• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.117-124
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• 2007

Objective: The aim of this study was to compare the efficacy of slow freezing with vitrification method for cryopreservation of mouse pronuclear stage embryos. Methods: Mouse pronuclear embryos obtained from superovulated mice and classified into 2 groups of slow freezing and vitrification. Slow freezing solution consisted of 1.5 M PROH, 0.1 M sucrose, while vitrification solution consisted of 40% ethylene glycol, 18% Ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline supplemented with 10% SSS. Recovery and survival rates after thawing and development rates to hatching balstocyst stage were compared between two groups. Results: After freezing and thawing, recovery rate of slow freezing group was 93.8%, whereas vitrification group was 66.5% (p<0.01). Survival rate of recovered embryos were similar between two groups as 83.2% in slow freezing and 87.6% in vitrification. Embryo development rates to 2-cell stage after 24 hrs (77.0% vs 59.1%), 4-cell after 48 hrs (72.6% vs 53.3%), blastocyst after 96 hrs (53.1% vs 40.1%) of thawing were significantly higher in vitrification group than those of slow freezing group, respectively. Conclusion: The vitrification method may provide better developmental competence of frozen-thawed embryos than that of slow freezing method for cryopreservation of mouse pronuclear stage embryos.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.4
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• pp.463-470
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• 1998

To improve muscle quality and prolong freshness of sashimi, the effects of freezing-thawing condition on physicochemical and rheological properties of plaice muscle were investigated. Muscle tested were frozen with quick freezing (liquid nitrogen gas) or slow freezing ($-15^{\circ}C$ air). Transition time of zone of ice crystal formation was within 10 minute for quick freezing and 110 minute for slow freezing. Time required for thawing to $0^{\circ}C$ in muscle temperature by various thawing methods was shortest with $25^{\circ}C$ tap water, followed by $15^{\circ}C$ tap water, $10^{\circ}C$ tap water, $25^{\circ}C$ air, $5^{\circ}C$ tap water and $0^{\circ}C$ cold water. Breaking strength of muscle was higher in quickly frozen sample than in slowly frozen sample. According to sashimi term, changes in breaking strength of muscle did not show any difference in quickly frozen sample, while showed significant difference in slowly frozen sample. The remaining content of ATP was not effected by freezing speed, and ATP content was apt to higher in quickly thawed sample than in slowly thawed sample. IMP was the majority of ATP and it's related compounds of sample after freezing and thawing. Collagen matrix was weakened markedly in slowly frozen sample than in quickly frozen sample.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.43-50
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• 2003

The objective of this study was to investigate the effect of cryopreservation by slow and rapid freezing on the sperm motility index, viability and morphology of post-thaw human spermatozoa. After rapid freezing and thawing, sperm motility index was significantly higher (MOT:47.40$\pm$20.06%, VCL : 38.12$\pm$15.58 $\mu$m/s, VSL : 28.19$\pm$14.10 $\mu$m/s, VAP:33.64$\pm$15.15 $\mu$m/s, and HYP 2.77$\pm$2.71%) than slow freezing and thawing(MOT : 43.39$\pm$ 18.79%, VCL .33.91 $\pm$ 13.50 Um/s, VSL . 19.98$\pm$0.88 $\mu$m/s, VAP : 24.60$\pm$11.72 $\mu$m/s, and HYP . 1.33$\pm$1.57% ; P<0.05). But sperm Linearity(LIN) was significantly lower(28.83 $\pm$ 10.35) comparing to the slow freezing method(34.64 $\pm$ 11.36 ; P<0.05). On the other hand, significant difference were not observed MAD, WOB, DNC and DNM by slow and rapid frozen-thawed methods. After rapid freezing and thawing, sperm viability was lower(60 $\pm$ 2.2%) than slow freezing method(62 $\pm$2.1%) and sperm morphology was higher(46$\pm$7.7%) than that(44: 8.3). But there was no significantly These results indicate that rapid freezing method was positive effect of sperm cryopreservation in human.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.75-84
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• 1999

