• Korean Journal of Veterinary Research
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• v.30 no.2
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• pp.231-241
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• 1990

The present study was carried out to investigate the viability of frozen-thawed aggregated mouse embryos and to produce the chimeras. Different phenotypic embryos were obtained by mating ICR female mice with ICR or CBA male mice. The aggregated morulae were produced following aggregation of embryos at 4-, 8- and 12- to 16-cell stage. The desirable stage for the aggregation of the mouse embryos was 8- to 16-cell stage. The post-thawed in vitro survival rates of aggregated embryos in glycerol, DMSO and ethylene glycerol were 51.5, 78.6 and 69.4%, respectively. Although the higher survival was obtained in DMSO, there were no significant differences in the survival rates among the three cryoprotectants. A total of 155 frozen-thawed aggregated embryos were transferred to 11 recipient mice, 3 out of 7 offsprings were born to overt chimera. These experiments have proven that mouse chimeras can be obtained from frozen-thawed aggregated embryos.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.339-347
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• 2003

The purpose of this study was to investigate the development of bovine nuclear transfer (NT) embryos cultured in serum-free conditions. Bovine NT embryos cultured in various culture conditions were compared blastocyst development, total cell number and apoptosis using TUNEL assay. In experiment 1, blastocyst rates of NT embryos were significantly higher (P<0.01) in FBS (22.0％) and BSA (26.6％) groups than in PVA (6.3％) group. Total cell number was significantly higher in FBS (78.4$\pm$19.4) and BSA (90.9$\pm$29.1) groups than in PVA group (46.0$\pm$0.0). Apoptotic cell number was significantly fewer in FBS (3.1$\pm$1.4) and BSA (1.7$\pm$1.4) groups than in PVA group (7.0$\pm$20.0) However, all of results were not different between the FBS and BSA group. In experiment 2, blastocyst rates of NT embryos were significantly higher (P<0.05) in fatty acid free-BSA (FAF-BSA) group (26.8％) than in fraction V-BSA group (11.2％). Total cell number were somewhat higher in FAF-BSA group (89.8$\pm$30.7) than in fraction V-BSA group (88.1$\pm$19.3). Apoptotic cell number were somewhat fewer in FAF-BSA (1.7$\pm$1.5) group than in fraction V-BSA group (4.2$\pm$2.9). These findings suggest that serum free condition were effective for the in vitro development of bovine NT embryos. Therefore, we concluded that fatty acid free-BSA has beneficial effect in development bovine NT embryos and can be use as a serum substitute.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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• pp.194-200
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• 2009

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of <20 and 20 ${\mu}m$ cumulus donor cells compared to the >20 ${\mu}m$ cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 $\mu{m}$ fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with <20 and >20 ${\mu}m$ donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of <20 and 20 ${\mu}m$ oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 ${\mu}m$ donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.275-282
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• 2000

Objective: This study was to determine the effect of different concentration of calcium III medium on the preimplantational development of zygotes and early 2 cell embryos. Methods: Female mice of ICR strain ($5{\sim}8$ weeks old) were superovulated and mated with fertile males. Zygotes or early 2-cell embryos were collected by flushing the oviducts $31{\sim}32$ hours after hCG injection. The embryos were cultured in various concentrations of $Ca^{2+}$ in medium or with EDTA, EGTA and $Ni^{2+}$. Result and Conclusion: Treatment of high concentraion of $Ca^{2+}$ (3.42 mM $(2X){\sim}17.l$ mM (10X)) in medium didn't develop well compared to the control. Low concentrations of $Ca^{2+}$ (0.214mM $(1/8X){\sim}0.855$ mM (1/2X) were deterimental to development beyond 2-cell stage. EDTA, $Ca^{2+}$ chelating agent was treated with ranged concentrations of EDTA (0.014 $mM{\sim}0.107$ mM) to medium contaning 1.71 mM $Ca^{2+}$ showed beneficial effect to development to blastocyst compared to the control. EGTA, extracellular $Ca^{2+}$ chelator, was treated with ranged concentrations of EGTA ($0.014{\sim}0.107$ mM) to the medium contaning 1.71 mM $Ca^{2+}$. There is no significant difference with the control. $Ni^{2+}$ (50 ${\mu}M$), T-type $Ca^{2+}$-channel blocker was treated to medium contaning low concentration of $Ca^{2+}$. It overcame 2-cell block significantly. Rate of degenerated embryos decreased and developmental rate to morula and blastocyst increased more than low $Ca^{2+}$ concentration alone. Further studies are needed for the overcoming effect of 2-cell block by $Ni^{2+}$.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.203-209
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• 2011

Objective: This study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined. Methods: Eight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes ($Rbm3$, $Birc5$, $Sod1$, $Sod2$, $Cirbp$, $Caspase3$, $Trp53$, $Hsp70.1$) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos. Results: There were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers ($107.0{\pm}21.0$ vs. $115.0{\pm}19.7$), or ICM cell numbers of blastocysts ($11.3{\pm}5.2$ vs. $11.1{\pm}3.7$). Cell numbers of blastocysts were significantly ($p$ <0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of $CirbP$ and $Hsp70.1$ were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences. Conclusion: These results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.89-93
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• 1995

In this study was demonstrate that retrovirus-mediated gene transfer is one of the promising alternatives to the conventional pronuclear DNA microinjection approach, especially in transferring the exogenous genes into the boving embryos. By co-culturing of zona of zona-free one-cell stage embryos with the retrovirus-producing cells for 24 hours followed by 6 days of culture in virus-free medium, we could get morulae and blastocysts expressing the E. coli LacZ genes which were transferred by our retrovirus vector. The results obtained in this study are summarized as follows : 1. Addition of 5$\mu\textrm{g}$/ml of polybrene in the embryo and virus-producing cell co-culture medium did not affect development of zona-free one-cell embryo. 2. Compared with the intact embryos removal of zona at one-cell stage before co-culturing with the virus-producing cells for one day caused only slight decrease of embryo develpment. 3. Co-culture of 625 zona-free one-cell stage embryos with the virus-producing cells resulted in 65(10.4%) morulae or blastocysts, and 12.3%(8/65) of the morulae or blastocysts were E. coli LacZ positive.

• Korean Journal of Animal Reproduction
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• v.11 no.2
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• pp.127-131
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• 1987

This study was carried out to obtain the basic information on splitting and culture of mouse and bovine embryos. Two-, four-, eight-, cell and morula mouse embryos were digested with pronase, splitted in vitro by micro-glass needel with hand, and bovine embryos were splitted by micromanipulator. The splitted embryos were cultured under 5% of CO2 gas in air at 37$^{\circ}C$ for 48-72 hours. The results obtained in this study were summarized as follows: 1. The mouse and cattle were superovulated by 5IU of PMS and HCG, and 2500IU of PMS and 25mg of PGF2$\alpha$, respectively. The average number of embryos after superovulation were 32.5$\pm$8.2 and 7.5$\pm$3:1, respectively. 2. Out of total 122 embryos splitted, the successful splitting rate was 75.0%, 66.7%, 68.4% and 71.4% in 2-, 4-, 8- and morula embryos in mouse, respectively. There was no different splitting rate between mouse(71.4%) and bovine embryos(66.7%) in morula. 3. The successful culture rate of splitted embryos was 68.0% and 67.9% in mouse and bovine embryos, respectively.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.229-237
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• 2015

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p<0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.183-187
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• 2009

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at $39^{\circ}C$ in 5% $CO_2$, 5% $O_2$ for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.