• Clinical and Experimental Reproductive Medicine
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• v.51 no.1
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• pp.57-62
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• 2024

Objective: The purpose of this study was to use a mouse model to investigate the blastocyst formation rate in vitrified-warmed embryos derived from vitrified-warmed oocytes. Methods: Metaphase II oocytes obtained from BDF1 mice were vitrified and warmed, followed by fertilization with epididymal sperm. On day 3, a total of 176 embryos, at either the eight-cell or the morula stage, were vitrified-warmed (representing group 1). For group 2, 155 embryos at the same developmental stages were not vitrified, but rather were directly cultured until day 5. Finally, group 3 included day-5 blastocysts derived from fresh oocytes, which served as fresh controls. The primary outcome measured was the rate of blastocyst formation per day-3 embryo at the eight-cell or morula stage. Results: The rates of blastocyst formation per day-3 embryo were comparable between groups 1 and 2, at 64.5% and 69.7%, respectively (p>0.05). The formation rates of good-quality blastocysts (expanded, hatching, or hatched) were also similar for groups 1 and 2, at 35.5% and 43.2%, respectively (p>0.05). For the fresh oocytes (group 3), the blastocyst formation rate was 75.5%, which was similar to groups 1 and 2. However, the rate of good-quality blastocyst formation in group 3 was 57.3%, significantly exceeding those of group 1 (p=0.001) and group 2 (p=0.023). Conclusion: Regarding developmental potential to the blastocyst stage, vitrified-warmed day-3 embryos originating from vitrified-warmed oocytes demonstrated comparable results to non-vitrified embryos from similar oocytes. These findings indicate that day-3 embryos derived from vitrified-warmed oocytes can be effectively cryopreserved without incurring cellular damage.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.365-373
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• 1994

To investigate the effect of EDTA on the in vitro development of parthenogenetic eggs of ICR strain mice, those were cultured in 35mm culture dishes containing NaHCO3-BMOC-3 medium supplemented with 10, 50, 100, or 500$\mu$M of EDTA at 37$^{\circ}C$ for 96hrs. under the atmosphere of 5% CO2 and 95% air. EDTA supplementation of 10, 50, or 100$\mu$M to medium significantly(P<0.01) increase morula and blastocyst formation rate compared with controls in haploid(19.8, 25.9, 39.0% vs. 0.0%). And compared with 10, or 50$\mu$M of EDTA supplementation, significantly(P<0.01) higher morula and blastocyst formation rate resulted from EDTA supplementatin of 100$\mu$M. Both the nuclear number and diameter of blastocysts developed from parthenogenetic eggs were not affected by the morphological types when they were cultured, or the supplementary concentrations of EDTA. The nuclear number of blastocysts developed from haploid, diploid, and immediately cleavaged eggs was 44.8$\pm$1.2, 45.2$\pm$1.5, and 45.4$\pm$1.8, respectively. And the diameter of those eggs ranged 104.4$\pm$1.8, 104.3$\pm$1.2, and 103.8 1.3$\mu$m, respectively.

• Journal of Veterinary Clinics
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• v.10 no.2
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• pp.243-249
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• 1993

Although plenty of experiments for production of genetically identical twins have been reported, most of these reports were focused on microsurgical bisection of morula stage embryos, and little research has been performed for the production with frozen emb교os. The objective of these experiments were to assess the in vitro and in vitro developmental potential of blastomeres isolated from frozen-thawed 4-cell and 8-cell mouse embryos and to produce the identical multiplets from those embryos. The percentages of isolated 1/4, 2/4, 4/4, (control), 1/8, 2/8, 3/8, 4/8 and 8/8(control) blastomeres of 4-cell and 8-cell mouse emb교os which developed to normal blastocyst were 38.7%, 73.6%, 96.2(control), 15.1%, 40.1%, 65.9%, 88.3%, and 98.5%(control), respectively. The percentages of isolated 1/4, 2/4, 4/4(control), 1/8, 2/8, 3/8, 4/8 and 8/8(control) blastomeres of frozen-thawed 4-cell and 8-cell mouse embryos which developed to normal blastocyst were 35.6%, 68.5%, 100.0%(control), 16.1%, 50.6%, 71.7%, 90.9% and 100.0%(control), respectively. Normal 26 pups were obtained by transfer of 240 blastocyts developed 1, on 1/4 to 8/8 blastomeres. Those 26 pups obtained, 4 identical twins were produced from 2/4, 2/8, 3/8 and 4/8 blastomeres.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.293-301
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• 1996

