• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.245-252
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• 2002

This study was to evaluate the in vitro survival of bovine enucleated MII (eMII) oocytes according to minimum volume cooling (MVC) freezing method and activation timing, and their in vitro development after somatic cell nuclear transfer (SONT). in vitro matured bovine oocytes for 20 h were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst, and their 1st polar body and MII plate were removed by enucleation micropipette under UV filter. Also, eMII oocytes were subjected to activation after (group II) and before (group III) vitrification in 5 ${\mu}{\textrm}{m}$ ionomycin added CRlaa medium for 5 min. For vitrification, eMll oocytes were pretreated with EG10 for 5 min, exposed to EG30 for 30 sec and then directly plunged into L$N_2$. Thawing was taken by 4-step procedures at 37$^{\circ}C$. Survived eMII oocytes were subjected to SONT with cultured adult bovine ear cells. Reconstructed oocytes were cultured in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide and 2.5 $\mu\textrm{g}$/$m\ell$ of cytochalasin D added CRlaa medium for 1 h, and then in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide added CRlaa medium for 4 h. Subsequently, the reconstructed oocytes were incubated for 2 days and cleaved embryos were further cultured on cumulus-cell monolayer drop in CRlaa medium for 6 days. Survival rates of bovine vitrified-thawed eMII oocytes in group II (activation after vitrification and thawing) and III (activation before vitrification) were 81.0% and 84.9%, respectively. Fusion rates of cytoplasts and oocytes in group II and III were 69.0% and 70.0%, respectively, and their results were not different with non-frozen NT group (control, 75.2%). Although their cleaved rates (53.4% and 58.4%) were not different, cytoplasmic fragment rate in group II (32.8%) was significantly higher than that in group III (15.6%)(P<0.05). Also, subsequent development rate into >morula in group II (8.6%) was low than that in group III(15.6%). However, in vitro development rate in group III was not different with that in control (24.8%). This result suggested that MVC method was appropriate freezing method for the bovine eMII oocytes and vitrified eMII oocytes after pre-activation could support in vitro embryonic development after SONT as equally well as fresh oocytes.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.263-271
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• 2006

The purpose of this study was undertaken to compare ability of frozen-thawed sperm characteristics between two strains (miniature pig and Duroc). The semen was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle. The semen was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. The frozen-semen straw were thawed at 20, 37 and $50^{\circ}C$ for 1 min, 45 sec and 10 sec within water-bath. The semen sample were evaluated at 0, 3, 6, 9, and 12 h after incubation at $37^{\circ}C$ for analysis of sperm ability. Abnormality of spermatozoa in miniature pig was significantly (p<0.05) higher than that in Duroc at 0, 9 and 12 h of post-thawing incubation after frozen-thawing. The percentage of F-patterned spermatozoa in miniature pig was significantly (p<0.05) lower, while the percentage of AR (acrosome reacted spermatozoa) pattern was higher in the miniature than in the Duroc. On the other hand, there was no significant difference in the viability of spermatozoa thawed at different temperature ($20^{\circ}C\;and\;37^{\circ}C$) between two species, but the viability in miniature pig was higher (p<0.05) than in Duroc when sperm was thawed at $50^{\circ}C$. In conclusion, this study suggest that suitable freezing method for miniature pig semen is required for increasing post-thawing viability and fertilization capacity.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1529-1535
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• 1999

This study was done for the extraction of carotenoid from Phaffia rhodozyma in combination with PEF and other methods. PEF treatment conditions were $30{\sim}80\;kV/cm,\;100{\sim}1000\;Hz\;and\;100{\sim}1000\;{\mu}s$. In order to increase permeability of yeast cell wall, various methods such as freezing-thawing, mechanical treatment, solvents, permeabilizing agents, and yeast cell wall lytic enzyme were used before PEF treatment. The combination of PEF $(50\;kV/cm,\;300\;Hz,\;1000\;{\mu}s)$ and conventional methods such as solvent and freezing-thawing pre-treatment had no effects on the extraction of carotenoid pigments. The extent of extracted carotenoid by the PEF $treatment(50\;kV/cm,\;300\;Hz,\;1000\;{\mu}s)$ combined with yeast cell wall lytic enzyme and mechanical pre-treatment increased 52% and 69.8% more than the sum of that by each treatment, respectively. Permeabilizing agents, especially Tween 20 and capric acid, enhanced the extraction efficiency of carotenoid pigments from P. rhodozyma cells. These results indicated the feasibility for the continuous extracting carotenoid pigments from P. rhodozyma by PEF combined with other permeabilizing methods.

