• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.191-200
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• 2000

Objective: This study was carried out to compare the effects of the stepwise exposure treatments on the morphological normality, fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing and to use as a fundamental data for the cryopreservation of human oocytes. Materials and Methods: The morphological normality and fertilization rates of the vitrified and ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were observed. After choosing the 3step exposure treatment groups, we observed the morphological normality and fertilization, blastocyst formation rate of the vitrified and ultra-rapid frozen mouse mature oocytes. Results: The morphological normality and fertilization rates of the vitrified mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 75%, 85%, 88% and 58%, 61 %, 54% respectively. There were no significant differences among treatments(p>0.05). The morphological normality and fertilization rate of the control was 92% and 65%. There were no significant differences in fertilization rate among control and treatments (p>0.05). The morphological normality and fertilization rates of the ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 83%, 83%, 84% and 75%, 63%, 56% respectively. There were no significant differences among treatments (p>0.05). The morphological normality and fertilization rate of the control was 95% and 67%. There were no significant differences among control and treatments (p>0.05). The morphological normality and fertilization rate of the vitrified or ultra-rapid frozen mouse mature oocytes after 3step exposure treatment were 69% and 75%, respectively. The blastocyst formation rate was 60% and 57%. The results did not differ significantly between vitrification and ultra-rapid freezing (p>0.05). Conclusion: As known in the above results, there were no significant differences in the fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing among the control and treatments. It is suggested that vitrification and ultra-rapid freezing method were effective for the cryopreservation of mouse mature oocytes.

• Journal of Animal Science and Technology
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• v.55 no.5
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• pp.417-425
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• 2013

We sought to provide a method for freezing and preserving primordial germ cells, or an avian germ cell of a bird, as a material for developmental engineering or species preservation. The aim of this study was to compare the efficacy of slow freezing with a vitrification method for the cryopreservation of chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonad of day 5.5-6 day (stage 28) cultured chick embryos, using the MACS method, were classified into two groups: slow freezing and vitrification. We examined the viability of PGCs after Cryopreservation. Four freezing methods were compared with each other, including the following: Method 1: The PGCs were frozen by a programmed freezer in a plastic straw, including 2.0 M ethylene glycol (EG) as cryoprotective additive (slow freezing) Method 2: The PGCs were vitrified in a plastic straw, including 8.0 M EG, plus 7% polyvinylpyrrolidone (PVP) (rapid freezing). Method 3: The slow freezing was induced with a cryotube including 2.0 M EG Method 4: The PGCs were frozen in a cryotube including 10% dimethyl suloxide (DMSO) (rapid freezing). After freezing and thawing, survival rates of the frozen-thawed PGCs from Method 1 to 4were 76.4%, 70.6%, 80.5% and 78.1% (p<0.05), respectively. The slow freezing ($-80^{\circ}C$ programmed freezer) method may provide better survival rates of frozen-thawed PGCs than the vitrification method for the cryopreservation of PGCs. Therefore, these systems may contribute to the cryopreservation of a rare avian species.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.121-129
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• 2001