In the present study, mouse follicular oocytes and 2-cell embryos(late -zygote stage embryos included) were cultured on the electric pad for electric stimulation in the culture incubator. In addition, follicular oocytes and embryos were tested for maturation and development under higher temperature condition($39^{\circ}C$).Mouse follicular oocyte maturation were not affected by anion electric stimulation and there is no significant difference in GBVD and MI between the control and experiment group after 4hr culture. In the embryo culture, it was found that more morula and blastocyst were found in the electric stimulation group rather than the control(96hr). This may seem to be caused with cytoplasmic $Ca^{2+}$ transient rise by electric stimulation(anion pad). On the other hand higher temperature incubation ($39^{\circ}C$) on the anion pad caused all the embryos degenerated within $12h{\sim}24hr$ culture. This was quite different from large animal embryos(bovine, pig, sheep), in which beneficial effect of high temperature incubation for oocyte maturation and embryo development were found.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.173-180
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• 1994

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse 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. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.85-92
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• 1999

This study was designed to determine effect of cryoprotectant kinds and cell stages on OPP vitrification method in mouse embryos. The freezing speed, cryoprotectants and cell stage could affect of embryo viability following various vitrification methods. The vitrification solution used were consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose solution in holding medium (D-PBS supplemented with 5% FCS: HM) (EFS) or 16.5% ethylene glycol , 16.5% dimethyl sulfoxide, 0.5 M sucrose in HM (EDS). The embryos were collected from oviduct at 18 h after hCG injection and then washed and cultured in mHTF medium until use. In experiment 1, the blastocysts were vitrified by OPP straw to determine the optimal vitrification solution of EFS or EDS. The post-thaw survival rates at re-expanded stage rates were significantly different between EFS and EDS (95.0 vs 100%), but at hatching stage was not different between EFS and EDS (90.0 vs 95.0%). respectively. In experiment 2, zygotes, 2-, 4-cell, morula and blastocysts were vitrified by OPP method to determine the acceptable of early stage embryos. The development rates to expanded blastocyst in zygote (70.0%) were significantly lower rather than those in 2-, 4- 8-cell, compacted morula or blastocyst (89.7, 90.0, 92.8, 97.6 or 97.5%), respectively. However, the cell number of post-thaw developed to expanded blastocyst in blastocyst and control blastocyst stage (39.6$\pm$2.81, 35.7$\pm$2.98) were significanty higher than those in zygote, 2-, 4-, 8-cell, compacted morula (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 or 30.8$\pm$2.93). In experiment 3, the zygotes were exposed in VSl for 1, 2, and 3 min to the optimal exposed time. The cleavage rates (91.6, 88.5, 88.9%) and develop mental rates to blastocyst (83.3, 74.3 and 69.4%) depends on the exposed time in VSl were not significantly different among 1, 2, or 3 min, respectively. The cell number also were not significantly different among exposed time in VS1. respectively. These results indicate that OPP method could be useful for vitrification either EFS or EDS vitrification solution. The post-thaw survival rates at zygote were significantly lower than those at 2-, 4-, 8-cell, morula or blastocyst, respectively. The zygote stage were more sensitive rather than late stage embryos. The exposing time in VS1 for 1 min was better than that for 2 or 3 min, even it was not significantly different. The OPP vitrification method could be useful of mouse embryos either with EFS or EDS vitrification solution.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.55-62
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• 1994

