• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Korean Journal of Food Science and Technology
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• v.30 no.5
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• pp.1209-1213
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• 1998

The gamma-radiation sensitivity of four kinds of Escherichia coli O157:H7 was investigated in frozen cells $(-18^{\circ}C)$ with 0.1 M phosphate buffer and inoculated cells in beef. The maximum populations were observed at 12 hr when E. coli O157:H7 was incubated in the tryptic soy broth at $37^{\circ}C$. In the case of the frozen cells at logarithmic phase, the $D_{10}$ and $12D_{10}$ values of four kinds of E. coli O157:H7 were $0.09{\sim}0.15\;kGy$ and $1.08{\sim}1.80\;kGy$, and inactivation factors were $13.33{\sim}22.22$ and $20.00{\sim}33.33$ at radiation doses of 2 and 3 kGy, respectively. The radiosensitivity of inoculated E. coli O157:H7 in beef showed the $D_{10}$ value of $0.30{\sim}0.47\;kGy$, the $12D_{10}$ value of $3.60{\sim}5.64\;kGy$, and inactivation factor of $4.26{\sim}10.00$. The radiosensitivity of the frozen cells was higher than that of the inoculated E. coli O157:H7 in beef. Gamma irradiation at doses within the range of 1.5 to 3 kGy is considered to be an effective method to control E. coli O157:H7 in beef.

• Archives of Plastic Surgery
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• v.35 no.4
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• pp.385-392
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• 2008

Purpose: The most effective methods of harvesting, preparing, and injecting autologous fat grafts have been inconsistent and conflicting. With its limitation as resorption in fat grafting, handling various techniques affect adipocyte survival, and is crucial to optimizing its long-term survival. To improve graft survival, re-implantation of cryopreserved adipocytes was developed. In addition, adipocytes do not induce immune rejection in response to non-self lymphocytes in a mixed lymphocyte reaction. The purpose of this study is to analyze the changes in cryopreserved adipocytes so as to determine the most efficient long-term storage period, and to analyze the changes in cryopreserved allografted adipocytes so as to determine the efficacy of cryopreserved adipocytes allografting. Methods: Fat tissues were harvested from the inguinal and retroperitoneal fat pad of mice. After the centrifugation of the harvested fat tissues, they were disintegrated with collagenase. The adipocytes were obtained by centrifugation of the disintegrated fat tissues. The adipocytes were treated as follows: (1) They were examined for weight and then frozen at $-20^{\circ}C$(n=25). For four months, each five frozen samples were taken and examined for weight and histologic changes in the 1st week, the 1st month, the 2nd month, the 3rd month, and the 4th month, respectively. (2) The adipocytes were immediately frozen at $-20^{\circ}C$(n=125). For four months, five frozen samples were taken, and allografted in the same time period as above. Finally, for four months, five cryopreserved allografted adipocytes were taken and examined for histologic changes in the same time period as above. Results: (1) Significant weight changes and histologic findings with inflammatory and destructive changes were observed in the cryopreserved adipocytes in three months. (2) Significant fat necrotic changes in the histologic changes with Hematoxylin and eosin stain were observed in the cryopreserved allografted adipocytes since the first week, independent of the freezing period. Conclusion: The study results show that the adipocytes that were cryopreserved for more than three months underwent obvious weight reductions and necrotic changes, and the adipocytes that were allografted without freezing were viable for four months, but the cryopreserved allografted adipocytes had obvious necrotic changes since the first week regardless of the freezing period.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.59-64
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• 2006

This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until $5^{\circ}C$ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with $LN_2$. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of $LN_2$ for 10 min). Sperm viability increased by holding at $-102^{\circ}C$ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in $37^{\circ}C$ group than in $52^{\circ}C$ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at $-102^{\circ}C$ for 10min before plunging into $LN_2$ is a convenient and easy freezing method for boar semen.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.2
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• pp.119-125
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• 2016

Objective: The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Methods: Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. Results: A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Conclusion: Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.71-76
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• 1998

This study was carried out to examine whether the developmental capacity of bovine immature oocytes frozen ultra-rapidly using electron microscope (EM) grids and EFS30 can be obtained. 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. As criterior of oocyte viability, the rates of maturation, fertilization and embryonic development were determined. The results obtained in this experiment were summarized as follows: When ultra-rapidly frozen immature oocytes were thawed, 43.2% of them were survived. The rates of maturation (84.1%) and normal 2 pronuclei formation (57.5%) of frozen immature oocytes were not significantly different when compared to those of control (92.5, 65.0%). In addition, the rates of $\geq2$-cell (65.0%) and blastocyst formation (30.8%) of freezing group were not significantly different when compared to those of control (73.7, 35.7%). These results demonstrate that developmental capacity of frozen-thawed bovine immature oocytes can be successfully obtained when survived from the ultra-rapid freezing method using EM grid and EFS30.