This study assessed development in vitro of pronuclear(PN) stage embryos cryopreserved by the method of either vitrification or slow freezing, by using of different cryoprotectants, and equilibration and cooling rate, in rabbit. Ethyleneglycol- ficoll- sucrose(EFS) or ethyleneglycol- polyvinylpyrrolidone - galactose- (EPG-I) for vitrification, and EPG- II for slow freezing as cryoprotectant were used. The pronuclear embryos were exposed to EFS for 0 to 5 min and diluted with D-PBS and/or pre-dilution with 0.5 M sucrose. To examine the viability of frozen-thawed embryos, PN embryos were co-cultured with bovine oviductal epitherial cell(BOEC) for 5 days to hatching blastocyst stage in 39 $^{\circ}C$ 5% $CO_2$incubator. The results obtained were as follows: The dilution with 0.5 M sucrose and D-PBS after the exposure to EFS for 1.0 min resulted in no significant(P＜0.05) decrease in the development of PN embryos to hatching blastocyst(72.0%), compared with controls. The development of PN embryos cryopreserved to hatching blastocyst was not significantly (P＜0.05) different between EFS for 1.0 min(72.0%), EPG-I for 1.0 min(72.0%) and EPG-II for 30 min(66. 7%). The post-thaw development of PN embryos to hatching blastocyst was similarly very low as 6.1% and 11.5% in vitrification with EFS and slow freezing with EPG-II, respectively. The incidence of post-thaw zona-crack in PN embryos cryopreserved by slow freezing with plunging to liquid nitrogen at －35$^{\circ}C$ was signicantly(P＜0.05) higher(25.0%), compared with －85$^{\circ}C$ (1.9%). These results indicated that the rabbit PN embryos could be cryopreserved with either vitrification or slow freezing procedure, and frozen PN embryos could be successfully developed in vitro to haching blastocyst. but the post-thaw development of cryopreserved PN embryos was still very low under the present conditions.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.263-268
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• 2000

The development of single blastomeres isolated from 2-, 4- and 8-cell mouse embryos and the ability of such blastomeres to survive slow freezing were studied. Of 223, 60 and 188 single blastomeres isolated from 2-, 4- and 8-cell mouse embryos, respectively, 111 blastomeres (49.8%) from 2-cell embryos, 12 blastomeres (20.0%) from 4-cell embryos and blastomeres (16.5) from 8-cell embryos developed into blastocysts after culture for 96 hrs. The recovery rate was 54.2% (65/120), 46.4% (13/28) and 24.3% (17/70) of blastomeres derived from 2-, 4- and 8-cell embryos following freezing and thawing and the survival of frozen-thawed blastomeres was 27.1% (16/59), 36.4% (4/11) and 17.6% (3/17), and respectively. The apparently six normal fetuses were obtained from frozen-thawed blastomere from 2-cell embryos after transferring into the recipients. These results indicate that mouse btastomeres isolated from preimplatation stage embryos can survive storage in liquid nitrogen following slow freezing.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.41-50
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• 2004

연구 목적: 본 연구는 인간배아줄기세포 동결에 minimum volume cooling (MVC) 초자화 동결방법이 유용하게 이용될 수 있는지의 여부를 조사하고자 실시하였다. 연구재료 및 방법: 인간배아줄기세포 콜로니는 0.05% collagenase 처리와 기계적 처리에 의해 작은 clumps로 자른 다음 동결 방법에 따른 효율을 비교 검토하고자, i) 대략 40-50개의 clumps를 10% DMSO가 들어있는 동결액에 $5{\sim}10$분간 처리하여 1ml cryo-vial에 넣고 slow-cooling용 cryo-module에 장착하고 -80C에서 overnight한 후 $LN_2$에 침지하여 완만동결을 실시하거나 ii) 10% ethylene glycol (EG)이 들어 있는 동결액에서 5$\sim$10분 처리하고 30% EG과 0.5 mol sucrose가 들어 있는 동결액에서 30초간 처리하여 본 연구를 위해 개발된 MVC straw에 10 clumps씩 적재한 다음 $4{\sim}5$ MVC straw를 $LN_2$가 들어있는 cryo-vial에 넣어 MVC 초자화동결을 실시하였다. 융해 후 생존율을 조사하였고 배아줄기세포의 특성을 유지하고 있는지의 여부를 조사하였다. 결 과: 융해 후, 인간배아줄기세포의 생존율은 완만동결을 실시했던 군 (20.0%) 보다 MVC 초자화동결을 실시했던 군에서 (76.0%) 유의하게 높게 나타났다. 또한 회복과정에서 완만동결을 실시했던 군에서는 세포성장이 매우 더디고 안 좋은 반면, MVC 초자화동결을 실시했던 군은 배아줄기세포의 증식이 동결을 실시하지 않은 세포와 같은 상태로 2주 이후부터 빠르게 전환되고 회복되는 것을 확인할 수 있었다. 이와 더불어 MVC 초자화동결－융해 후 회복된 배아줄기세포에서 정상 핵형, alkaline phosphatase acitivity, SSEA-4와 TRA-1-60 염색 및 Oct-4 발현을 확인하였으며 체외분화의 특성도 확인하였다. 결 론: 새로이 개발된 MVC 초자화동결을 이용하면 인간배아줄기세포는 고유의 특성을 잃지 않고 성공적으로 동결될 수 있다.

