• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.35-42
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• 1996

This study was carried out to investigate the effect of straw loading method and thawing protocol on the in vitro development of in vitro produced mouse blastocysts cryopreserved by vitrification. Three loading types of straw I, Il and III on loading and sealing method were made for vitrification. The ability of the solution on straw loading methods to remain vitreous during warming was tested by exposed in air for 1 to 10 s sec. and then plunged the vitrified straws into water bath at 25°C. Embryos to be vitrified were equilibrated to the 20% EG for 5min. and exposed in EFS 40 for 1min. The plug ends of Straw I and Straw II were sealed with straw powder and Straw III was treated straw powder, followed by heat sealing and then plunged into LN$_2$. The results obtained in these experiments were summarized as follows; 1) Straw I embryo column mostly changed from transparent to opaque upon thawing without exposure in air for 3-6 sec. Straw II embryo column was I improved partially but was not remained completely vitreous during warming. However, when Straw lll loading method was used, the embryo column was remained vitreous completely. 2) High survival rates and development rates of each groups (middle blastocysts and hatching blastocysts) of vitrified embryos were obtained by using Straw III loading method than Straw I method (P<0.05). And the range of s standard error was low in Straw lll method. 3) When the embryos vitrified-frozen were placed in air for 3, 5 and l0sec. and then warmed rapidly in water bath at 25$^{\circ}C$, the survival rates after 24h of culture were 72.7-87.1% and the development rates to hatching stage after 48h of culture were 34.0-48.4%. There were no significantly differences according to exposure time in air during warming. In conclusion, the present results showed that highly survival and low standard error of vitrified-frozen mouse bIastocysts were obtained by using straw lll loading, double sealing and appropriate 2 step warming method.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.207-214
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• 1996

To examine the developmental capacity of manipulated embryos after ultrarapid refreezing and thawing, mouse embryos were biopsied at 4-cell stage, frozen twice at 4-cell and morula stages, respectively, and then transferred to rec-ipients. Single blastomeres were biopsied from 4-cell embryos by a modified aspiration method. Biopsied 4-cell embryos were equilibrated into freezing medium at room temperature for 2.5 min, loaded into 40 $\mu$I of freezing medium in 0.25 ml plastic straw and then directly immersed into liqiud nitrogen. Freezing medium for 4-cell embryos consisted of 4.0 M ethylene glycol and O.25 M sucrose in dPBS supplemented with 6 mg/lm BSA. Morulae were frozen into freezing medium containing 5.0 M glycerol instead of ethylene glycol. Thawing was conducted by agitating each straw in 3TC water for 20 sec. The c content of each straw was expelled into 0.5 ml of dilution medium, which consisted of 0.25 M sucrose and 3 mg/ml BSA in dPBS. The thawed embryos were rehydrated in dilution medium for 10 min, washed 3 times with dPBS and then cultured in M16 medium at 37$^{\circ}C$, 5% CO$_2$ in air. Blastocysts that developed from frozen or refrozne biopsied embryos were transferred to recipients on Day 3 of pseudopregnancy, respectively. In vitro and in vivo developmental rates of the biopsied and intact 4~cell embryos after freezing and thawing were 78 (10l/130) and 25% (10/40), and 91 (114/125) and 30% (12/40), respectively. Although the rates of in vitro development of biopsied and intact embryos to blastocyst stage were significantly different after freezing and thawing (P

• Journal of the Society of Disaster Information
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• v.9 no.4
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• pp.449-461
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• 2013