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.7
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• pp.994-1001
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• 2012

Quality changes of pre-processed garlic, peeled and chopped, were analyzed during storage at $-18^{\circ}C$ or $2^{\circ}C$ for 30 days and at $20^{\circ}C$ for 3 days only for chopped garlic. As storage time increased, Hunter L values decreased and a, b values increased, indicating browning regardless of the pre-process type and storage temperature. Decay and sprouting rates of peeled garlic during storage at $2^{\circ}C$ significantly increased while those of peeled garlic were maintained during storage at $-18^{\circ}C$. Weight loss of peeled garlic during storage was greater at $-18^{\circ}C$ than at $2^{\circ}C$. Hardness of peeled garlic rapidly decreased by half from 1.04 kg to 0.58 kg by freezing, and it did not significantly change during the storage period. Viable numbers of total aerobic bacteria of peeled and chopped garlic did not significantly change during the storage period at $2^{\circ}C$ but were reduced at $-18^{\circ}C$. Total aerobic bacterial count of chopped garlic stored at $20^{\circ}C$ slightly increased during the storage period. Pyruvic acid content of chopped garlic was almost 2.5 times higher than that of peeled garlic at the initial stage (463.87 ${\mu}mol/g$ and 190.52 ${\mu}mol/g$, respectively). As storage time increased, pyruvic acid content of peeled garlic increased while that of chopped garlic decreased. These results indicate that pre-process type and storage temperature affected the quality changes of garlic during storage.

• 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 Poultry Science
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• v.40 no.3
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• pp.207-216
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• 2013

This study was conducted to establish the method for preserving PGCs that enables long-term storage in liquid nitrogen for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of freeze-thaw treatment on viability of PGCs in chickens. PGCs were collected separately from a germinal gonad of an early embryo of 5.5~6 day (stage 28) of Isa brown, Korean Oge (KO), White Leghorn and Commercial breeds. PGCs separated from a germinal gonad of an early embryo of 5.5~6 day (stage 28) are suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The PGCs were then purified using magnetic activated cell sorting (MACS) method. The viability of PGCs after thawing was $87.4{\pm}0.4%$ and $89.4{\pm}0.2%$ with the 10% EG treatments with no significant difference between the Isa brown and Commercial breeds. The viability of PGCs after freeze- thawing was significantly higher for Isa brown ($87.4{\pm}0.4%$) and Commercial breeds ($89.4{\pm}0.2%$) than Korean Oge (KO) ($77.6{\pm}1.1%$) and White Leghorn ($76.2{\pm}0.9%$)(p<0.05) using 10% EG cryoprotectant. This study established a method for pre- serving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at agermplasm repository and ease of entry into a data base. 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.

• Journal of Conservation Science
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• v.9 no.1
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• pp.40-47
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• 2000

Dimension stability was examined after PEG pretreatment and post freeze-drying treatment in order to determine the PEG(#3350) concentration and solvent for pre-treatment of freeze-drying of highly-degraded waterlogged ash woods(Fraxinus spp.; ca. 5,700 BP) excavated from peat lands at Pyungtack, Kyounggi-do. At the low concentration (<30-40%) of PEG soaking in both water and t-butanol, the weight increases abruptly, but at high concentration (>50%) gradually, consequently, taking longer treatment time. PEG loading was higher in t-butanol solution than in water. However, the best dimesional stability was obtained from freeze-drying after lower PEG solution (40% in water) soaking. Low dimensional stability, found in the samples treated with higher PEG solutions (60%-70% in t-butanol), might come from incomplete freezing and excess PEG absorbing moisture. The samples air-dried after 70% PEG treatment had collapse defects. In conclusion, the use of low concentration (about 40% in water) PEG solution was the most suitable pretreatment for freeze drying of highly-degraded waterlogged ash woods.

