• 한국전자현미경학회:학술대회논문집
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• 한국현미경학회 2005년도 제36차 춘계학술대회 및 제3회 HVEM 이용자 워크샵
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• pp.47-48
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• 2005

• 한국가축번식학회지
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• 제8권2호
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• pp.116-121
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• 1984

These experiments were carried out to examine the effect of rapid thawing (500$^{\circ}C$/min) on the survival of 8-cell mouse embryos cooled slowly (0.5-1$^{\circ}C$/min) to precooling temperatures between -10 and 070$^{\circ}C$ before direct transfer ofembryos to -196$^{\circ}C$, and to compare the survival of embroys thawed slowly (20$^{\circ}C$/min) and rapidly (500$^{\circ}C$/min) after in vitro culture. In addition, the sensitivity of 8-cell mouse embroys to the rate of addition and removal of cryoprotectant at room temperauture, and the effect of developing stages on the survival of embryos frozen-thawed slowly were investigated. The results obtained were summarized as follows: 1. Embryos were survived in rapid thawing (500$^{\circ}C$/min) only when slow cooling was terminated at relatively high subzero teperaure (-20 to -60$^{\circ}C$). The highest survival rate(77.0%) in in vitro culture of embryos thawed rapidly was obtaeined after transfer to -196$^{\circ}C$ from -40$^{\circ}C$. 2. For the survival of embryos in slow thawing (20$^{\circ}C$/min.), slow cooling to lower subzero temperature (-50$^{\circ}C$ and below) was required before transfer of embryos to -196$^{\circ}C$. These results indicate that embryos transferred to -196$^{\circ}C$ from high subzero temprature contain much interacellular ice to damage them during slow warming but to permit survival of embryos after rapid warming. 3. The Survival rate (72.7%) of 8-cell mouse embryos after rapid addition and removal of cryoprotectant, DMSO at room temperature was similar to that (83.9%) after slow addition and removal of cryoprotectant at same temperature. 4. The survival rates of 1-, 2-, 4- and 8-cell embryos frozen-thawed slowly were 26.7, 76.4, 70.0 and 83.9%, respectively.

• 한국자원식물학회지
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• 제17권2호
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• pp.75-81
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• 2004

본 연구는 산림 수목 종자를 대상으로 다양한 초저온 동결 조건을 적용하고, 초저온 저장 후에 나타나는 종자의 발아 특성 및 유묘의 생장 특성 조사하기 위해 수행되었다. 유리화 과정을 기초로 한 초저온 저장은 다릅나무 종자의 발아율을 크게 감소시켰으나, 초저온 저장 전 동결보호제 처리와 초저온 저장 후의 빠른 해빙은 종자의 발아율 감소를 완화시켰다. 초저온 저장 전 동결보호제 노출 시간은 종자의 발아율에 영향을 주었다. 즉 동결보호제 노출시간이 길어짐에 따라 종자의 발아율은 감소하였다. 그러나 초저온 저장 기간은 종자의 발아율에 영향을 주지 않았다. 또한 동결보호제의 노출 시간과 초저온 저장 기간은 유묘의 생장 특성에 영향을 주지 않았다. 따라서 수목 종자의 초저온 저장은 조직의 손상이나 변형이 없이 장기간 저장할 수 있는 현지 외 보존 기술로 적절하다고 판단된다. 그러나 산림 종자의 보다 효율적인 초저온 저장을 위해서는 각 수종의 종자 특성에 맞는 동결보호제 및 처리 기술이 개발되어야 할 것으로 판단된다.

• 한국균학회지
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• 제26권1호통권84호
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• pp.91-96
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• 1998

국내에서 채집, 분리한 12종 버섯류의 균사를 대상으로 하여 멸균 증류수를 이용한 최적 보존 방법을 탐색하였다. 온도, cryoprotectant의 종류 및 농도 등의 조건을 달리하여 12개월간 보존 후 생존을 및 생장속도를 측정한 결과, 보존 온도는 상온과 $5^{\circ}C$가 최적으로 대상 균주 거의 모두가 생존하여 91.7%의 생존율을 보였다. 균사의 생장속도는 저온인 $-75^{\circ}C$에 보존하였을 때 가장 높았으며, 이 온도에서 보존시 polyethylene glycol를 cryoprotectant로 첨가하였을 때 최대의 생존율이 관찰되었다. 향기갈색깔대기버섯(H. ferrugineum)을 대상으로 하여 계대배양으로 보존한 균주와 $5^{\circ}C$에서 보존한 균주의 세포내 laccase의 활성을 조사한 결과, 효소의 활성양상이 동일하게 나타났다.