Objective: The aim of this study is to compare the efficiency of a method for the cryopreservation of mouse blastocyst.. Methods: Mouse embryos were obtained at 2-cell stage and cultured to blastocyst stage in T6 medium supplemented with 10% fetal bovine serum. Morphologically normal blastocysts were collected and randomly divided to one control and four experimental groups. In control group, blastocysts were cultured in vitro continuously for additional two days. In group 2, blastocysts were exposed to vitrification solution (ethylene glycol) only without cryopreservation (exposure only group). In group 3, 4 and 5, blastocysts were cryopreserved by slow-freezing procedure with glycerol (slow-fteezing group) or by vitrification procedure using EM grids (EM grids group) and cryoloop (cryoloop group), respectively. Frozen blastocysts were thawed and cultured for additional two days. Twenty four hours after thawing, some blastocysts were fixed and stained with Hoechst 33342 (bisbenzimide) and the number of nuclei in each blastocysts were counted to confirm the survival of bias to cysts in experimental groups. Results: Survival rate and hatching rate of the blastocysts in slow-freezing group (24 h: 72.4% and 66.0%, 48 h: 63.2% and 64.6%) and EM grids group (24 h: survival rate 77.3%, 48 h: 70.1% and 71.4%) were significantly lower ($X^2$-test p<0.05) than those of control group (24 h: 93.4% and 86.0%, 48 h: 88.5% and 90.7%). In contrast, the survival rate and hatching rate of the blastocysts in cryoloop group (24 h: 84.1% and 84.1%,48 h 79.3% and 87.7%) is well compared with those in the control group. The mean (${\pm}SD$) cell number of blastocyst in the exposure only ($89.2{\pm}11.5$), EM grids ($85.0{\pm}10.3$) and cryoloop ($89.0{\pm}11.0$) groups, except slow-freezing group ($79.0{\pm}10.0$), were not significantly different from that of control group ($93.1{\pm}13.9$) 24 h after thawing (Student's t-test). Conclusion: This study demonstrates that higher survival rate of vitrified-thawed mouse blastocyst can be obtained using cryoloop as the embryo container at freezing rather than slow-freezing or vitrification using EM grids. The results of this study suggest that vitrification using cryoloop (with ethylene glycol) may be a preferable procedure for mouse blastocyst cryopreservation and could be applied to the human blastocyst cryopreservation.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.225-230
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• 2000

This study was designed to investigate effect of cryoprotectant kinds and cell stages on the viability of bovine embryos cryopreserved by vitrification. The oocytes were collected from ovarian follicles of Korean native cows. The follicular oocytes were cultured in TCM-199 medium containing hormone and 10%(v/v) FCS for 24~48hrs in a incubator with 5% $CO_2$, in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 7~10 hrs with spermatozoa capacitated by preincubation. The vitrification solutions of EFS and EDS were consisted of 40%(v/v) ethylene glycol, 18%(v/v) Ficoll and 0.3M sucrose, and 20%(v/v) ethylene glycol, 16.5%(v/v) DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10% FCS, respectively. The embryos were exposed to EFS or EDS at $25^{\circ}C$ and loaded into OPP straw for 30 sec. The plug end of each straws was heat-sealed and straws was slowly immersed into liquid nitrogen(L$N_2$). The results obtained were summarized as follows : 1 . The rates of cleavage and hatching of embryos frozen with vitrification, rapid and slow freezing methods were 67.5%, 27.5% and 42.5%, 20.0% and 52.5%, 25.0%, respectively And rates of cleavage and hatching of embryos frozen with vitrification method were significantly(p<0.05) higher than those in other methods, and the rates were lower than those in control group(82.5% and 37.5%). 2. The rates of cleavage and hatching of embryos were significantly(p<0.05) different between EFS(47.5% and 22.5%) and EPS(52.5% and 27.5%), and the rates were lower than those in control group(82.5% and 37.5%). 3. After vitrification freezing of bovine embryos at zygote, 2 cell, 8 cell, morulae and blastocyst stage, the rate of cleavage and hatching were 25.0% and 15.0%, 32.5% and 20.0%, 37.5% and 20.0%, 52.5%, 27.5%, 47.5% and 25.0%, respectively. And developmental rates to the expended blastocyst stage of embryos frozen at zygote stage was significantly(p<0.05) lower rather than those in 2, 8-cell and morulae stage.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.251-255
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• 2007

This study was carried out to examine on developmental competence of Hanwoo embryos cultured in vitro according to culture conditions and freezing methods. The in vitro developmental competence to blastocyst stage at Day 8 of culture in SOF was significantly (p<0.05) higher than that in CR1aa (30.3% vs. 18.4%). The in vitro developmental rate of morula and blastocysts cultured in group culture was significantly (p<0.05) higher than that in individual culture (41.4% and 36.0% vs. 21.1% and 10.5%, respectively). The cell number of Day 8 blastocysts in group culture was significantly (p<0.05) higher than that in the individual culture ($120.1{\pm}12.8\;vs.\;94.1{\pm}12.1$, respectively). The survival rates of frozen-thawed balstocysts that were exposed in 1.5 M ethylene glycol or 1.5 M ethylene glycol containing 0.1 M sucrose were 77.5% and 78.7%, respectively. The survival rates of blastocysts cultured for 48 h in slow freezing and vitrification was not significantly different (73.3 and 74.0%). In conclusion, in vitro developmental competence of bovine embryos was influenced on the culture medium (SOF) and culture method (Group culture). Survival rate of frozen-thawed of bovine embryos was not influenced on freezing solutions and freezing methods.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.271-281
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• 1996

