• Clinical and Experimental Reproductive Medicine
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• v.28 no.3
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• pp.191-198
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• 2001

Objective : This study was conducted to examine the effect of vitrification on the survival and in vitro development of mice 1-cell zygotes. Method: Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. The 1-cell zygotes were also subjected to a slow freezing-thawing method to compare with vitrification method. Solution composed of ethylene glycol (6.0 M, 5.0 M, 4.0 M) and sucrose (1.0 M) were used as cryopropectant. The experiments employed the method loading the embryos on electron microscope grids. Results: I. The effects of exposure in vitrification solution. 1-cell zygotes were non-toxic at all concentrations of the vitrification solution showing the survival rate between 88.1% and 97.5%. Development into 2-cell was more successful in the higher concentrations of the vitrification solution. Therefore, higher concentrations of the vitirification solution do not seem to cause any problems in vitrification procedure. II. The effects of vitrification method. 1-cell zygotes showed the survival rate between 78.8% and 92.4%. The lowest and the highest survival rate was observed in the 6.0 M and 4.0 M vitrification solution, respectively. 2-cell development rates varied from 77.6% to 91.3%. Blastocyst development rate was shown highest in 5.0 M and the lowest in 4.0 M solution. Therefore, the highest 2-cell and blastocyst development rate was observed in 5.0 M solution. III. Comparison of vitrification and slow freezing-thawing method on 1-cell zygotes. This experiment showed that 1-cell zygotes had the highest survival and development rates in 5.0 M vitrification solution. Vitrified group of 1-cell zygotes, in the 5.0 M vitrification solution, were compared with the group processed in slow freezing-thawing method. The development rate into 2-cell and blastocyst as well as the survival rate were higher in the vitrified group than in the slowly freezed group. Conclusion: 1. The results demonstrate that the best cryoprotectant is a 5.0 M vitrification solution for 1-cell zygotes. 2. Vitrification method significantly increases the survival rate of the 1-cell zygote and its development into 2-cell and blastocyst. Equilibration and exposure time during the vitrification was remarkerbly short in this experiment. Total time, from the exposure to vitirification solution to storage in the liquid nitrogen, was taken only 90 seconds. In contrast, the slow freezing-thawing method have taken more than four hours. Taken together, we presume that the overall time used for the procedure contributes to the results as an important parameter. 3. The loading of 1-cell zygotes on the EM grid is technically more simple and takes less time than the straw or cryo vial method.

• Composites Research
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• v.37 no.3
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• pp.215-218
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• 2024

Liquid crystalline thermosetting epoxy oligomer DD-A was synthesized with Diglycidyl ether of 4,4'-dihydroxy-α-methylstilbene (DGE-DHMS) and aniline in a ratio of 2:1 and cured with a catalytic curing agent, 1-Methyl Imidazole. The gelation times and vitrification times were measured to create Time-Temperature-Transition Diagrams with liquid crystalline phase changes. It was found that the gelation and vitrification times were decreased as the concentration of curing agent increased, and the vitrification curve showing a typical S-shape was confirmed.

• Journal of Forest and Environmental Science
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• v.36 no.4
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• pp.274-280
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• 2020

As the importance of biological resources increases, the conservation technology is becoming important for rarities. This study was conducted to establish an efficient cryopreservation conditions for the Deutzia paniculata, endangered plant species, by using both cryopreservation methods of vitrification and encapsulation. As a result, the sucrose pretreatment seed viability showed up to 30.7% in the treatments. The cryoprotectant treatment improved the viability of the seeds, and was found to be excellent in the vitrification method using PVS3. The vitrification method had over 10% higher germination rate than the seeds preserved by encapsulation. In addition, the germination rate showed a significant difference according to the cryopreservation treatment time, and the germination rate of seeds decreased very much as the long time became longer. Plants germinated from preserved seed in liquid nitrogen showed poor growth compared to untreated, and good growth in PVS3 120 minutes. In addition, the growth of germinated plants by liquid nitrogen treatment time was better in the vitrification method. These results are expected to be useful for long-term preservation of D. paniculata, endangered plants.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.635-641
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• 1995

The purpose of this study was to investigate the effects of developmental stage and equilibration time on survival of rabbit embryos following freezing by vitrification. Adult New Zealand White female rabbits were superovulated with PMSG and hCG. The 8-cell stage embryos were collected from 40 to 45 hours after hCG injection by flushing oviducts with Dulbecco's phosphated buffered saline and in vitro cultured in TCM-199 containing 10% fetal calf serum(FCS). Each embryos developed in vitro to 16-cell, compact morula and blastocyst was cryopreserved and cultured following thawing to examine their developmental potential to expanded blastocyst stage in vitro. The frozen-thawed-cultured embryos were stained with Hoechst 33342, and their nuclei were counted using a fluorescence microscope. On the toxicity test of EFS solution as cryopreservation, the survival rates of 8-cell stage embryos was decreased in reverse to increasing of exposure time over 5 minutes. The post-thaw survival rates of embryos on equilibration times was significantly(P<0.05) higher for 2 min. than for 5 or 10 minutes. From morula to blastocyst of rabbit embryos was more suitable than 8-cell stage for cryopreservation by vitrification. The higher post-thaw survival rate of embryos can be achieved by keeping the cryoprotectant at $4^{\circ}C$ than at $20^{\circ}C$. The mean number of nuclei per embryo following freezing by vitrification and in vitro culture to expanded blastocyst at compacted morula and blastcyst was not significantly differ from fresh blastocyst.

