• Title/Summary/Keyword: Freezing method

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Studies on Rapid Freezing and Thawing of Porcine Embryos III. Factors affecting the survival rate of porcine embryos cryopreserved and diluted by one-step straw method (돼지 수정란의 급속 동결 융해법에 관한 연구 - 돼지 동결 수정란에 대한 1단계 Straw법이 배의 생존성에 미치는 영향)

  • 김상근;김무강;서길웅
    • Journal of Embryo Transfer
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    • v.7 no.1
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    • pp.13-19
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    • 1992
  • This study were carried out to investigate the effective concentration of cryoprotective agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen porcine embryos. The porcine embryos foflowing dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined by FDA test. The results are sunnnarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen4hawing in the freezing medium with a various concentration of glycerol, DMSO and propanediol added 0.25M sucrose were higher survival rate than those of sucrose concentration of 0.50M. 2. The survival rates of porcine embryos after ultrarapid ftozen4hawing in the freezing medium added 0.25M and 0.SOM sucrose were higher survival rate than those of sucrose concentration of 0.75M and 1.00M. 3. The temperature thawed at 2$0^{\circ}C$ and 3$0^{\circ}C$ resulted in a significantly higher embryos survival rate after 72 hrs in culture than did at 35$^{\circ}C$. 4. The equilibration time on the survival rate of porcine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time(10~20 min.).

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Comparison of Vitrification and Slow Freezing-thawing Method on 1-cell Zygotes (생쥐 1-세포기 수정란의 동결방법에 있어서 초자화동결과 완만동결의 비교)

  • Lee, Ji-Hyang;Han, Hyuck-Dong;Koo, Hye-Young
    • 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.

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Fertility preservation in pig using ovarian tissues by vitrification method

  • Hwang, In-Sul
    • Journal of Animal Reproduction and Biotechnology
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    • v.37 no.2
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    • pp.106-112
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    • 2022
  • Cryopreservation of porcine ovarian tissue by vitrification method is a promising approach to preserve genetic materials for future use. However, information is not enough and technology still remains in a challenge stage in pig. Therefore, the objective of present study was to determine possibility of vitrification method to cryopreserve porcine ovarian tissue and to confirm an occurrence of cryoinjuries. Briefly, cryoinjuries and apoptosis patterns in vitrified-warmed ovarian tissue were examined by histological evaluation and TUNEL assay respectively. In results, a damaged morphology of oocytes was detected among groups and the rate was significantly (p < 0.05) lower in vitrification group (25.8%) than freezing control group (67.7%), while fresh control group (6.6%) showed significantly (p < 0.05) lower than both groups. In addition, cryoinjury that form a wave pattern of tissues around follicles was found in the frozen control group, but not in the fresh control group as well as in the vitrification group. Apoptotic cells in follicle was observed only in freezing control group while no apoptotic cell was found in both fresh control and vitrification. Similarly, apoptotic patterns of tissues not in follicle were comparable between fresh control and vitrification groups while freezing control group showed increased tendency. Conclusively, it was confirmed that vitrification method has a prevention effect against cryoinjury and this method could be an alternative approach for cryopreservation of genetic material in pigs. Further study is needed to examine the viability of oocytes derived from vitrified-warmed ovarian tissue.

Numerical Analysis for Cooling and Freezing Processes with Subcooling (과냉각을 동반한 동결과정의 수치해석)

  • Yoon, J.I.;Kim, J.D.;Kim, S.G.
    • Korean Journal of Air-Conditioning and Refrigeration Engineering
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    • v.8 no.4
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    • pp.451-462
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    • 1996
  • In this study, which focuses on ice storage, a fundamental study in cooling and solidification was performed, including the interesting phenomena of density inversion, supercooling and dendritic ice. A numerical study was performed for natural convection and ice formation considering existence of subcooling and dendritic ice were analyzed numerically by using finite difference method and boundary fixing method. In the mesh, the solid fraction was introduced with adding as a term to the energy conservation equation. A flow in the dendrite was modelled as a flow in a porous medium, and the momentum conservation equation was modified to incorporate resistance forces involved in flows through porous media. A numerical solution of the time dependencies of dendrite area and dense ice front was successfully obtained, and the numerical results were good agreement with experimental results. Based on this methodology, a discussion was made of phenomena and characteristics of cooling and freezing processes under various conditions.

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Mock-Up Test On Anti-Freezing Method with Double bubble Sheets Subject to Cold weather Banking (이중버블시트를 이용한 동상방지공법의 동절기 성토공사 Mock-up 실험)

  • Hong, Seak-Min;Son, Ho-Jung;Oh, Chi-Hyun;Han, Min-Cheol;Han, Cheon-Goo
    • Proceedings of the Korean Institute of Building Construction Conference
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    • 2011.11a
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    • pp.33-34
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    • 2011
  • In this study, using the double bubble sheet to anti-freezing method in winter the soil embanking Mock up as a part of the development process was carried out. As results, two layers of the double bubble sheet effect 12.6℃~13.8℃ temperature difference of out door temperature that proved superior insulation and thermal performance of the double bubble sheet.

