• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.113-113
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• 2003

This study was conducted to investigate the effect of vitrification solution(VS) on in vitro developmental competence of immature porcine oocytes. The immature porcine oocytes were exposed to the following vitrification solution, at RT. 1) EFS sol. : 20% ethylene glycol (EG) 3 min, 40% EG + 18%(w/v) Ficoll(MV70, 000) + 0.3 M sucrose 30 sec, 2) GE sol. : 10% glycerol 5 min, 10% G + 20% EG 5 min, 25% G +25% EG 30 sec, 3) EG sol : 1.5M EG 2.5 min, 5.5 M EG + 1.0 M sucrose 30 sec. Oocytes were immediately transferred into 1.0 M, 0.5 M, 0.25 M, 0125 M, 0 M sucrose solution for 2.5 min each at RT. After removal of VS, immature oocytes were matured in vitro and subsequently all oocytes were subjected to IVF followed in vitro culture for 7 days. Maturation rates of oocytes were 38.8%, 44.5%, 22.4% and 57.6%, in EFS, EG, GE and Control, respectively, maturation rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.01). Fertilization rates of oocytes in Control was significantly higher than other treated groups(P<0.05), but no difference were observed among treated groups. Polyspermic rates were no significant difference among four groups. Cleavage rates of oocytes were 21.9%, 47.1%, 19.0% and 65.9%, in EFS, EG, GE and Control, respectively, cleavage rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.05), but blastocyst formation rates were no significant difference among four groups. These results suggested that the use of EG solution could be a great challenge for reaching a successful vitrification of immature porcine oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.24-30
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• 2011

Objective: The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen ($SN_2$). Methods: Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into $SN_2$or liquid nitrogen ($LN_2$). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed. Results: The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using $SN_2$ were increased in both the EG only and EG+DMSO groups. Conclusion: A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, $SN_2$ may improve the efficiency of vitrification by reducing cryoinjury.

• Development and Reproduction
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• v.15 no.4
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• pp.281-289
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• 2011

The objective of this study was to investigate the effective cryoprotectants for the cryopreservation of porcine mesenechymal stem cells (pMSCs). In order to understand the effectiveness of various cryoprotectants on pMSCs, we studied the most commonly used cryoprotectants; dimethyl sulfoxide (DMSO), ethylene glycol (EG), DMSO and EG. pMSCs were isolated from bone marrow matrix of piglet (2 month) and characterized by alkaline phopshatase (AP) activity, colony forming, and differentiation to adipocyte. In slow cooling cryopreservation, the pMSCs were exposed to cell medium containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% DMSO, 1.5M EG and 5% DMSO/0.75M EG, respectively, and freezed to $-1^{\circ}C$/min from $25^{\circ}C$ up to $-80^{\circ}C$ in a cryo-container. The proportion of viable cells and the growing rates in fresh pMSCs were significantly (P<0.05) higher than those of other groups, but did not differ between the cryopreserved groups. The expression of Sox-2 and Nanog gene was increased by extending culture time in cryopreserved groups. The expression of Bax gene in cryopreserved groups was similar with fresh pMSCs. Moreover, the gene expression of adipocyte-specific marker as well as chondrogenic/osteogenic factors in cryopreserved groups was similarly to fresh pMSCs. Taken together, our results suggested that all these cryoprotectants of 10% DMSO, 1.5M EG and 5% DMSO/0.75M EG could be used for cryopreservation of the pMSCs.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.797-804
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• 2006

The present study was undertaken to investigate the post-thawed survivability of bovine embryo depending on different dose of ethylene glycol and sucrose. Ovaries were collected at local slaughterhouse and the cumulus-oocyte-complexes aspirated from ovaries were in vitro matured, fertilized and cultured at 39°C in an atmosphere of 5% CO2 incubator. For conventional slow-freezing, d 7 or 8 expanded blastocysts were collected. Embryos were equilibrated in 1.5 M and 1.8 M ethylene glycol(EG) with 0.1 M and 0.3 M sucrose in Dulbecco's phosphate-buffered saline(D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25ml-straw and placed directly into cooling chamber of programmable freezer precooled to 󰠏7°C, after 2 min, the straw was seeded, maintained at 󰠏7°C for 8 min, and then cooled to 󰠏35°C at 0.3°C/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 sec and exposed to 37°C water for 20 sec. Straws were then removed from 37°C water. Rates of blastocyst survive and hatching were evaluated at 24 to 72 h post-warming. No difference of the survivability was shown between 1.5 M and 1.8 M EG (71 and 70%, respectively). Addition of 0.1 M sucrose to 1.5 M and 1.8 M ethylene glycol in the freezing solution did not differ significantly embryo survival (74 and 77%, respectively), whereas survival rates was higher(89%) in freezing solution contained 0.3M sucrose to 1.8M EG compared with 0.3M sucrose to 1.5M EG group(71%). However, there was no difference in the overall total cell number between the two groups (122±1.8 vs 131±1.4, respectively). In conclusion, the results suggest that 0.3 M sucrose in 1.8 M EG may be optimal condition for freezing and thawing methods with in vitro produced embryos and may be applied to on-farm conditions for embryo transfer.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.155-162
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• 2012