• Development and Reproduction
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• v.18 no.2
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• pp.117-125
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• 2014

Vitrification method is widely used in oocyte cryopreservation for IVF but the birth rates are lower than that of the fresh oocyte. One of the known main reasons is structural instability of meiotic spindle and chromosome systems of mature oocyte. To get the best way for keeping competence of matured oocytes, we studied the best conditions for vitrification focused on equilibration times. The mature oocytes were underwent vitrification with current popular method and analyzed the survival rates, microtubule stability and DNA integrity. The survival rates of recovered oocyte are almost same between groups and are more than 93%. The structural configuration of meiotic spindle was well kept in 10 min equilibration group and the stability rate was almost same with that of control. The chromosomal breakdown was observed in all experimental groups, but the chromosomal stability was higher in 10 min equilibration group than the other groups. The 10 min equilibration group showed best condition compared with the other groups. Based on these results, the equilibration time is one of the key factors in successful keeping for competence of mature oocyte. Although, more fine analysis about the effects of physical stress on oocyte during vitrification is needed to define the optimal condition, it is suggested that the optimal equilibration time to get competent oocyte in mouse is 10 min. Information acquired this study may provide insight into intracellular structural events occurring in human oocytes after vitrification and application for cryopreservation of human oocyte.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.201-207
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• 2011

The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.

• Korean Journal of Plant Resources
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• v.19 no.6
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• pp.665-669
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• 2006

Somatic embryos do not survive at exposure to liquid nitrogen temperatures without cryoprotective treatments. A simplified technique which simultaneously induces and cryoprotects embryogenic calli using plant vitrification solution 2 (PVS2) followed by dehydration was developed for the cryopreservation of Soap berry genetic resources. Vitrification is a way of removing the moisture in vegetation through PVS2. The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% Dimethylsulfoxide (w/v) in B5 medium containing 0.4M sucrose. Two tests were done. The one was to eliminate moisture at $0^{\circ}C$ and the other at $25^{\circ}C$. In both cases the best results came out at a vitrification time of $10{\sim}20$ minutes. It was also found that the survival rate was higher at $0^{\circ}C$ than at $25^{\circ}C$. In particular, the survival rate reached more than 80%. Water-damaged embryos turned brown and stoped growth, but energetic embryos took on a milky hue and show a very vigorous growth rate. Successful cryopreservation of somatic embryos of soapberry can be used to establish in vitro genebanks for long-term conservation of Soapberry genetic resources to complement field genebanks and other in vitro methods already being used.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.317-323
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• 1993

This study was carried out to study effects of the addition level of acetamide and non-permeable cryoprotectants(Ficoll, sucrose) in VS(20% glycerol＋10% ethyleneglycol) and equilibration time on the survival of vitrified mouse morulae. The results are summarized as follows: 1. When 10, 15 and 20% of acetamide were added to the new vitrification solution(20G 10E), FDA-scores of embryos were 4.4(control), 4.4(10%), 3.6(15, 20%), respectively. The addition of acetamide did not affect the survival of forzen-thawed morulae(P<0.05). 2. The survival rate betwen 5 min(3.5) and 10 min(4.6), 10 min(4.6) and 20 min(3.2) of equilibration in 10% sucrose, and 20 min(3.2) and 5 min(4.0), or 10 min(4.3) in 20% sucrose were significantly different(P<0.05). The highest survival(4.6) rate was obtained in mouse morulae equilibrated in VS(20G 10E) containing 10% sucrose for 10 minutes. 3. FDA-score of morulae frozen in the new vitrification solution containing 0, 10, 20 and 30% Ficoll was 4.5, 4.2, 4.4 and 4.6, respectively and had no significant effect among concentrations of Ficoll(P>0.05). The development rate after culture(24h) was 89%(20% Ficoll) and 93%(30% Ficoll), respectively.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.49-57
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• 1991

This study was carried out to investigate the effect of equilibration time, precooling and straw loading method on the post-thaw survival rates of mouse embryos cryopreserved by vitrification method. The effect of the vitrification procedure on the damage of the embryos was also examined by the straining of nuclei using Hoechst 33342. The results obtained were summarized as follows; 1. The equilibration in Medium-1 for 10 minutes was considered to be the optimum equilibration time. Embryos equilibrated in Medium-1 for 10 minutes(81.0%) showed significantly(P<0.05) higher survival rates than those equilibrated for 5 minutes(40.0%) or 15 minutes(74.1%). 2. The survival rate of embryos cryopreserved using the double Medium-2 column(81.0%) was significantly(P<0.01) higher than that using the single Medium-2 column, whish was considered to be due to the double Medium-2 column method being more reliable for preventing contamination of diluent solution of 1M sucrose. 3. The survival rate of compacted morula stage embryos cryopreserved with the precooled and non-precooled Medium-2 was not significantly(P<0.05) different. 4. The number of blastomeres of embryos at late blastocyst stage after culture of mouse morulae for 24 to 28hours was significantly(P<0.05) decreased by freezing embryos using vitrification(53.3${\pm}$1.6 vs 41.4${\pm}$1.5), which was considered to be due to the damage of embryos during vitrification and the delay of embryo development by handling in vitro. 5. The vitrification procedure is considered to be a very simple and efficient method for cryopreservation of embryos at early developmental stage.

• 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.