The effect of superovulation (PMSG, FSH) on the ovarian response of matured cows were tested. The survival rates of bovine embryos and ovarian oocytes frozen by slow, rapid freezing and vitrification were investigated. A total of 15 heads of cow were devided into 3 groups by injection dose of GTH (PSMG, FSH). Each group was superrovulated with injections of 2, 500, 3, OOOJU PSMG and 40mg FSH followed by injection of 30mg PGF2a. Embryos were non-surgically recovered from superovulated cows 6~7days after estrus. The recovered embryos were frozen in 10% glycerol + 10% sucrose by slow and rapid freezing. Ovarian oncytes were frozen in 20% g]ycerol+l0% ethylene glycerol + 30% Ficol + 10% sucrose by vitrification and the survival of frozen embryos and ovarian oncytes were judged by FDA-test. The results are summarized as follows; 1. Estrus after the injection of 2500, 3000 I.U. PMSG and 4Omg FSH were 32.8, 35.0 and 43.4 and the duration of estrus were 18.6, 18.8 and 22.4 hours respectively. 2. The average sizes of the left ovaries were 5.4cm (2, 500 IU PMSG), 5.1cm (3, OOOIU PMSG) and 6.4cm (FSH), and the right were 6.2cm (2, 5001U PMSG), 5.7cm (3, OOOIU PMSG) and 7.&m (FSH) respectively. There were significant differences in the right overies among treatments (P<0.05). 3. The average number of ovarian follicles in the left ovaries were 4.8 (2, 500 IU PMSG), 5.2(3, 000 IU PMSG) and 7.8 (FSH) respectively. There were significant difference in the right ovaries among treatments (P<0.05). 4. In the average numbers of ovulation points in the left ovaries were 3.0 (2, 5001U PMSG), 3.2 (3, OOOIU PMSG) and 4.4 (FSH) respectively, and the right were 7.2 (2, 5001U PMSG), 7.8(3, 000IU PMSG) and 11.4 (FSH). There were significant differences in the right ovaries among treatments (P<0.05). 5. The numbers of the recovered embryos were 20 (2, 5001U PMSG), 19 (3, 000 IU PMSG) and 21 (FSH) respectively, and oncytes and degenerted oncytes were 6.5 and 11.0 Estrus periods of post parturation were 52.4days (2, 5001U PMSG), 69.8days (3, OOOIU PMSG) and 62.4days (FSH) respectively. 6. The FDA score of cow morulae frozen by slow freezing, sernirapid frezing and vitrified freezing was higher in slow (3.1) and vitrified freezing (3.0) than that in semirapid freezing (1.28). The FDA-scores of cow, pig and rabbit ovarian oocytes frozen in 20% glycerol + 10% ethylene glycol + 30% Ficoll + 10% sucrose by vitrification were higher in cows (3.3) than both in pigs (2.6) and rabbits (2.3).

• Clinical and Experimental Reproductive Medicine
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• v.37 no.4
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• pp.267-291
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• 2010

Life can be kept in suspended animation either before fertilization at oocyte stage or after fertilization at different stages of embryonic development for a variety of reasons. It not only has potential applications in fertility preservation and management in human but also has important roles in the preservation and management of animal genetic resources, low-cost international movement of selected genetics, and rapid dissemination of germplasm through assisted reproductive technologies (ART) and genetic engineering. Currently, slow-freezing and vitrification are the two approaches by which oocytes and embryos can be cryopreserved for long-term storage. Both of these methods have their own advantages and disadvantages but allow the cryopreservation of oocytes and embryos with comparable efficiency. Vitrification of oocyte and embryos, although proven successful just 13 years after slow-freezing, is generally considered an emerging technology and appears to slow gain acceptance in both animal and human ART despite having controversial storage and contamination issues. In this manuscript, we discuss the basic techniques of oocyt/embryo cryopreservation and review the current status and recent developments in vitrification.