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Effect of Pre-Treatment Methods before Cooking on Mineral Retention in Siraegi (Raddish Leaves) (조리전 전처리 방법에 따른 시래기의 무기성분의 변화)

  • 박세원;유양자
    • Korean journal of food and cookery science
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    • v.13 no.5
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    • pp.635-638
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    • 1997
  • Dried raddish leaves were prepared by using three different pre-treatment methods (shady sun-drying, freezing after blanching, and shady sun-drying after blanching). Then, the retention of minerals in dried raddish leaves was determined. It was shown that the retention of most minerals (Na, K, Fe, Ca, Mg) except P was higher when shady sun-drying method was used. The retention of P was shown to be the lowest when freezing after blanching method was used.

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Pattern Mapping Method for Low Power BIST (저전력 BIST를 위한 패턴 사상(寫像) 기법에 관한 연구)

  • Kim, You-Bean;Jang, Jae-Won;Son, Hyun-Uk;Kang, Sung-Ho
    • Journal of the Institute of Electronics Engineers of Korea SD
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    • v.46 no.5
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    • pp.15-24
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    • 2009
  • This paper proposes an effective low power BIST architecture using the pattern mapping method for 100% fault coverage and the transition freezing method for making high correlative low power patterns. When frozen patterns are applied to a circuit, it begins to find a great number of faults at first. However, patterns have limitations of achieving 100% fault coverage due to random pattern resistant faults. In this paper, those faults are covered by the pattern mapping method using the patterns generated by an ATPG and the useless patterns among frozen patterns. Throughout the scheme, we have reduced an amount of applied patterns and test time compared with the transition freezing method, which leads to low power dissipation.

Rapid freezing versus Cryotop vitrification of mouse two-cell embryos

  • Inna, Namfon;Sanmee, Usanee;Saeng-anan, Ubol;Piromlertamorn, Waraporn;Vutyavanich, Teraporn
    • Clinical and Experimental Reproductive Medicine
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    • v.45 no.3
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    • pp.110-115
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    • 2018
  • Objective: To compare our in-house method of embryo freezing with Cryotop vitrification in terms of immediate survival, subsequent cleavage and blastocyst formation, and cell numbers in blastocysts. Methods: Two-cell mouse embryos were randomly allocated into three groups: a non-frozen control group (group 1, n = 300), a group that underwent Cryotop vitrification (group 2, n = 300), and a group that underwent our in-house freezing method (group 3, n = 300). Results: There were no significant differences between groups 2 and 3 in the immediate survival rate (96.3% vs. 98.6%, respectively; p= 0.085), the further cleavage rate (91.7% vs. 95.0%, respectively; p= 0.099), or the blastocyst formation rate (80.7% vs. 78.6%, respectively; p= 0.437). The cell numbers in the blastocysts from groups 1, 2, and 3 were comparable ($88.99{\pm}10.44$, $88.29{\pm}14.79$, and $86.42{\pm}15.23$, respectively; p= 0.228). However, the percentage of good-quality blastocysts in the Cryotop vitrification group was significantly higher than in the group in which our in-house method was performed, but was lower than in the control group (58.0%, 37.0%, and 82.7%, respectively; p< 0.001). Conclusion: At present, our method is inferior to the commercial Cryotop vitrification system. However, with further improvements, it has the potential to be useful in routine practice, as it is easier to perform than the current vitrification system.

Simplified Slow Freezing Program Established for Effective Banking of Embryonic Stem Cells

  • Kim, Gil Ah;Lee, Seung Tae;Lee, Eun Ju;Choi, Jung Kyu;Lim, Jeong Mook
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.3
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    • pp.343-349
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    • 2009
  • This study was designed to simplify a cryopreservation program for embryonic stem cells (ESCs) by selection of cooling method and cryoprotectant. Commercially available mouse E14 embryonic stem cells (ESCs) were cryopreserved with various protocols, and morphology and viability of the frozen-thawed ESCs and their reactive oxygen species (ROS) production were subsequently monitored. Post-thaw colony-formation of ESCs was detected only after a slow freezing using dimethyl sulfoxide (DMSO) by stepwise placement of a freezing container into a $-80^{\circ}C$ deep freezer and subsequently into -$196^{\circ}C$ liquid nitrogen, while no proliferation was detected after vitrification. When the simplified protocol was employed, the replacement of DMSO with a mixture of DMSO and ethylene glycol (EG) further improved the post-thaw survival. ROS generation in ESCs frozen-thawed with the optimized protocol was not increased compared with non-frozen ESCs. The use of fresh mouse embryonic fibroblasts as feeder cells for post-thaw subculture did not further increase post-thaw cell viability. In conclusion, a simplified slow-freezing program without employing programmable freezer but using DMSO and EG was developed which maintains cell viability and colony-forming activity of ESCs during post-thaw subculture.

Establishment of the Convenient Boar Semen Freezing Method and Assessment of Viability in Frozen/Thawed Boar Semen (돼지 정액의 간편 동결 방법 확립과 동결 정액의 융해 후 생존성 평가)

  • Kim Seong-Kon;Jang Hyun-Yong;Park Dong-Heon;Park Chun-Keun;Cheong Hee-Tae;Kim Choung-Ik;Yang Boo-Keun
    • Reproductive and Developmental Biology
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    • v.30 no.1
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    • pp.59-64
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    • 2006
  • This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until $5^{\circ}C$ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with $LN_2$. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of $LN_2$ for 10 min). Sperm viability increased by holding at $-102^{\circ}C$ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in $37^{\circ}C$ group than in $52^{\circ}C$ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at $-102^{\circ}C$ for 10min before plunging into $LN_2$ is a convenient and easy freezing method for boar semen.