• The Korean Journal of Cytopathology
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• v.16 no.2
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• pp.93-97
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• 2005

Merkel cell carcinoma (MCC) is a rare primary cutaneous neuroendocrine carcinoma which commonly presents as a skin nodule, and can develop into regional lymph node metastases, as well as spread systematically. Here, we describe the cytological features of a touch imprint of MCC that arose on the face of a 62-year-old female. This touch imprint was acquired from an incisional biopsy specimen which had been submitted for frozen section. The touch preparation produced a highly cellular imprint of loosely cohesive groups of small- to medium-sized malignant cells exhibiting uniform round to oval nuclei, delicate nuclear membranes, fine chromatin, small nucleoli, and scanty cytoplasm, with occasional paranuclear button-like inclusions. We applied cytokeratin 20 to the touch imprint for immunochemistry, allowing us to visualize the tumor cells with paranuclear dot-like positivity. Both the cytological and immunocytological features were quite distinct.

• KSBB Journal
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• v.14 no.4
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• pp.389-395
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• 1999

Bifidobcterium spp. can provide human being with several beneficial physiological. Therefor, there has been a considerable interest in products Bifidobcterium spp. dietary supplements or as starter cultures for probiotic products that may assint in the improvement of health on the human. But indusrial applications have been limited because Bifidobcterium spp. are sensitive to acidic pH due to organic acid produced by themselves and various conditions. The objective of this study was to establish new method for improvement of Bifidobcterium viability by entrapment im calcium alginate beads. We have a plan to select the most suitable polymer through the comparison with acid tolerance oxygen tolerance and theological properties of polymer. Increase of the viable number of Bifidobcterium induced increasing acid tolerance and oxygen tolernce trough the development of entrapment technique. The 4%, 3030mm diameter) sodium alginate beads led to the best survivability under acid condition. Especially, addition of 6% mannitol, 6% glycerol or 6% sorbitol to the sodium alginate helped a beneficial effect on viability against acid, bile salt, hydrogen peroxide and cold strage. The number of viability of entrapeede cells by retreatment was 96 fold higher than non-entrapeed cells after 5 hours of storage under pH 3 acidic condition. These experimental data clearly demonstrate that a whole cell immobilization by entrapment in calcium alginate beads is an important survival mechanism enable to withstand environmental stresses as the acidic condition, hydrogen peroxide toxicity and frozen state.

• Korean Journal of Head & Neck Oncology
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• v.32 no.2
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• pp.19-22
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• 2016

Adenosquamous carcinoma (ASC) of the larynx is very rare malignancy which has well defined two distinctive pathological features, an adenocarcinoma and a squamous cell carcinoma (SCC). Diagnosis of ASC by endoscopic biopsy is challenging due to small amount of harvested tissue. ASC has a worse prognosis than SCC with an early lymph node metastasis and a distant dissemination. We experienced a rare case of vocal fold ASC which was initially misdiagnosed as atypical squamous cell proliferation at frozen biopsy. We reported this case with a literature review.

• Proceedings of the SAREK Conference
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• 2006.06a
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• pp.74-79
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• 2006

Good cold start characteristics are essential for satisfactory operation of fuel cell vehicles. In this study, the melting process has been numerically investigated for a water tank frozen in cold weather The 2-D model of the tank containing ice and plate heaters was assumed and the unsteady melting process of the ice was calculated. The enthalpy method was used for the description of the melting process, and a FVM code was used to solve the problem. The feasibility study compared with other experiment showed that the developed program was able to describe the melting process well. From the numerical analysis carried out for different wall temperatures of the pate heaters, some important design factors could be found such as local overheating and pressurization in the tank.