This study was carried out to test the effects of fluorescein diacetate(FDA 0${mu}ell$/ml, 0.5${mu}ell$/ml, 5${mu}ell$/ml, 10${mu}ell$/ml or 0${mu}ell$/ml, 5${mu}ell$/ml in PBS) treated before culture on the survival of vitrified mouse morulae in vitrification solution(20% glycerol＋glycerol＋10% ethylene glycol＋30% ficoll＋10% sucrose). The results were summarized as follows; 1. The survival rate of FDA-tested fresh mouse morulae after 24 hours culture was over 96%((P4.8) in the control or treatment groups with various levels of FDA. Because the rate of mouse morulae which developed to hatched blastocysts was higher with the various levels of FDA treatment(67%) than control(50%), it was considered that toxicity of FDA did not affect the survival of mouse morulae. 2. When mouse morulae were FDA-tested in FDA 0(control), 0.5, 5, and 10${mu}ell$/ml treatment after vitrification, the development rate to expanded blastocyst were 66, 82, 64 and 76%, and FDA scores were P4.2(84%), P4.7(94%), P4.2(84%) and P3.9(78%), respectively. There were no significant differences between control and FDA treatments, but there were significant difference between 0.5${mu}ell$/ml)94%) and 10${mu}ell$/ml(78%) treatment(P<0.01). 3. The survival rates of cultured mouse morulae according to FDA-scores(P0=non-fluorescence; P1~P5=according to their fluorescence) after vitrification were P5;92%, P4;67%, P3;42% and P2.P0;0%, respectively. 4. Implantation rates of morulae stage embryos cultured into early blastocysts and implanted into uterine hornes vitrification were 14 and 11% embryos treated control(0${mu}ell$/ml) and FDA 5${mu}ell$/ml and the normal fetus development was 2% embryos for both treatments. Results of this percent study indicated that toxicity of FDA does not affect not only the survival of fresh and vitrified mouse morulae but also the development rate and implantation of fetus after transfer as well. The development rates of mouse morulae with the FDA score of P5, P4 and P3 were 92, 67 and 42%, respectively, it was considered that FDA-test was fit for the judge of survival.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.4
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• pp.189-196
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• 2019

Objective: We aimed to evaluate the effects of different oxygen conditions (20% [high O₂], 5% [low O₂] and 5% decreased to 2% [dynamic O₂]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture. Methods: The high-O₂ and low-O₂ groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O₂ group, mouse embryos were cultured from the one-cell to the morula stage under 5% O₂ for 3 days, followed by culture under 2% O₂ to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets. Results: The blastocyst formation rate was significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The total cell number was significantly higher in the dynamic-O₂ group than in the low-O₂ and high-O₂ groups. Additionally, the apoptotic index was significantly lower in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. Conclusion: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.239-243
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• 2001

Selection of oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. Compared with slow freezing method, the vitrification has various advantages such as avoiding intracellular ice crustal formation. In our previous, we attempted to employ a vitrification method using ethylene glycol and an electron microscope grid for cryopreservation of mouse oocytes. However, A high incidence of spindle and chromosome abnormalities was detected in thawed oocytes after vitrification. We examined whether the addition of a cystoskeleton stabilizer Taxol $^{TM}$, to the vitrification solution could promote the post-thawed survival and subsequent development of stored oocytes. More oocytes developed to the 4-cell (44.7% vs. 69.7%), 8-cell (31.8% vs. 64.2%), morula (24.7% vs. 54.3%), and blastocyst (20.3% vs. 49.2%) stages after the addition of Taxol$^{TM}$ to the cryoprotectant than after no addition. 21 and 26 mouse pups were born after transfer of blastocyst derived from oocytes vitrified without and with Taxol. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of ICR morose oocytes.tes.