• 대한수의학회지
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• 제35권3호
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• pp.635-641
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• 1995

The purpose of this study was to investigate the effects of developmental stage and equilibration time on survival of rabbit embryos following freezing by vitrification. Adult New Zealand White female rabbits were superovulated with PMSG and hCG. The 8-cell stage embryos were collected from 40 to 45 hours after hCG injection by flushing oviducts with Dulbecco's phosphated buffered saline and in vitro cultured in TCM-199 containing 10% fetal calf serum(FCS). Each embryos developed in vitro to 16-cell, compact morula and blastocyst was cryopreserved and cultured following thawing to examine their developmental potential to expanded blastocyst stage in vitro. The frozen-thawed-cultured embryos were stained with Hoechst 33342, and their nuclei were counted using a fluorescence microscope. On the toxicity test of EFS solution as cryopreservation, the survival rates of 8-cell stage embryos was decreased in reverse to increasing of exposure time over 5 minutes. The post-thaw survival rates of embryos on equilibration times was significantly(P<0.05) higher for 2 min. than for 5 or 10 minutes. From morula to blastocyst of rabbit embryos was more suitable than 8-cell stage for cryopreservation by vitrification. The higher post-thaw survival rate of embryos can be achieved by keeping the cryoprotectant at $4^{\circ}C$ than at $20^{\circ}C$. The mean number of nuclei per embryo following freezing by vitrification and in vitro culture to expanded blastocyst at compacted morula and blastcyst was not significantly differ from fresh blastocyst.

• 한국수정란이식학회지
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• 제19권1호
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• pp.27-34
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• 2004

This study evaluated the efficiency and compared with different materials of loading vessels for vitrification-plastic/glass, copper grid and nylon. The loading method, vitrification, cryop-reservation and warming method of the oocytes were examined. The loading samples prepared in manual or company-made and sterilized, loaded the COCs selected on each samples and cultured for maturation during 40 hours, and then exposed sequentially to ethylene glycol solution. Thawing method was reversely treated and exposed for warmed oocytes. After oocytes were thawed, fertilized and cultured in vitro for 3-4 hours, rates of development and morphological appearance were examined. The results were as summarized: ㆍOPS from company-made or hand-made of the hematocrit micropipettes, NLS from fishing line and EMG from company-made for EM were used for loading oocytes, respectively. ㆍThe efficiency of freezing method and loading convenience were orderly higher in OPS, NLS and EMG. The optimal capacity per vessel was orderly lowered in NLS, EMG and OPS, respectively. ㆍAfter oocytes were warmed, the recovery rate, morphology and rate of development were orderly higher in OPS, NLS and EMG, respectively. ㆍIn conclusion, OPS has the advantages of achieving a little more survival and preserving results than other two loading methods.

• Nuclear Engineering and Technology
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• 제51권3호
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• pp.746-754
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• 2019

Vitrification of radioactive liquid waste (RLW) provides a feasible solution for isolating radionuclides from the biosphere for an extended period. In vitrification, base glass and radioactive waste are added simultaneously into the melter. Determination of heat and mass transfer rates is necessary for rational design and sizing of melter. For obtaining an assured product quality, knowledge of reaction kinetics associated with the thermal decomposition of waste constituents is essential. In this study Thermogravimetry (TG) - Differential Thermogravimetry (DTG) of eight kinds of nitrates and two oxides, which are major components of RLW, is investigated in the temperature range of 298-1273 K in the presence of base glasses of five component (5C) and seven component (7C). Studies on thermal behavior of constituents in RLW were carried out at heating rates ranging from 10 to $40\;K\;min^{-1}$ using TG - DTG. Thermal behavior and related kinetic parameters of waste constituents, in the presence of 5C and 7C base glass compositions were also investigated. The activation energy, pre-exponential factor and order of the reaction for the thermal decomposition of 24% waste oxide loaded glasses were estimated using Kissinger method.

• 한국자원식물학회지
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• 제36권6호
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• pp.562-570
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• 2023

The droplet-vitrification technique for cryopreservation has proven successful across a diverse range of germplasm, ensuring safe and effective long term preservation. In this study, we investigate an effective cryopreservation protocol using the droplet-vitrification technique for shoot tips of Freesia hybrida cultivars 'Sunny Gold' and 'Sweet Lemon'. To determine optimal conditions for Freesia cryopreservation, we employed a carefully selected standard procedure along with additional treatments and alternative solutions. For 'Sunny Gold', the highest regrowth rate of 24% was achieved when shoot tips underwent dehydration with PVS3 solution for 120 minutes before direct immersion in liquid nitrogen (LN) for 1 hour, coupled with a standard protocol involving a two-step preculture with 0.3 M - 0.5 M sucrose, loading with C4 for 40 minutes, and unloading with 0.8 M sucrose for 40 minutes. In the case of 'Sweet Lemon,' regrowth of cryopreserved shoot tips was observed with dehydration treatments, including PVS2 (A3) for 60 minutes and PVS3 (B1) for 60 minutes, as well as longer exposure. The results reflect the distinct sensitivity of shoot tips to chemical toxicity and osmotic stress in these two genotypes. This study provides valuable evidence to consistently enhance the effectiveness of cryopreservation methods for the long-term conservation of Freesia germplasm.