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY ($69.00%{\pm}4.18$; EG and $63.00%{\pm}9.75$; 7% G) than 8% LDL ($57.00%{\pm}5.70$; EG and $52.00%{\pm}4.47$;G). Treatment of 4% LDL + 5% EY-EG ($66.85%{\pm}5.06$) has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG ($64.65%{\pm}6.10$) among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

• KSBB Journal
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• v.13 no.5
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• pp.518-523
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• 1998

A large quantity of ethylene glycol(EG) is remained in the effluent after pretreating polyester weight-loss wastewater physicochemically in the fist stage and must be treated biologically in the second stage. Therefore, an excellent EG-utilizing bacteria strain was isolated from the natural system and the optimal culture conditions of the strain were investigated. The optimal culture conditions of temperature, pH, and nitrogen source were found to be 35$^{\circ}C$, 7.5 and ammonium chloride, respectively, when CODCr removal efficiency was more than 90%. The growth of stains and EG removal efficiency was slightly improved by adding elements such as niacin and biotin. With increasing inoculation size in a batch culture, the removal efficiency of EG was conspicuously increased. Growth rate was inhibited when the initial concentration of EG was more then 30g/L. The strain was identified as Pseudomonas sp. based on morphological and biological characteristics and named as Pseudomonas sp. EG1.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.253-258
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• 2012

This study was carried out to effects of ethylene glycol concentration, sucrose and culture day of in vitro production embryo on slow-down freezing in Hanwoo. 6, 7, 8 and 9 day embryos produced in vitro were frozen using 1.8M EG+0.1M sucrose, 1.8M EG+0.5% BSA and 1.5M EG+0.1M sucrose media. Survivability was confirmed after frozen-thawed 24 and 48h and ICM, TE cell number were counted by Hoechst 33342 and PI staining after frozen-thawed 24h. As a result, 1.8M EG+0.1M sucrose group was most significantly (p<0.05) higher compared with the other treatment groups on survivability, TE and total cell number after frozen-thawed 24h ($94.2{\pm}2.6%$, $94.67{\pm}3.4$ and $129.67{\pm}5.5$). ICM number did not found significant (p<0.05) differences between the three treatment groups. in 6, 7, 8 and 9 day of embryos using three types of freezing media, frozen-thawed, 1.8M EG+0.1M sucrose groups with embryos cultured 8 day was significantly (p<0.05) highest survivability to $98.3{\pm}1.7%$ after frozen-thawed 24h. 1.5M EG+0.1 sucrose group with embryos cultured 9 day was significantly higher survivability than group of embryos cultured 8 day after frozen-thawed 24 and 48h. In conclusion, 1.8M EG+0.1M sucrose media is considered to be effective to cryopreservation of embryos cultured 8 and 9 day.

• Research in Plant Disease
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• v.19 no.2
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• pp.108-113
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• 2013

Wilt damage on tomato plants caused by Ralstonia solanacearum has been increased as the areas of tomato cultivation increased during the warm seasons. Also, the tomato rootstocks used to prevent the disease occurrence are not effective in the highly prevailing regions. Therefore, bacterial wilt resistant eggplant rootstock EG203, collected from AVRDC, was tested for its effect to deter the Ralstonia solanacearum wilt disease in the greenhouses at Buyeo Tomato Experiment Station from 2003 to 2005, and at Gumi, Kyungpook province from 2009-2011. Planting of eggplant rootstock EG203 should be done three weeks before the planting of tomato scions so that they can have similar stem diameter (2.5-3.0 mm) and can be easily grafted. Both insertion and inarching grafting showed 93-96% success rates. In the greenhouse tests at Buyeo Tomato Experiment Station from 2003 to 2005, eggplant rootstock EG203-grafted tomatoes showed the disease occurrence of 4.3%. On the other hand, non-grafted or other commercial rootstock-grafted tomatoes showed disease occurrence of 58.0% and 25.0-36.7%, respectively. In the greenhouse tests at Gumi, Kyungpook province in 2009, the disease occurrence on the EG203-grafted and non-grafted tomatoes was 2-5% and 20-80%, respectively. In 2010, at Gumi, Kyungpook province, when the wilt disease occurred slightly, the tomatoes grafted with tomato rootstocks B-blocking and Chung-gang, and eggplant rootstock EG203 showed similar disease severities, but EG203-grafted tomatoes formed lately cluster, resulting in the reduction of yield compared to tomato-grafted tomatoes. In 2011, at Gumi, Kyungpook province, when the wilt disease occurred severely, the tomato rootstocks 'B-blocking' and Chung-gang and eggplant rootstock EG203-grafted tomatoes showed disease occurrences of 60-85% and 0-1%, respectively. Therefore, it was concluded that tomato rootstocks 'B-blocking' and 'Chung-gang' are more useful in the areas contaminated with low levels of pathogen and eggplant rootstock EG203 is more useful in the areas contaminated with high levels of pathogen.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.390-399
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• 2012

Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, $pYES2M{\alpha}$-egIV, and $pYES2M{\alpha}$-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants $IpYES2M{\alpha}$-xegIV was higher than that of transformant IpYES2-xegIV or $IpYES2M{\alpha}$-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that $MF{\alpha}$ signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of $35^{\circ}C$ to $65^{\circ}C$. The optimal reaction condition for EGIV enzyme activity was at the temperature of $55^{\circ}C$, pH of 5.0, 0.75 mM $Ba^{2+}$, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant $IpYES2M{\alpha}$-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.