• Journal of Acupuncture Research
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• v.21 no.5
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• pp.59-68
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• 2004

Objective : This study was designed to evaluate the effect of electroacupuncture with various scales on symptoms of idiopathic Parkinson's disease. Methods : Subjects were voluntarily recruited by newspapers and internet. All the subjects are confirmed as idiopathic parkinson's disease by a neurologist. The acupuncture therapy was performed twice a week for 8 weeks. Acupuncture points for EA group were GB34, and LR3, electrical stimulation frequency was 120Hz, duration 15 minutes, and intensity is up to pain threshold according to patients. The patient's symptoms were assessed before, after 4 weeks and after 8 weeks treatments by unified Parkinson's disease rating scale(UPDRS), modified Hoehn-Yahr(H-Y) stage, Schwab & England activity of daily living and freezing of gait questionnaire(FOGQ). Results : UPDRS I, II, III, IV and total UPDRS scores were significantly improved after 4 weeks(p<0.05) and after 8 weeks(p<0.05) compared to the pre-treatment. The scores of ADL were significantly improved after 4 weeks(p<0.05) and after 8 weeks(p<0.05). There were significant changes in H-Y stage after 4 weeks(p<0.05), but There were no significant changes in H-Y stage after 8 weeks. There were significant changes in FOGQ scale after 4 weeks(p<0.05) and after 8 weeks(p<0.05). Conclusion : This study suggests that electroacupuncutre treatments can be applicable to improve symptoms in the patients with idiopathic Parkinson's disease.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.05a
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• pp.12-12
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• 2010

We developed droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification, and applied to germplasm collections of garlic, potato, lily as well as cell lines, including hairy roots, somatic embryos. To establish a garlic cryobank, four Korean garlic field collections at Danyang, Suwon, Mokpo and Namhae were cryopreserved last five years. The protocol applied consisted of preculture for 3-4 days at $10^{\circ}C$ on solid MS medium with 0.3M sucrose, loading for 40 min in liquid medium with 35% PVS3, dehydration with PVS3 for 150 min, cooling in $5{\mu}l$ droplets of PVS3 placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated($40^{\circ}C$) 0.8M sucrose solution for 30s. A total of over 900 accessions of garlic were stored in liquid nitrogen for long-term conservation using unripe inflorescences, cloves or bulbils. Twelve alternative plant vitrification solutions were designed by modifying cryoprotectant concentrations from the original PVS2 and PVS3. The results suggest that PVS2-based vitrification solutions with increased glycerol and sucrose and/or decreased DMSO and EG concentrations can be applied for medium size explants which are tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.

• The Korean Journal of Nuclear Medicine Technology
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• v.13 no.1
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• pp.116-119
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• 2009

Background: Preanalytical factors can affect reliability of hormone assay results. Adrenocorticotropic hormone (ACTH) in blood is considered highly unstable because of proteolytic degradation, so storage of blood samples on ice until analysis is recommended. In clinical practice, however, this procedure may present logistical problems because most samples for ACTH measurement must be shipped from the place of sample collection to the laboratory. Therefore, we studied the impact of time and temperature before plasma separation and analysis on the results of ACTH assays. Methods: A total number of 22 patients were enrolled in this study. We obtained 2 blood samples. ACTH concentrations were 35~126 pg/mL. ACTH concentrations were measured by immunoradiometric assay (IRMA) using commercial kits (CIS Biointernational, Gif-sur-Yvette, France). Results: ACTH levels showed a significant difference between the samples of $22^{\circ}C$ EDTA and $4^{\circ}C$ EDTA. Measured ACTH concentrations significantly decreased with time before freezing at $-20^{\circ}C$. ACTH levels showed no significant difference between the groups of after storage for 24 hr without centrifugation at $22^{\circ}C$ and $4^{\circ}C$. Conclusion: We recommend that blood samples be obtained on pre-chilled EDTA collection tubes. The shortest possible time between sample collection and processing is always the best laboratory practice.