• Reproductive and Developmental Biology
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• 제28권3호
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• pp.173-179
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• 2004

본 연구는 돼지 미성숙 난포란의 유리화 동결액에 노출시 독성과 삼투압 스트레스가 가장 적은 유리화 동결액을 조사하기 위하여 실시하였으며, 그 결과를 요약하면 다음과 같다. 미성숙 난포란을 EFS(40% ethylene glycol +18% ficoll +0.3 M sucrose) 용액, ES(5.5 M ethylene glycol +1.0 M sucrose) 용액, GE(10% glycerol +20% ethylene glycol) 용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙하여 관찰한 체외성숙율은 ES 용액과 대조구 에서 EFS 용액과 GE 용액보다 유의적으로 높았으며(P<0.05), 유리화 동결액의 독성에 의한 난포란의 퇴화율은 대조구, ES 용액, EFS 용액, GE 용액 순으로 유의적으로 낮은 퇴화율을 나타내었다(P<0.05). 미성숙 난포란을 EFS 용액, ES용액, GE용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙, 체외수정하여 관찰한 정상 수정율은 대조구가 세 종류의 유리화 동결액보다 유의적으로 높았고(P<0.05), 유리화 동결액 간에는 유의적인 차이가 없었다. 다정자 침입율과 난자당 침입한 평균 정자수는 모든 처리구간에 유의적인 차이가 없었다. 미성숙 난포란을 EFS 용액, ES 용액, GE 용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙, 체외수정, 체외 배양하여 관찰한 수정 3 일째의 분할율은 ES 용액과 대조구가 EFS 용액과 GE 용액보다 유의적으로 높았으며(P<0.05), 수정 7일째의 배반포 형성율은 모든 처리구간에 유의적인 차이가 없었다. 배양기간 동안 나타난 퇴화율은 대조구가 세 가지 유리화 동결액에 비해 유의적으로 낮았고(P<0.05), ES 용액은 EFS 용액과 GE 용액보다 유의적으로 낮은 퇴화율을 나타내었다(P<0.05).

• 한국자원식물학회지
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• 제31권6호
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• pp.684-694
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• 2018

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: 'Frost Eureka limon' and 'Cook Eureka limon', using a droplet-vitrification method. The shoot tips that were excised from in vitro grown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at $25^{\circ}C$ and incubated in a loading solution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at $25^{\circ}C$. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at $0^{\circ}C$ or PVS3 at $25^{\circ}C$. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in $\text\tiny{^1/_2}$ MS for 30 min at $25^{\circ}C$. Shoot tips were post-cultured overnight on survival medium and then micrografted onto 'trifoliate orange' (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% for cryopreserved shoot tips of 'Frost Eureka limon' and 'Cook Eureka limon', respectively, when pre-cultured in 0.3 M and 0.5 M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at $0^{\circ}C$. We also investigated whether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoot tips. The viability of cooled samples, following culturing on woody plant media (WPM) containing $\text\tiny{^1/_4}$ ammonium nitrate overnight before micrografting, was the highest (70.3%) in 'Frost Eureka limon'. The study described here is a cost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruit plants and for breeding disease resistance.

• 한국자원식물학회지
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• 제31권6호
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• pp.675-683
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• 2018

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of Chrysanthemum morifolium (Ramat.) cvs. 'Borami' and 'Yes morning'. The shoot tips of Chrysanthemum were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explants were treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed to dehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on $NH_4NO_3$-free MS medium for first 5 days was increased to two-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growth regulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemum germplasm.

• 한국수정란이식학회지
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• 제8권1호
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• pp.31-36
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• 1993

The post-thaw survival of mouse embryos of the various developmental stages was determined after cryopreservation by vitrification in a solution containing ethylene glycol, Ficoll and sucrose (EFS). All the embryos were equilibrated for 2 minutes just prior to freezing. The number of blastomeres during in vitro development was counted by nuclei higher rates of post-thaw survival were obtained from the embryos of 2-cell(92.2%), 8-cell(77.2%) or morula stage(90.0%) than those of blastocyst stage(62.7%). The number of blastomeres per embryo following in vitro culture for 24 hours was significantly(P<0.05) smaller as 66.0f22.3 in vitrified and thawed morulae than fresh morulae(91.7$\pm$12.2).

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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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.

• Clinical and Experimental Reproductive Medicine
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• 제39권4호
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• pp.153-160
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• 2012

Objective: The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates. Methods: Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 ${\mu}g/mL$ Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups. Results: Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05). Conclusion: The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation.