• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.83-83
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• 2002

The purpose of this study was to investigate the warming temperature and exposed time on the post-thaw survival rate and viability of bovine blastocyst cryopreserved by GMP vitrification. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into the GMP straws and immersed into LN$_2$within 20 to 25 sec. The warming rate was increased 2 times of warming temperature for improvement of post-thaw survival rates. The frozen embryos were warmed either at 35 or 70$^{\circ}C$ for 1 or 2 sec and then diluted in sucrose solution. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM: TCM199 supplemented with 10% FCS) and TCM-199 for each 5 min, respectively, and then cultured in TCM199 for 24 h. The rate of re-expanded blastocyst was significantly different fer 35 and 70$^{\circ}C$ warming temporature (76.4 vs. 89.3%; P<0.05). The rate of re-expanded blastocyst at 70$^{\circ}C$ for 1 sec was significantly higher than that for 2 sec (91.1 vs. 70.9%; P<0.05). The number of nuclei counted were significantly different among control, 35 and 70$^{\circ}C$ (121${\pm}$8.5 vs. 104${\pm}$11.7 vs. 114${\pm}$10.3; P<0.05). These results indicated that the increasing of warming rate can provide high survival rates of bovine IVP blastocysts. Especially, the best viability of post-thaw blastocyst could be thaw at 70$^{\circ}C$ for 1 sec. The warming temperature and exposed time far warming was considered to be limiting factors to the viability of bovine IVP embryos. he purpose of this study was to investigate the warming temperature and expose.

• Journal of Chest Surgery
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• v.39 no.6 s.263
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• pp.427-433
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• 2006

Background: Current vascular prostheses are considered still inadequate for reconstruction of small-diameter vessels. To evaluate the potential use of xenograft vessels as small diameter arterial grafts, we implanted porcine vessels in goats. The grafts were treated with two different processes, freezing and acellularization, before implantation, and gross inspection as well as microscopic examination followed after a predetermined period. Material and Method: Bilateral porcine carotid arteries were harvested and immediately stored at $-70^{\circ}C$ within tissue preservation solution. One of them was designated as frozen xenograft vessel. The other one was put on acellularization process using NaCl-SDS solution and stored frozen until further use. Grafts were implanted in the place of carotid arteries of the same goat. The grafts have remained implanted for 1, 3, and 6 months in three animals, respectively. Periodic ultrasonographic examinations were performed during the observation period. After explantation, the grafts were analyzed grossly and histologically under light microscope. Result: All animals survived the experimental procedure without problems. Ultrasonographic examinations showed excellent patency of all the grafts during the observation period. Gross examination revealed nonthrombotic, patent lumens with smooth surfaces. Microscopic examinations of the explanted grafts showed cellular reconstruction at the 6-month stage in both grafts. Although more inflammatory responses were observed in the early phase of frozen xenografts, there was no evidence of significant rejection. Conclusion: These findings suggest that porcine xenograft vessels, regardless of pre-implantation processes of acelluarization or freezing, can be acceptably implanted in goats, although short duration of observation in a small number of animals may limit this study.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.1-9
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• 1998

This study was carried out to confirm whether the developmental capacity of bovine mature oocytes frozen ultra-rapidly using electron microscopic(EM) grids and EFS30 can be obtained, and whether the cryoprotectants and the freezing method used in this study effect detrimentally to the bovine oocytes by indirect immunocytochemistry. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. The results obtained in this experiment were summarized as follows: When the effects of cryoprotectant and freezing procedure on the microtuble, micrfilament and chromatin morphology of oocytes were evaluated using indirect immunocytochemistry, the results of freezing as well as exposure group were not different with that of the control oocytes. When the fertilization abnormality after ultrarapid freezing of bovine mature oocytes was examined by Hoechst staining, the rates of total penetration(96.7, 9.0%), normal two pronuclei formation(74.6, 68.9%) and mean number of sperm / oocyte(1.50, 1.44) were not different between control and freezing group. In addition, when the developmental capacity of frozen-thawed of oocytes(85.5%) was survived, 74.5% of them were cleaved and 31.4% of cleaved embryos were developed to blastocyst. These data were similar to those of the control(76.0%, 34.6%) and exposure(74.5%, 33.0%) except survival rates. Also, when the total cell number of blastocysts produced from the each treatment at day after IVF was examined by hoechst staining, there were not different among groups. There results demonstrate that developmental capacity of frozen-thawed bovine mature oocytes can be successfully obtained by ultra-rapid freezing method using EM grid and EFS30 solution.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.197-205
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• 2013

This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for preservation of the species. The purpose of this study was to compare the effects of Ethylene Glycol (EG) and Propylene Glycol (PG) on viability of cryopreserved PGCs with vitrification in Korean Native Chicken (Ogye), and to fine should be find or to the optimal protocol for PGCs freezing. One of the important components of cryopreservation process is cryopreservation medium that plays a vital role in preventing cellular injury during freeze-thawing. Cryoprotective agents have been known to improve cell viability after freeze-thawing. PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents. Gonads were harvested from stage 28 chick embryos and pooled in groups of 10E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments: 2.5% EG, 5% EG, 10% EG, 2.5% PG, 5% PG, 10% PG, and 0% cryoprotectant as a control. Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. After freezing and thawing, survival rates of the frozen-thawed PGCs from the 0, 2.5, 5, 10 and 15% EG plus FBS treatment were 44.24%, 64.51%, 85.63%, 80.51% and 73.52% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05)(85.63% vs 66.81%). Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGCs in liquid N at a germplasm repository and ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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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.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.43-49
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• 1990