As the hydrofluoric acid leak in Gumi-si, Gyeongsangbuk-do or hydrochloric acid leak in Ulsan, Gyeongsangnam-do demonstrated, chemical related accidents are mostly caused by large amounts of volatile toxic substances leaking due to the damages of storage tank or pipe lines of transporter. Safety assessment is the most important concern because such toxic material accidents cause human and material damages to the environment and atmosphere of the surrounding area. Therefore, in this study, a hydrofluoric acid leaked from a storage tank was selected as the study example to simulate the leaked substance diffusing into the atmosphere and result analysis was performed through the numerical Analysis and diffusion simulation of ALOHA(Areal Location of Hazardous Atmospheres). the results of a qualitative evaluation of HAZOP (Hazard Operability)was looked at to find that the flange leak, operation delay due to leakage of the valve and the hose, and toxic gas leak were danger factors. Possibility of fire from temperature, pressure and corrosion, nitrogen supply overpressure and toxic leak from internal corrosion of tank or pipe joints were also found to be high. ALOHA resulting effects were a little different depending on the input data of Dense Gas Model, however, the wind direction and speed, rather than atmospheric stability, played bigger role. Higher wind speed affected the diffusion of contaminant. In term of the diffusion concentration, both liquid and gas leaks resulted in almost the same $LC_{50}$ and ALOHA AEGL-3(Acute Exposure Guidline Level) values. Each scenarios showed almost identical results in ALOHA model. Therefore, a buffer distance of toxic gas can be determined by comparing the numerical analysis and the diffusion concentration to the IDLH(Immediately Dangerous to Life and Health). Such study will help perform the risk assessment of toxic leak more efficiently and be utilized in establishing community emergency response system properly.

• 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.

• Journal of Veterinary Clinics
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• v.32 no.3
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• pp.255-258
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• 2015

Gastric dilatation and volvulus (GDV) is an acute and life-threatening disease most commonly affecting large- and giant-breed dogs. However a 17-year-old Shih-tzu (4 kg, spayed female) was hospitalized for acute GDV. Repeated unproductive retching, lethargy, and excessively enlarged abdomen were observed. Physical examination indicated that the patient had suffered from hypothermia ($36.5^{\circ}C$), tachycardia (240 bpm), slowed capillary refill time (> 2 sec.), and pale mucous membrane. Grade III murmur with normal lung sound was auscultated. Abdominal palpation revealed that tympanic regions existed in both the left and right sides. Systolic blood pressure decreased gradually from 220 to 40 mmHg within 4 hours. In blood analysis, slight azotemia was observed by blood urea nitrogen (BUN; 29.1 mg/dl) and creatinine (1.6 mg/dl). Blood lactate concentration (8.13 mmol/l) was severely elevated. Additionally, dilatation and volvulus of the stomach was observed by radiograph. Supportive oxygen, heat, fluid, and drugs were administered with gastric decompressions (e.g., gastrocentesis and nasogastric tube). However the patient entered into comatose status with uncontrollable systolic blood pressure, despite the administration of dobutamine intravenously. The case was closed by euthanasia, considering welfare and age. We finally diagnosed the patient as a GDV, thus this is the first GDV case report in small-breed dog such as Shih-tzu.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.50 no.6
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• pp.406-410
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• 2005

This study was conducted to establish the efficient protocols of the germination and cryopreservation of dehisced Korean ginseng seeds for long-term germ­plasm conservation. $GA_3$ and BA treated on seed for 24 hr facilitated germination at 5 and $10^{\circ}C$. Germination percentage of desiccated seeds was decreased under moisture content (MC) of below $7.2\%$. Dehisced ginseng seeds were dried under airflow of laminar floor cabinet and seed drying room. The high levels (more than $90\%$) of germination after cryogenic exposure were obtained after drying under vertical airflow of laminar floor for 12-30 hours (MC $10.6{\~}7.2\%$). Decrease in germination percentage of ginseng seeds due to desiccation damage and freezing injury was observed at MC of below $7.2\%$ and of above $12.1\%$, respectively. Therefore, MC of ginseng seeds need to be controlled with a range of $8{\~}10\%$ to avoid damages from both desiccation and freezing.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.65-70
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• 2012

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at $-7^{\circ}C$ to $-35^{\circ}C$ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen-thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/0.5 ml, and 43.6 % frozen at 20 embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.379-384
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• 1996

