• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.75-81
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• 2004

Objective: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. Methods: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1, 2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-$\beta$-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). Results: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. Conclusion: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

• 한국수정란이식학회지
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• 제12권2호
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• pp.141-149
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• 1997

This study was carried out to investigate the effect of vitrification and slow freezing methods on the post-thaw developmental rate of rabbit zygotes. After exposing rabbit zygotes in EFS solution for 0.5, 1, 2, 3 and S min at room temperature, they were washed with 0.5 M sucrose solution, D-PBS and TCM-199 and then cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) to examine whether the cryoprotectant induced injury during the various exposure periods. The embryo development rates to hatched blastocyst after exposing in EFS solution for 3 and 5 min(40.0 and 16.7%) were significantly lower than in 0.5, 1 and 2 min(63.0, 72.0 and 54.5%), respectively. The post-thaw development rates to hatched blastocyst were significantly(P<0.05) higher in in vivo morula with intact mucin coat(85.2%) and mucin seperated morula(77.8%) than those of in vitro morula(58.5%) and zygote(5.9%), hut no difference was shown between in vitro morulae and mucin separated morula. The cryoprotectant dilution procedures showed no effects on the post-thaw development rates to hatched blastocyst under the present culture conditions. The post-thaw development to hatched blastocyst in the rabbit zygotes was not significantly different between the slow freezing(12.8%) and vitrification(5.9%). These results indicated that the rabbit frozen zygotes could he successfully developed in vitro to hatched blastocysts, though their developmental rate was very low, compared with morula stage embryos, in either vitrification or slow freezing procedure under the present conditions.

• Integrative Medicine Research
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• 제6권1호
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• pp.12-18
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• 2017

Cryopreservation is a process that preserves organelles, cells, tissues, or any other biological constructs by cooling the samples to very low temperatures. The responses of living cells to ice formation are of theoretical interest and practical relevance. Stem cells and other viable tissues, which have great potential for use in basic research as well as for many medical applications, cannot be stored with simple cooling or freezing for a long time because ice crystal formation, osmotic shock, and membrane damage during freezing and thawing will cause cell death. The successful cryopreservation of cells and tissues has been gradually increasing in recent years, with the use of cryoprotective agents and temperature control equipment. Continuous understanding of the physical and chemical properties that occur in the freezing and thawing cycle will be necessary for the successful cryopreservation of cells or tissues and their clinical applications. In this review, we briefly address representative cryopreservation processes, such as slow freezing and vitrification, and the available cryoprotective agents. In addition, some adverse effects of cryopreservation are mentioned.

• 한국수산과학회지
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• 제44권6호
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• pp.665-670
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• 2011

The rotifer Brachionus plicatilis is one of the most important food organisms in aquaculture. The resting eggs produced by mictic female rotifers are easily stored and hatched, making them useful as the starter for the mass culture of rotifers in marine larval culture. This study examined the optimum preservation method for resting eggs to ensure a high hatching rate. To produce resting eggs, the marine rotifer B. plicatilis was cultured with Nannochloris oculata (KMMCC 16). The resting eggs were harvested and cryopreserved using 5% and 10% methanol (MeOH), dimethylsulfoxide (DMSO), and glycerol as cryoprotectant agents (CPAs). The cryopreservation comprised slow or rapid freezing and the resting eggs were stored for one month in liquid nitrogen ($-196^{\circ}C$). The resting eggs were also dried at different temperatures (30, 40, and $50^{\circ}C$) and for different times (1, 2, and 3 h). In general, the hatching rates of the resting eggs preserved with CPA were higher than those without CPA and the slow freezing method was better than the rapid freezing method. However, the optimum CPA concentration for the hatching rate of the resting eggs varied with the freezing method and kind of CPA, and the CPA also affected the viability of the resting eggs. Dried resting eggs had a high, rapid hatching rate over 80%. The moisture content of the resting eggs cryopreserved in liquid nitrogen affected the hatching rate. Drying at $30^{\circ}C$ for 1 hour resulted in a high hatching rate of the resting eggs. In conclusion, drying at $30^{\circ}C$ for 1 hour and preservation in liquid nitrogen with the slow freezing method, without CPA, is recommended for a high hatching rate (ca. 95%) of rotifer resting eggs.