To improve the freezing techniques of animal embryos using vitrification solution as a cryoprotectant rabbit embryos, by cell stages, dehydration temperature and dehydration temperature and dehydratin time, were frozen-thawed and cultured. Following are the main results obtained. 1. The damage rate of zona pellucida after thawing was higher(13.6%) when the cell stage of embryos was less than 4 cells than when the cell stage was 8~16 cell or morula. The damage rate was higher when the dehydration temperature was 4$^{\circ}C$ than -3$0^{\circ}C$ or -50~-8$0^{\circ}C$. The zona pellucida was damaged more when dehydrated for 5 min than when dehydrated for 10~15 min. 2. After being cultured for 72 hours, 5.3% of 4 cell(or less) embryos were developed to morula, while 86.4% of morula embryos were developed further. 3. More percentage of embryos(73.2%) was developed when dehydrated at -3$0^{\circ}C$ than when dehydrated at 4$^{\circ}C$ at -5$0^{\circ}C$~-8$0^{\circ}C$. 4. The hatching rate was higher when dehydrated for 5 min. When the embryos were dehydrated for 10~15 min and cultured for 24 hours, they were not even developed or development was not good in later stages.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.9-16
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• 1990

This study was carried out in order to investigate effects of cryoprotectant concentration and equilibration time on survival of ultrarapidly frozen 2-cell mouse embryos. Mouse 2-cell embryos, following dehydration by exposure to DMSO and sucrose, were directly immersed into liquid nitrogen and thawed in 37$^{\circ}C$ water. Viability was defined by development rate to the blastocyst stage after in vitro culture for 72 hours. The results are summarized as follows ; 1. When 0.25M of sucrose was added into the freezing medium at various concentrations of DMSO and dilution medium, higher development rate of embryo was obtained in 3.0M DMSO concentrations (82.6%). However, when sucrose concentraitons of 0.25 and 0.5 M were added to the freezing medium with 3.0 M DMSO and dilution medium, development rate of embryos were 81.7% and 24.1%, respectively. 2. In the equilibration time at room temperature, higher development rate was attained after short period of time (2.5min) in 3.0 M DMSO＋0.25 M sucrose (85.9%). 3. The development rate of embryos at in vitro 2-cell, in vitro 2-cell, solution control and untreated control was 84.6%, 90.9%, 89.9%,, and 89.7%, respectively.

• The Korean Journal of Food And Nutrition
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• v.12 no.2
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• pp.113-118
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• 1999

Ten specimens of pufferfish Lagocephalus gloveri(called Gumeun-mibog in Korean) were purchased at a fish market in Pusan Korea. The pufferfishes were immediately frozen packed in ice boxes transpor-ted to our laboratory and then dissected in to four parts. The tissues were homoginized after adding with chloroform: methanol mixture solution and storaged at cool and dark place to extract total lipid. The total lipid contents were 29,34∼36.54% in liver 4.95∼6.11% in intestine 1.08∼1.60% in skin and 0.23∼0.38% in muscle of the pufferfish respectively. The contents of DHA and EPA were higher in the total lipids of livers showing 15.99% DHA and 3.04% EPA. The other fatty acids in the total lipids of liver were mainly composed of palmitic acid(16:0) palmitoleic acid(16:1) stearic acid(18:0) and oliec acid(18:1) Furthermore the contents of neurtral lipids were 95.45% and those of phospholipids and glycolipids were 1.45 and 3.09% respectively. Main fattty acids of the neutral lipid were composed of palmitic acid(16:0) stearic acid(18:0) oleic acid(18:1) oleic acid(18:1) EPA(20:6) and DHA(22:6) The contents of DHA and EPA were 16.62 and 2.41% respectvely. From these results of toxicity in the raw liver the tissue was judged to be nontoxic before and after extracting of total lipid.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.470-476
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• 1992

Upon lyophilization of Nocardia mediterranei, the effects of cryoprotectants, cell concentration and drying time on viability were examined, The data were treated by computer according to response surface analysis. As a result, the maximum value of presumed viability was 39.3% under the optimal conditions of 1l.6%(v/v) sucrose, $1.16{\times}10^{11}$(CFU/ml) cell concentration, and drying time for 6.18 hrs. We also used the starter cell of rehydrated solution after lyophilization in industrial production, obtained the fermentation pattern and the purity of rifamycin B which were the same with control (FVM) and it is possible for us to use N mediterranei as a starter cell after the storage of lyophilization for 18 months.

• Journal of Ginseng Research
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• v.14 no.3
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• pp.353-356
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• 1990

The isolation of volatile compounds by vacuum-distillation with freeze-drying was tested 1 with fresh ginseng roots. The roots were frozen at-8$0^{\circ}C$; they were dried at-4$0^{\circ}C$ tinder vacuum(40 tory), for 24 hours; and the ice condensed at the silrface of condenser in the freeze-dryer was thauved at room temperature. The ether extract of the resulting aqueous solution was analyzed by gas chromatography (GC) equipped with a flame ionization detector (FID) or a nitrogen-phosphorils detecto(NPD) and by gas : chromatography/mass spectrometry(GC/MS). More than forty peaks were observed in the CG(FID) profile. and more than ten peaks were observed in the GC(NPD) profile. Among them, thirteen components 1including one aldehyde, four hydrocarbons, two esters, folly alcohols, and two vyrazines were identified: six components the molesuiar ions of which were m/z, 204 were estimated to be a series of azulene compounds; and the other components unidentified were estimated to have molecular weights of lower than 254. Therefore, the freeze-drying technicue is thought to be usefu1 for the isolation of volatile compounds of such low molecufilar weights from vegetables, fruits and biological fluids as well as fresh ginseng roots under the tested conditions.