• 한국자원식물학회지
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• 제19권6호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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• pp.283-289
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• 2000

Objective: The study was performed to compare the survival rate and the development of day 2 mouse embryos which had freezing procedures done. Methods: We used three different vitrification solutions (EFS, VS14, DPS) and a ultrarapid freezing solution (UFS) for cryopreservation of day 2 mouse embryo. Results: We tested toxicity by exposing embryos to vitirification solutions and a ultrarapid freezing solution. The survival rates are 100%, 97.8%, 95.6% and 100% (EFS, VS14, DPS and UFS). After cultured for 96 hours, hatching rates of each group are 93.5% (no freezing), 95.6% (EFS), 86.4% (VS14), 93.0% (DPS), and 93.0% (UFS). There is no significant differences among groups. The survival rates after thawing cryopreserved embryos are 80.2%, 91.7%, 69.5%, 0% and 91.8% (slow freezing, EFS, VS14, DPS and UFS). Also cultured for 96 hours, the hatching rates are 93.5% (no freezing), 84.1% (slow freezing), 93.9% (EFS), 48.5% (VS14) and 70.1% (UFS). Conclusion: The survival rates of vitrification in EFS solution and ultrarapid freezing are higher than slow freezing (p<0.05). The hatching rate of vitrification in EFS solution cultured for 96 hours is highest, so vitrification of day 2 mouse embryos in EFS solution considered as more effective for cryopreservation.

• 한국수정란이식학회지
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• 제18권1호
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• pp.1-14
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• 2003

본 연구는 미성숙, 성숙 단계의 돼지 난포란이 유리화 동결에 의한 동결보존이 가능한지를 조사 하고자 실시하였다. 난포란은 세포질 내 지방구를 분극시키기 위해 원심분리를 실시하였고, 미세조작기를 이용하여 지방구를 제거하였다. 돼지 난포란을 CB 처리하여 원심분리 후 지방구를 제거한 지방제거구(Delipated), CB 처리 후 원심분리만 하여 지방구를 분극시킨 원심분리구(Centrifuged), 아무처리도 하지 않은 대조구(Control)를 EM grid를 이용한 유리화 동결, 융해 한 후 생존율과 체 외 발생율을 조사하였다. 그 결과를 요약하면 다음과 같다. 1. 미성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시한 돼지 난포란의 생존율은 각각 15.1%, 0%, 0%로 지 방제 거구에서만 유의적으로 높은 생존율을 나타내었다(p<.01). 2. 성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시한 돼지 난포란의 생존율은 각각 12.2%, 0%, 0%로 지방제거구에서만 유의적으로 높은 생존율을 나타내었다(P<.01). 3. 미성숙 시기에 지방제거 후 유리화 동결을 실시하여 생존한 난자치 핵 성숙율은 37.5%로 동결하지 않은 난포란의 성숙율 68.9%보다 유의적으로 낮은 것이었다(P<.01). 4. 미성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시하여 생존한 난자의수정 후 배 발달율은 12.5%, 0%, 0%로 지방 제거구에서만 배 발달을 나타었으나, 세 처리간의 유의차는 없었고, 이것은 동결하지 않은 난포란의 배 발달율 56.1%보다는 유의 적으로 낮은 것이었다(P<.05). 5. 성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시하여 생존한 난자의 수정 후 배 발달율은 25.0%, 0%, 0%로 지방제거구에서만 배 발달을 나타내었으나 세 처리간의 유의차는 없었고, 이것은 동결하지 않은 난포란의 배 발달율 67.9%보다는 유의적으로 낮은 것이었다(P<.05).

• 한국수정란이식학회지
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• 제23권4호
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• pp.269-274
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• 2008

The purpose of this study was to determine the effects of Taxol pre-treatment to in vitro matured bovine oocytes, and sucrose and trehalose added to vitrification solution on spindle morphology and embryonic development following cryopreservation. Bovine oocytes were collected from ovaries and matured in tissue culture medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS), 0.05ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone and $1{\mu}g/ml$ estradiol for 22h in $39^{\circ}C$, 5% $CO_2$, TCM 199-HEPES containing 20% FBS was used as basic medium (BM) to prepare vitrification solution. Oocytes were pre-treated with $1\;{\mu}M$ Taxol in maturation medium for 15 min prior to vitrification. Oocytes were exposed to 1.6 M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM and then were exposed to 3.2 M EG, 2.6 M DMSO and 0.5 M sucrose in BM or 3.2 M EG, 2.6 M DMSO and 0.5 M trehalose in BM. Oocytes with cumulus cells and oocytes without cumulus cells were considered as control 1 and control 2, respectively and held in TCM 199-HEPES at $39^{\circ}C$. Oocytes were frozen using modified solid surface vitrification and were stored in cryotubes in liquid nitrogen for more than 1 week. Frozen oocytes were thawed in TCM 199-HEPES containing 0.5 M, 0.25 M and 0.1 M sucrose in BM for 2 min, respectively or 0.5 M, 0.25 M and 0.1 M trehalose in BM for 2 min, respectively. Immunoflurorescence staining of oocytes was performed to assess spindle morphology and chromosome configuration of oocytes. The rates of cleavage and blastocyst were examined following in vitro fertilization. Normal spindle morphology rate of oocytes pre-treated with Taxol prior to vitrification was not higher than that of other vitrified groups. Taxol pre-treatment did not increase cleavage and blastocyst formation rates, although control groups showed significantly higher rates (p<0.05). Percentages of normal spindle and embryonic development were not significantly different among vitrified groups regardless of type of sugar. In conclusion, Taxol pre-treatment of oocytes before cryopreservation did not reduce the damage induced by vitrification and subsequently did not improve embryonic development following vitrification. Trehalose may be used as an alternative non-permeating cryoprotectant in vitrification solution.