• 한국수산과학회지
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• 제31권4호
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• pp.463-470
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• 1998

본 연구는 생선회 맛의 관능적 품질향상 및 생선회로서의 품질수명을 연장시키기 위하여 우리나라에서 고급횟감으로 널리 소비되고 있는 넙치를 시료로 하여 동결속도 및 해동방법에 따른 근육의 물리화학적 및 조직학적 변화를 살펴보았다. 1. 넙치를 chunk 형태로 급속동격 (액체질소동결) 및 완만동결 ($-15^{\circ}C$ 동결하였을때 최대 빙결정생성대의 통과시간은 각각 10분이내와 110분이었다. 해동방법에 다른 해동소요시간은 $25^{\circ}C$ 수도수. $15^{\circ}C$ 수도수 $10^{\circ}C$ 수도수. $25^{\circ}C$ 공기. $5^{\circ}C$ 수도수. $0^{\circ}C$ 수도수 순으로 짧았다. 2. 근육의 파괴강도는 급속동결하여 급속해동한 것이 완만동결하여 완만해동한 것에 비하여 높았으며. 조리형태에 따른 파괴강도는 급속동결한 것은 조리형태에 따라 큰 차이를 나타내지 않았으나, 완만동결한 것은 slice형태로 처리한 것이 높은 값을 나타내었다. 4. ATP의 잔존량은 동결속도에 큰 영향을 받지 않았으며, 급속해동한 것이 완만해동한 것에 비하여 높은 경향을 나타내었다. 그리고 IMP가 동결-해동후의 ATP관련화합물의 대부분을 차지하고 있었다. 5. 근육의 표본조직을 광학현미경 (LM)으로 관찰한 결과 즉살직후에는 근섬유간의 간격이 관찰되지 않았으며. 급속동결하여 해동방법을 달리한 근육에 있어서도 근섬유간의 간격이 거의 관찰되지 않았다. 그러나 완만동결하여 해동한 것은 근섬유간의 간격이 현저하게 관찰되었다.

• 한국임상수의학회지
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• 제17권2호
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• pp.420-430
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• 2000

These studies were preformed to investigate the freezing conditions to achieve good post-thaw viability of spend and the practical methods of artificial insemination frozen canine semen. Semen were collected from nine male dogs which had been proved to be fertile in the past and the semen were treated for freezing procedure. Post-thaw motility and viability of canine sperm were evaluated to investigate individual tolerance of freezing, difference among freezing extenders, dif-ference among freezing equipments and freezing conditions, difference between fast and slow cooling rate, difference according to different glycerol concentration, effect of seeding on post-thaw viability, difference according to cutting part of straw, difference according to thawing temperatures, and dif-ference according to media added to thawed semen. Thawed semen for insemination were added with equal volnme of canine capacitation medium (CCM) and the volume of semen and the number per insemination were adjusted as 2-3 ml and $20-30 {\times}10^7,$ respectively. The semen were inseminated in vagina using balloon catheter and en17ryos were cellected from 9 to 11 days after the second Al to d determine fertilization.

• 한국콘크리트학회:학술대회논문집
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• 한국콘크리트학회 1996년도 가을 학술발표회 논문집
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• pp.148-153
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• 1996

Concrete with ground granulated blast-furnace slag can be affected by frost attack because the reaction of hydration is slow at the early age. In this study, therefore, the freezing and thawing test has been carried out to investigate the freezing and thawing resistance on concrete with ground granulated blast-furnace slag. The freezing and thawing test has been performed on concrete a blended cement, which was substituted by ground granulated blast-furnace slag with 4 kinds of ratio (non-admixture, 20%, 40% and 60%). And also tested on concrete added the AE agents to the concrete of same mix proportion to search the improvement effects about the resistance. As a result, the freezing and thawing resistance showed a tendency of reduction in proportion to the increase of the substitution ratio. For non-AE concrete, resistances of the freezing and thawing were very poor as the durability index indicated less than 5.8%. For AE concrte, resistance of the freezing and thawing were excellent as the durability index indicated more than 80.9%.

• Clinical and Experimental Reproductive Medicine
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• 제28권3호
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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.