• 한국수정란이식학회지
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• 제24권4호
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• pp.275-279
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• 2009

The beneficial effect of glycerol as a cryoprotectant, especially for sperm cryopreservation, has been shown in many studies. However, glycerol is toxic to living cells, and boar sperm in particular show greater sensitivity to glycerol than sperm from other domestic animals. Amides have been studied as alternative cryoprotectants for freezing stallion sperm. Sperm frozen in methylformamide or dimethylformamide as cryoprotectants show similar motility when thawed compared with sperm frozen in glycerol. We evaluated the cryoprotective effects of dimethylformamide on boar sperm freezing. To test the effect of amides, the concentration of boar semen was adjusted to $10^9sperm/mL$, and seminal plasma was removed using Hulsen solution. After centrifugation, the pellet was diluted in modified-Modena B extender. Lactose-egg yolk (LEY) extender was used as the cooling extender. The freezing extender was madeed aaddition of the optimal amount of glycerol and amides to LEY-Glycerol-Orvus ES Paste extender, and this extender was used for the second dilution. Diluted sperm were frozen in liquid nitrogen using the 0.5 mL straw method. Sperm frozen in extender with glycerol as a cderol were compared with those frozen in extender including the different amides. Sperm were tested for motility, viability, the sperm chromatin structure assay, and normal apical ridge after thawing. The percent of motile sperm diluted in glycerol was as high as that in the stallion study (61%). Dimethylformamide showed positive effects on sperm quality and was better than glycerol. Methylformamide provided similar sperm quality as glycerol. Therefore, dimethylformamide is useful for reducing cryoinjury in boar sperm and is expected to be useful as an alternative cryoprotectant.

• 한국수정란이식학회지
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• 제27권2호
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Asian-Australasian Journal of Animal Sciences
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• 제15권4호
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• pp.485-493
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• 2002

Different factors may affect the sensitivity of porcine oocytes during cryopreservation. The effect of two methods (cooling and vitrification), four cryoprotectants [glycerol (GLY), 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol (EG)] and two vitrification media (1 M sucrose (SUC)+8 M EG; 8 M EG) on the developmental capacity of porcine oocytes at the germinal vesicle (GV) stage or after IVM at the metaphase II (M II) stage were examined. Survival was assessed by FDA staining, maturation and cleavage following IVF and IVC. A toxicity test for different cryoprotectants (GLY, PROH, DMSO, EG) was conducted at room temperature before cooling. GV and M II-oocytes were equilibrated stepwise in 1.5 M cryoprotectant and diluted out in sucrose. The survival rate of GV-oocytes in the GLY group was significantly lower (82%, p<0.01) than that of the other group (92 to 95%). The EG group achieved a significantly higher maturation rate (84%, p<0.05) but a lower cleavage rate (34%, p<0.01) than the DMSO group and the controls. For M II-oocytes, the survival rates for all groups were 95 to 99% and the cleavage rate of the GLY group was lower than the PROH-group (21 vs 43%, p<0.01). After cooling to $10^{\circ}C$, the survival rates of GV-oocytes in the cryoprotectant groups were 34 to 51%, however, the maturation rates of these oocytes were low (1%) and none developed after IVF. For M II-oocytes, the EG group showed a significantly higher survival rate than those of the other cryoprotectant groups (40% vs 23-26%, p<0.05) and the cleavage rates of PROH, DMSO and EG group reached only 1 to 2%. For a toxicity test of different vitrification media, GV and M II-oocytes were equilibrated stepwise in 100% 8 M EG (group 1) and 1 M SUC + 8 M EG (group 2) or equilibrated in sucrose and then in 8 M EG (SUC+8 M EG, group 3). For GV-oocytes, the survival, maturation and cleavage rates of Group 1 were significantly lower than those in group 2, 3 and control group (p<0.05). For M II-oocytes, there were no differences in survival, maturation and cleavage rates between groups. After vitrification, the survival rates of GV and M II-oocytes in group 2 and 3 were similarly low (4-9%) and none of them matured nor cleaved after in vitro maturation, fertilization and culture. In conclusion, porcine GV and M II-oocytes do not seem to be damaged by a variety of cryoprotectants tested, but will succumb to a temperature decrease to $10^{\circ}C$ or to the process of vitrification, regardless of the cryoprotectant used.

• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.55-63
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• 2001

Objectives: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Materials and Methods: Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. Results: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1 %,79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The developmen1 rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. Conclusions: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

• Journal of Pharmaceutical Investigation
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• 제25권3호
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• pp.249-264
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• 1995

Although liposome has many advantages as a pharmaceutical dosage form, its application in the industrial field has been limited because of some problems such as preparation method, reproducibility, scale-up, stability and sterilization etc. Liposomes prepared by microemulsification method had defined size, narrow size distribution, reproducibility and high entrapment efficiency. For enhancing the stability, the dry form of liposome was recommended. These types of liposome are proliposome and freeze-dried liposome. The liposome must have some properties for preparing of freeze-dried liposome; small size $(50{\sim}200\;nm)$, narrow size distribution and cryoprotectant. In this experiment, the liposomes containing 5-Fluorouracil(5-FU) and its prodrug(pentyl-5-FU-1-acetate; PFA, hexyl-5-FU-1-acetate; HFA) were made with soybean phosphatidylcholine, cholesterol, stearylamine(SA) and dicetyl phosphate(DCP) employing hydration method or microemulsification method using $Microfluidizer^{TM}$. Both or liposome types were MLV and MEL. After preparation, freeze drying and rehydration were performed. In the process of freezing, trehalose(Tr) was added as a cryoprotectant. Their evaluation methods were as follows; entrapment efficiency, mean particle size and size distribution, dissolution test, retain of entrapment efficiency and turbidity after freeze-drying. The results are summarized as belows. The entrapment efficiency of 5-FU was dependent on total lipid concentration and cholesterol content but that of PFA and HFA was decreased when cholesterol was added. When DCP and SA were added, entrapment efficiency was decreased. As the partition coefficient of drug was increased, entrapment efficiency was increased. Under the same condition, entrapment efficiency of MEL is similar to that of MLV. The mean particle size and size distribution of MEL were smaller than those of MLV. Dissolution rates of drug from both liposome types were comparatively similar. Dissolution rates of drugs with serum and liver homogenate were faster than without these material. After preparation of liposome, free drug was removed efficiency by Dowex 50W-X4. When liposome was freeze-dried and then rehydrated in the presence of Tr, characteristics of liposome were maintained well in MEL than MLV. Tr Was used successfully as a cryoprotectant in the process of freeze drying and the optimal ratio of Tr:Lipid was 4:1(g/g).

• 한국가축번식학회지
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• 제16권1호
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• pp.47-53
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• 1992

This study was carried out to investage the effect of cryoprotectant concentration and equilibration time on volume change and in vitro development of intact and bisected mouse embryos by rapid freezing. When compacted morulae were rapidly frozen in 3.0 to 4.0 glycerol or DMSO with 0.25M sucrose solution, the superior(P<0.05) post-thaw survival rate was obtained at the glycerol concentration of 4.0M(89.4%) than 3.0M(71.4%) or 5.0M(42.4%), but at the DMSO concentration of 3.0M(84.5%) than 4.0M(51.1%) or 5.0M(0.0%). The optimal equilibraton time for rapid freezing of ZP-free or bisected morulae in 4.0M glycerol with 0.25M sucrose was found tobe 3 minutes. The minimal volume of compacted morulaewhich corresponded with 61 to 62% of pre-equilibrated embryo volume was obtained from equilibration for 3 minutes in both 3.0 and 4.0M glycerol solutions with 0.25M sucrose.