• 한국발생생물학회지:발생과생식
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• 제18권2호
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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.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.103-108
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• 2007

This study was carried out to investigate the effects of cryoprotectants, warming solution and removal of lipid on open pulled straw vitrification (OPS) method of porcine embryos produced by nuclear transfer (NT) of fetal fibroblasts. All solutions used during vitrification were prepared with holding medium consisting of 25 mM Hepes buffered TCM199 medium containing 20% fetal bovine serum (FBS) at $38.5^{\circ}C$. The blastocysts derived from NT with or without lipid were vitrified in each medium of different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Also, blastocysts after cryopreservation were warmed into different concentrations of sucrose in warming solution. The optimal concentrations of cryoprotectants in vitrification solution were 10% DMSO + 10% EG in vitrification solution 1 (VS1) and 20% DMSO + 20% EG in vitrification solution 2 (VS2). The optimal concentrations of sucrose were 0.3 M sucrose in warming solution 1 (WS1) and 0.15 M sucrose in warming solution 2 (WS2). lipid removal from oocytes before NT enhanced the viability of NT embryos after vitrification. Our results show that use of the OPS method in conjunction with lipid removal provides effective cryopreservation of porcine nuclear transfer embryos.

• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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• pp.229-236
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• 2002

Objective : The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. Methods : Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at $37^{circ}C$ for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at $4^{circ}C$ overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. Results: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05). Conclusion: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.251-256
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• 1999

The present study was conducted to evaluate whether vitrification could be used for ovarian tissue preservation. The important issue here is that the vitrification is very simple, easy, and economical compared to the conventional cryopreserving method that using automatic freezing instrument. Human ovarian cortical tissues were cryopreserved by vitrification with 5.5 M ethylene glycol and 1.0 M sucrose as cryoprotectant. Three points of temperature ($4^{\circ}C$, room temperature, and $37^{\circ}C$) and two points of duration (5 or 10 minutes) for cryoprotectant treatment were examined to determine the best condition for vitrification of the human ovarian cortical tissues. After thawing, viability of the isolated primordial follicles was examined by dye-exclusion method. Histological appearance of tissues before and after the cryopreservation was evaluated. There was no toxic effect of the 5.5 M ethylene glycol on the primordial follicles. However, when the tissues were treated with cryoprotectant at $37^{\circ}C$ for 10 minutes and exposed to liquid nitrogen, it seems likely that there is certain deleterious effects on the viability of the primordial follicles. The highest viability of the primordial follicles was obtained with the treatment of cryoprotectant at room temperature for 10 minutes. Follicles and oocytes survived after freezing and thawing had the similar normal shapes as was seen in the specimens before cryopreservation. It would be useful to apply vitrification in establishing ovarian tissue banking for clinical purposes.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.325-333
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• 1997

The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VS11 and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VS11 is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.

• Clinical and Experimental Reproductive Medicine
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• 제32권2호
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• pp.91-99
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• 2005

Objective: To define an appropriate vitrification condition of preantral follicle that yields high survival and to evaluate growth and ovulation rate of mouse follicles during in vitro culture after vitrification. Methods: Preantral follicles were isolated mechanically from mouse ovaries that were surgically recovered from mice aged 14 days. Retrieved preantral follicles were placed in EG (Ethylene Glycol) for 2, 5, 10 minutes and transferred to EFS-40 (40% EG, 18% Ficoll-70, 0.5 M sucrose) for 0.5, 1, 2 minutes. And then, preantral follicles were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing was carried out at room temperature. After defining the most appropriate vitrification condition that yields high survival, in vitro growth and ovulation rate of follicles were evaluated. Results: Appropriate vitrification condition that yield high survival rate ($83.2{\pm}2.1%$) of preantral follicle was EG for 5 minutes and EFS-40 for 0.5 minutes. In vitro survival rate of the vitrified preantral follicles were $85.5{\pm}0.5%$, $67.9{\pm}0.8%$ and $40.2{\pm}0.5%$ on day 2, 6 and 10. And in vitro growth of the vitrified preantral follicles were $107.1{\pm}16.1{\mu}m$, $117.1{\pm}18.4{\mu}m$, $178.4{\pm}45.6{\mu}m$ and $325.4{\pm}54.4{\mu}m$ on day 0, 2, 6 and 10. Although in vitro survival rate and growth of vitrified preantral follicles were lower than that of non-vitrified preantral follicles, the patterns of survival and growth were similar in vitrified and non-vitrified preantral follicles. The ovulation rate of antral follicles that was grown from vitrified preantral follicles was $32.6{\pm}1.2%$. Conclusion: Vitrified preantral follicles could be grown to antral sizes, and mature oocytes that can be used for IVF-ET programs were produced successfully. These data suggest that cryopreservation of preantral follicle by vitrification can be used for the preservation of the fertility.