This study was undertaken to investigate the sister chromatid exchange (SCE) frequency and embryonic development after exposure to cryoprotectants and vitrification of mouse zygotes. Mouse IVF zygotes were cryopreserved by vitrification using vitrification solution, EFS40 (40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). After mouse zygotes were exposed to EFS40 at $25^{\circ}C$ for 30 sec., they were immediately plunged into $LN_2$ or cultured for cryoprotectant toxicity test without freezing. The results obtained in these experiments were summarized as follows: After thawing, survival rates to the 2-cell stage of zygotes exposed to or vitrified in EFS 40 (98.5%, 95.2%) were not significantly difference compared with that of control (100%). However, the developmental rates upto blastocyst and hatching blastocyst in vitrified groups (66.7, 50.0%) were lower than those of control (93.9, 81.8%) or exposed group (94.0, 78.8%) (p<0.05). When the influence of vitrification and exposure to cryoprotectant on the in vitro development of mouse zygotes was assessed by the SCE frequency, the SCE frequency in exposed ($20.2{\pm}2.1$) to or vitrified embryos ($21.4{\pm}3.2$) was higher than that in control embryos ($16.8{\pm}1.5$). These results suggest that the frequency of SCE was increased after cryoprotectant exposure or Vitrification although developmental rates of zygotes upto blastocysts and /or hatching blastocysts were not afected by cryoprotectant.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.163-169
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• 2013

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells (PGCs) into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. In the present study, fertilized eggs were incubated for about 5.5 days to obtain embryos at stage 28. The whole embryo was collected from the germinal gonad using a fine glass micro pipette under a microscope. The PGCs were then purified using MACS method. Two commercially available cryoprotectants (A and B) were used to preserve the PGCs, and EG were used as a control. The average recovery rate of PGCs after thawing was 35.5% and 60.5% with the A and B treatments, respectively. There was no significant difference between B treatments and control, which showed an average recovery rate of 52.8%. However, the recovery rate obtained using A cryoprotectant (35.5%) was significantly lower than using treatment control and B. The average viability of the PGCs after thawing were 77.9% and 77.4% for cryoprotectants A and B, respectively, and the control were was 81.6%. There was no statistically significant difference between the two treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos. Further experiments to produce germline chimera from PGCs preserved using this techniques are strongly recommended.

• Journal of Korean Society of Forest Science
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• v.43 no.1
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• pp.31-34
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• 1979

In this study, enzymatic hydrolysis of the substrate of the waste wood of Cortinellus edodes was investigated using crude cellulase preparation of Trichoderma viride Pers. ex. Fr. SANK 16374. The crude cellulase was produced by the submerged culture process and produced in the culture fluid was salted out quantitatively by the use of ammonium sulfate. Reducing sugar was determined by the dinitrosalisylic acid (DNS) method. 1. The chemical composition of the waste wood was crude protein 2.26%, c. fat 2.57%, c. fibre 44.60%, c. ash 5.58% and lignin 13.62%. In amino acid composition, no cystine and methionine was showed, but trace amount of Vitamin A, $B_1$, and $B_2$, niacine and chloride were detected. (Table 1) 2. As heat treatment of the substrate was found to produce the highest reducing sugar yield being reacted for 48hr. with T.v cellulase, the substrate was heated to $190{\pm}5^{\circ}C$. for 45 min. either before or immediately after milling. 3. The substrate heated and ball milled at $190{\pm}5^{\circ}C$. for 45 min. the reducing sugar yield reached to 11.5%. 4. The substrate without any treatment was found to produce the highest reducing sugar yield being reacted 72hr. with T. v cellulase, the reducing sugar yield reached to 10.1%. 5. The rate of reducing sugar per each treated substrate was decreased by the order of the substrated, heated and then ball milled at $190{\pm}5^{\circ}C$. for 45 min. (11.5%)> without any treatment (10.1)> ball milled and heated at $190{\pm}5^{\circ}C$. for 45 min. (6.9%). 6. Saccharification of waste wood has been shown to be possible by heat treated and milling the substrate in contact with cellulase. And it is likely to be recommended that the waste wood may be valuable for raw materials of saccharification.