• 대한생식의학회:학술대회논문집
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• 2005.06a
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• pp.126-126
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• 2005

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.98-98
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• 2003

Recently, human embryonic stem (hES) cells have become very important resources for ES cell basic research, cell replacement therapy, and other medical applications; thus, efficient cryopreservation methods for these cells are needed. This study examined whether a newly developed minimum volume cooling (MVC) vitrification method, which was tested through cryopreservation of sensitive bovine oocytes, can be used for freezing hES cells. Feeder-free cultured hES cell (MB03) colonies were mechanically dissected into several small clumps following enzymatic treatment. We compared the freezing efficiency of a slow-cooling method using a cryo-module (0.4-0.6C/min, 20-30 clumps/vial) and MVC vitrification using a modified 0.5-ml French mini-straw designated as a MVC straw (>$20,000{\circ}C$/min, 10 clumps/straw) After thawing, in vitro survival of hES cell clumps was higher for MVC-vitrified cells (80.8%, 97/120) than for slow-cooled cells (38.2%, 39/102). Further, the proliferation rate of surviving MVC-vitrified cells was similar to that of control hES cells from 2 weeks after thawing. In addition, vitrified-thawed hES cells demonstrated a normal karyotype, were positively immunostained for surface marker antibodies (AP, SSEA-4 and TRA-1-60) and the Oct-4 antibody, and could differentiate into all three embryonic germ layer cells in vitro. This result demonstrates that hES cell clumps can be successfully cryopreserved by a newly developed MVC vitrification method without loss of human cell characteristics.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.76-76
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• 2001

The objective of this study was to optimize the vitrification method of in vitro produced bovine embryos. Thus, in vitro produced embryos at 8 cell, morula and blastocyst stages were vitrified on electron microscope grids (EM grids) or in open pulled straws (OPS) with EG5.5 (5.5 M ethylene glycol, 1.0 M sucrose and 10% FBS in m-DPBS medium) freezing solution and their survival rates after thawing were compared. The embryos on EM grids or in OPS were briefly exposed to EG5.5 freezing solution and plunged directly into liquid nitrogen within 30 to 35 sec. Post-thawed embryos were serially diluted in 0.5, 0.25 and 0.125 M sucrose in m-DPBS, each for 1 min, and then cultured in CRI aa medium supplemented with 10% FBS. Embryonic survival rate was assessed as re-expanded and hatched rates of those embryos after warming. The rates of re-expansion embryos did not significantly different between EM grid (8 cell: 42.10%, morula: 66.66% and blastocyst: 77.08%) and OPS (8 cell: 47.36%, morula: 61.90% and blastocyst: 83.33%) methods. In addition, the hatched rates in EM grid (8 cell: 31.57%, morula: 57.14% and blastocyst: 72.91%) were similar to those in OPS (8 cell. 34.21%, morula: 50.00% and Blastocyst: 77.08%). Interestingly, even at the same blastocyst stage, the in vitro survival of day 7 embryos (EM grid: 79.48 and OPS: 87.18%) was higher than those of day 8 embryos (EM grid: 72.10 and OPS: 82.06%). The total cell number of blastocyst developed in vitro after vitrification was examined with Hoechst 33342 staining to compare the embryo quality among different treatment groups. The total cell number of blastocyst was not significantly different between vitrified groups (EM grid: 162.4$\pm$8.0 and OPS: 158.4$\pm$7.1) and unvitrified control (168.0$\pm$5.6). These results indicate that both vitrification containers can provide the high rate of embryo survival. Moreover, the OPS container may not need a cap to protect the container from floating after immersion in L$N_2$. Therefore, this study suggest that bovine embryos can be cryopreserved easily, effectively and successfully by vitrification method using EM grid or OPS with EG5.5 freezing solution. In the future, the Pregnancy rate would be investigated after transfer of our vitrified embryos into the appropriated recipients.