• Bulletin of Food Technology
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• v.25 no.2
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• pp.116-128
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• 2012

식품동결에 의한 품질손상에 있어 크게 영향을 미치는 인자 중의 하나가 체적변화에 의한 조직 파괴이므로 식육 및 과채류의 냉동시 내부압력에 따른 조직파괴를 방지하기 위한 기초연구로 다양한 동결방법에 따른 내부압력 변화에 대하여 조사한 결과, 급속 및 완만동결시 우육의 내부압력 변화 경향은 동결 직전에 급격히 상승한 후 동결 초기부터 최대빙결정생성대를 통과하는 동안은 급격히 감소하다가 다시 온도가 내려감에 따라 압력변화는 증가와 감소를 반복하여 상승하는 경향을 보여 주었고, 동결시의 내부압력 크기는 약 8~10 psig 수준이며, 내부압력의 변화 경향은 정지공기식보다 침지식에서 약 1 psig 정도 크게 나타났다. 냉동냉장시에 일어나는 내부압력의 상하변화는 돈육의 품온차가 ${\pm}1^{\circ}C$일 때, 시료중량에 따라 1.84~2.32 psig 정도의 내부압력 차가 반복적으로 발생됨을 알 수 있었다. 또한, 돈육의 해동시 내부압력은 해동 개시 후 급격히 상승하여 5분 이내에 최고압력에 도달한 이후에는 서서히 하강하였으며, 해동시에 발생한 내부압력의 값은 동결시의 내부압력 값보다도 대부분 크게 나타났다. 또한, 균온처리한 우육의 동결시 내부압력 값은 약 1~4 psig 수준으로 균온처리하지 않은 우육에 비하여 매우 적게 나타났으며, 동결소요시간도 약 10~20% 정도 짧게 나타났다. 그리고 균온처리 및 다양한 동결방법에 따른 동결식육의 조직과 냉동냉장 중의 품질변화를 비교 검토한 결과, 동결방법에 따른 드립손실율은 우육의 경우 정지공기식으로 처리한 시료는 타 처리구에 비해 계속 높게 나타났으며, 돈육은 우육에 비해 상대적으로 드립손실량이 적게 나타났으나 송풍식으로 처리한 시료에서 저장 40일째, 7.39%로 가장 높게 나타났다. 동결 우육 및 돈육의 pH변화는 뚜렷한 차이를 나타내지 않았으며, VBN 및 TBA값에 있어서는 균온 처리한 시료가 완만 및 급속동결 처리한 타 시료에 비해 저장 200일까지 가장 변화가 없었다. 동결저장온도의 상하변동에 있어서는 온도변동 횟수가 증가할수록 pH 및 수분함량은 뚜렷한 변화가 없었으나 드립손실율, VBN 및 TBA값은 온도 변동횟수가 증가할수록 서서히 증가하는 경향을 보여 주었다.

• Journal of Animal Science and Technology
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• v.55 no.5
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• pp.417-425
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• 2013

We sought to provide a method for freezing and preserving primordial germ cells, or an avian germ cell of a bird, as a material for developmental engineering or species preservation. The aim of this study was to compare the efficacy of slow freezing with a vitrification method for the cryopreservation of chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonad of day 5.5-6 day (stage 28) cultured chick embryos, using the MACS method, were classified into two groups: slow freezing and vitrification. We examined the viability of PGCs after Cryopreservation. Four freezing methods were compared with each other, including the following: Method 1: The PGCs were frozen by a programmed freezer in a plastic straw, including 2.0 M ethylene glycol (EG) as cryoprotective additive (slow freezing) Method 2: The PGCs were vitrified in a plastic straw, including 8.0 M EG, plus 7% polyvinylpyrrolidone (PVP) (rapid freezing). Method 3: The slow freezing was induced with a cryotube including 2.0 M EG Method 4: The PGCs were frozen in a cryotube including 10% dimethyl suloxide (DMSO) (rapid freezing). After freezing and thawing, survival rates of the frozen-thawed PGCs from Method 1 to 4were 76.4%, 70.6%, 80.5% and 78.1% (p<0.05), respectively. The slow freezing ($-80^{\circ}C$ programmed freezer) method may provide better survival rates of frozen-thawed PGCs than the vitrification method for the cryopreservation of PGCs. Therefore, these systems may contribute to the cryopreservation of a rare avian species.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.4
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• pp.267-291
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• 2010

Life can be kept in suspended animation either before fertilization at oocyte stage or after fertilization at different stages of embryonic development for a variety of reasons. It not only has potential applications in fertility preservation and management in human but also has important roles in the preservation and management of animal genetic resources, low-cost international movement of selected genetics, and rapid dissemination of germplasm through assisted reproductive technologies (ART) and genetic engineering. Currently, slow-freezing and vitrification are the two approaches by which oocytes and embryos can be cryopreserved for long-term storage. Both of these methods have their own advantages and disadvantages but allow the cryopreservation of oocytes and embryos with comparable efficiency. Vitrification of oocyte and embryos, although proven successful just 13 years after slow-freezing, is generally considered an emerging technology and appears to slow gain acceptance in both animal and human ART despite having controversial storage and contamination issues. In this manuscript, we discuss the basic techniques of oocyt/embryo cryopreservation and review the current status and recent developments in vitrification.

• 대한생식의학회:학술대회논문집
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• 1999.11a
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• pp.46-46
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• 1999