• 한국식품조리과학회지
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• 제10권4호
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• pp.405-411
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• 1994

This study was conducted to know the quality of chou made with flour pastes which were stored at different conditions of quick freezing, slow freezing, cold and room temperature. Also, this study included investigation of the chou properties such as expansion, sensory evaluation, degree of gelatinization, and physical and structural properties of paste were observed. There were not significant differences m diameter, height, volume, appearance, hollow formation, and sensory evaluation between the chou made with the paste stored at freezing condition and chou directly baked after pasting. Quick and slow freezing storages did not significantly affect the properties of chou, and the same results were obtained among the chou made with pastes thawed at room temperature and in microwave ovenrange. The chou of pastes stored at room temperature and in microwave ovenrange. The chou of pastes stored at room temperature and stored in refrigerator showed lowed expansion and value of sensory evaluation than those of frozen pastes. The paste stored at room temperature had the lowest hardness and viscosity compared with the other storage conditions. According to the observation of light microscope. the lipid bodies of the paste of freezing storage smaller those of the room temperature and refrigerator storage. The expantion of chou made with paste stored at room temperature was greatly decreased due to the high coalescence of lipid bodies, and also the paste components such as lipid, starch granule gluten at room temperature had inferior dispersion condition. The general tendency of the degree of gelatinization of chou were low in all treatments of paste. The values were 23.5%~46.0% in freezing, 77.3% in room temperature, 68.7% in directly baked after pasting, and 61.0% in cold storage, respectively. The formation and the taste of chou made with frozen paste were similar to those of chou directly baked pasting.

• 한국수정란이식학회지
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• 제18권1호
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• pp.43-50
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• 2003

정자의 동결보존을 위한 새로운 기술개발 목적은 동결과정에서 최소한의 손상으로, 응해 후 최대한 높은 활력도의 정자를 얻는 것이다 정자가 난자와 수정하기 위해서는 적당한 생존성과 운동성을 유지해야 하는데, 가장 일반적인 방법으로는 정자의 진진 운동성과 첨체의 정상 여부 및 형태 검사방법 등이 있다 본 연구는 사람 정액을 동결보존 할 때 semi-programmable freezer를 이용한 완만동결 방법과, 액체질소의 vapor를 이용한 급속동결 방법이 응해 후 정자의 운동양상과 생존율 및 형태에 미치는 영향을 알아보기 위해 실시하였다. 동결-응해 후 정자의 MOT, VCL, VSL, VAP는 각각 급속 동결방법에서 47.40$\pm$20.06%, 38.12$\pm$15.58 $\mu$m/s, 28.19$\pm$14.10 $\mu$m/s, 33.64$\pm$15.15 $\mu$m/s로 완만 동결방법인 43.39$\pm$18.79%, 33.91$\pm$13.50 $\mu$m/s, 19.98$\pm$10.88 $\mu$m/s, 24.60$\pm$11.72 $\mu$m/s보다 유의적으로 높았으나(p<0.05), LIN은 완만동결 방법이 34.64$\pm$11.36으로 급속동결 방법인 28.83$\pm$10.35보다 더 좋은 성적을 나타내었다(p<0.05). 고 활력정자를 나타내는 HYP 역시 급속동결 방법에서 2.77$\pm$2.71%로 완만동결 방법의 1.33$\pm$1.57%보다 유의적으로 높게 나타났다(p<0.05). 그러나 정자의 운동양태를 나타내는 MAD, WOB, DNC, DNM에 있어서는 완만동결 방법이 급속동결 방법에 비해 조금 더 좋은 성적을 보였으나 유의적인 차이는 없었다. 동결-응해 후 정자의 생존율 및 정상형태의 정자는 완만동결 방법이 각각 62$\pm$2.1%, 44$\pm$8.3%, 급속동결 방법은 60$\pm$2.2%, 46$\pm$7.7%로 유의적인 차이가 없이 비슷한 결과를 보였다. 따라서 사람 정액의 동결 보존 시 많은 시간과 고가의 장비가 필요한 완만동결 방법보다는 짧을시간동안 액체 질소만으로 간단히 시행할 수 있는 급속동결 방법이 더 효과적이라고 사료된다.