scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

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.)

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

scholarly journals 43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
G.-S. IM ◽  
L. Lai ◽  
Z. Liu ◽  
Y. Hao ◽  
C.M. Murphy ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Electric Pulses ◽  
Developmental Rates ◽  
Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P&lt;0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P&lt;0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

26 EFFECT OF TREATMENT OF BOVINE DONOR CELLS WITH MOUSE EMBRYONIC STEM CELL EXTRACT ON THE DEVELOPMENT OF EMBRYOS AFTER NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 119
Author(s):  
S. Akagi ◽  
E. Mizutani ◽  
Y. Inaba ◽  
M. Kaneda ◽  
T. Somfai ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
In Vitro Development ◽  
Donor Cells ◽  
Effect Of Treatment ◽  
Vitro Development

The efficiency of somatic cell cloning is very low, probably because of incomplete reprogramming of the somatic cell nucleus. In recent studies, it is suggested that transient exposure of donor somatic cells to mouse embryonic stem cell (ESC) extract enhances pluripotency of the cells in vitro (Bru et al. 2008 Exp. Cell Res. 314, 1634–1642; Xu et al. 2009 Anat. Rec. 292, 1229–1234). In the present study, we examined the effect of treatment of donor cells with mouse ESC extract on the in vitro development of bovine NT embryos. First, in order to examine effect of treatment of donor cells with streptolysin O (SLO), which reversibly permeabilizes the plasma membrane, we compared the in vitro development of NT embryos using donor cells treated with 5 μg mL–1 SLO (SLO group) and untreated donor cells (control group). As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used. Fibroblasts were treated with 5 μg mL–1 SLO for 45 min, and then incubated for resealing in DMEM including 2 mM CaCl2 for 60 min. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). After in vitro culture for 8 days, blastocyst formation and cell number of blastocysts were examined. There were no significant differences between SLO and control groups in the fusion rate (80% and 72%, respectively), cleavage rate (60% and 65%, respectively), developmental rate to the blastocyst stage of NT embryos (31% and 28%, respectively), and blastocyst cell number (127 ± 6 and 112 ± 14, respectively). These results suggest that SLO treatment of donor cells has no negative effect on the in vitro development of NT embryos. Next, we examined the in vitro developmental ability of NT embryos using donor cells treated with mouse ESC extract (ES extract group). After SLO treatment for 45 min, permeabilized fibroblast cells were treated with mouse ESC extract for 45 min, and then incubated in DMEM including 2 mM CaCl2 for 60 min, and used for producing NT embryos. There were no differences between ES extract and control groups in the fusion rate (68% and 69%, respectively), cleavage rate (86.7% and 80.6%, respectively), and developmental rate to the blastocyst stage of NT embryos (39.8% and 43.5%, respectively). The cell number of NT embryos at the blastocyst stage in ES extract group (201 ± 30) was significantly (t-test; P < 0.05) higher than that in control group (140 ± 14). In conclusion, treatment of bovine donor cell with mouse ESC extract did not affect the in vitro developmental ability of NT embryos, but improved the quality of blastocysts.

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 Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
M. Fahrudin ◽  
K. Kikuchi ◽  
N. W. K. Karja ◽  
M. Ozawa ◽  
T. Somfai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gradient Centrifugation ◽  
Somatic Cells ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.

362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 269
Author(s):  
A. De Stefano ◽  
A. Gambini ◽  
D. Salamone
Keyword(s):  
Cumulus Cells ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Blastocyst Quality ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P < 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P < 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (>200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.

The role of the zona pellucida in the development of mouse eggs in vivo

Development ◽  
1970 ◽  
Vol 23 (3) ◽  
pp. 539-547
Author(s):  
Jacek A. Modliński
Keyword(s):  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Development In Vitro ◽  
Single Blastomeres ◽  
Mammalian Eggs ◽  
Mouse Eggs

Up to the present time the function and significance of the zona pellucida in the development of mammalian eggs has not been fully explained. Zona-free mouse eggs will develop in vitro from the 2-cell stage, or later, up to the blastocyst stage (Tarkowski, 1961; Mintz, 1962; Gwatkin, 1963). Single blastomeres isolated at the 2-cell (Mulnard, 1965), 4- and 8-cell stage (Tarkowski & Wróblewska, 1967) will also develop in vitro up to the blastocyst stage. Similar experiments on development in vitro of 1- and 2-cell rabbit eggs (Edwards, 1964) showed that in this species also cleavage can occur when the zona pellucida is absent, although the blastomeres exhibit a tendency to fall away from each other. Tarkowski's observations (unpublished) would appear to show, however, that naked 1-, 2- and 4-cell mouse eggs do not develop when transferred to the oviduct. A few hours after transplanting the naked eggs none could be recovered by flushing the oviduct, whereas eggs surrounded by zonae which were transplanted simultaneously were recovered.

Sign in / Sign up

Export Citation Format

Share Document