72 h of biomaterials treatment, about 25% inhibition of cell number, unchanged morphology of dividing cells, 6.31–0.16% increase of released LDH, about 10% inhibition of cell protein content and about 20% inhibition of DNA content was found. On the other hand, from the growth rates it resulted that NIH-3T3 cells, affected by tested biomaterials, divided about 20% slowlier than the control (untreated cells). Using the linear regression analysis we found out that deviations in measurements of cytotoxicity by four methods were as follows: less than 10% for cell number, protein and DNA content methods and 12.4% for released LDH method. Based on a good correlation of the cytotoxicity of biomaterials obtained from all end points we could conclude that fibroblast NIH-3T3 cell line was appropriate for measuring the basal cytoxicity of tested biomaterials.

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

Micromolar concentration of pentoxifylline improves development in vitro of hamster 8-cell embryos: conrmation of biological viability by embryo transfer

1997 ◽  
Vol 9 (7) ◽  
pp. 697 ◽  
Author(s):  
Rupasri Ain ◽  
P. B. Seshagiri
Keyword(s):  
Embryo Transfer ◽  
Embryo Development ◽  
Human Sperm ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development ◽  
Embryo Culture Medium ◽  
Cell Embryo ◽  
Continuous Presence ◽  
Development In Vitro

The inßuence of the sperm motility stimulant pentoxifylline (PF) on preimplantation embryo development in hamsters was evaluated. Eight-cell embryos were cultured in hamster embryo culture medium (HECM)-2, with or without PF (0· 0233·6 mM). There was 90%, 37% and 29% inhibition of blastocyst development by 3·6 (used for human sperm), 0·9 and 0 ·45 mM PF, respectively. However, 23 µM PF (exposed to hamster oocytes during IVF) signicantly (P < 0·05) improved blastocyst development (63· 6% v. 51· 8%); morulae development was, however, not curtailed by 0·45 mM or 0·9 mM PF (51·8%±6·0 or 50·5%±11·3, respectively). Post-implantation viability of PF-treated embryos was assessed by embryo transfer; 43% of 80 PF-treated embryos implanted compared with 40% of 79 control embryos. Of the 9 recipients, 6 females delivered pups (19, i.e. 16% of transferred embryos or 53% of implanted embryos). These data show that in hamsters, continuous presence of PF at 0·45-3·6 mM is detrimental to 8-cell embryo development whereas 23 µM PF improves the development of embryos to viable blastocysts which produce live offspring.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 250
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
V. P. Oliveira ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Electric Pulses ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

74 EXAMINATION OF ABNORMAL CELL DIVISION AND CHROMOSOME ABERRATION IN PIG PARTHENOTES AND CLONED EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 145
Author(s):  
S. J. Uhm ◽  
M. S. Kim ◽  
M. K. Gupta ◽  
H. Y. Lee ◽  
S. J. Park ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Aberration ◽  
Bovine Serum ◽  
Polar Body ◽  
Cell Number ◽  
Chromosome 1 ◽  
Hybridization Technique ◽  
Cell Stage ◽  
Abnormal Cell ◽  
Fetal Fibroblasts

Blastomere fragmentation is commonly observed in pig embryos and is associated with reduced blastocyst and pregnancy rates. This study examined the effect of the frequency of abnormal cell division and chromosome aberration on the embryonic developmental ability of pig parthenotes and nuclear transferred (NT) embryos. Pig immature oocytes cultured in TCM-199 supplemented with 10% pig follicular fluid, 0.2 mM pyruvate, 10 ng/mL epidermal growth factor (EGF), 5 �g/mL Folltropin V, 1 �g/mL estradiol-17�, and 25 �g/mL gentamycin for 44 h. Cumulus cells from matured oocytes were removed by vortexing for 1 min in TL-HEPES medium containing 0.1% hyarunonidase. Denuded oocytes were enucleated using 20 um micropipette in TCM-HEPES medium containing 7.5 �g/mL cytochalasin B (CB) and 10% fetal bovine serum, and were reconstructed with fetal fibroblasts by electrofusion (two DC pulses of 2.0 kV/cm for 30 �s). For production of parthenotes and reconstructed embryos, denuded oocytes were activated by a DC pulse of 1.0 kV/cm for 30 �s and then cultured for 4 h in NCSU23 with 10 �g/mL CB and 0.4% bovine serum albumin for inhibition of polar body extrusion. Subsequently, these oocytes were cultured in 50 �L of NCSU23 containing 0.4% BSA for 7 days at 39�C in a humidified atmosphere of 5% CO2 in air. The frequency of chromosome aberrations was evaluated using fluorescent in situ hybridization technique with a porcine chromosome-1 submetacentric specific probe. Data were analyzed by Student's t-test and ANOVA using SAS software as appropriate (SAS Institute, Inc., Cary, NC, USA). Parthenotes and NT embryos showed similiar cleavage rates (61.4 and 62.9%), but the blastocyst rate of parthenotes (18.4%) was significantly higher (P < 0.05) than that of NT embryos (10.4%). The frequency of chromosome aberration in NT embryos (39.8%) at the 4-cell stage on Day 3 of culture was significantly higher (P < 0.05) than that of parthenotes (21.9%). The percentage of fragmentation was significantly higher (P < 0.05) in NT embryos (51.7%) than in parthenotes (27.1%). Furthermore, the developmental rates of non-fragmented parthenotes (40.0%) and NT (22.9%) embryos to the blastocyst stage were significantly higher (P < 0.05) than those of fragmented parthenote and NT embryos (17.3 and 5.9% respectively). The total cell number of non-fragmented parthenote and NT embryos (34.4 � 10.0 and 29.7 � 7.5) were significantly higher (P < 0.05) than those of fragmented parthnote and NT embryos (22.3 � 9.6 and 18.4 � 6.2 respectively). Therefore, these results indicate that chromosomal abnormality and embryonic fragmentation could be associated with reduced developmental ability in pig NT embryos. This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.

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