• Journal of Embryo Transfer
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• v.17 no.2
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• pp.129-136
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• 2002

The purpose of this study was to investigate the comparison of viability of bovine blastocysts following glass micropipette (GMP) vitrification and the possibility of production of Korean Native Cow ("Hanwoo,"Bos taurus coreanae) following embryo transfer into Mongolia cows (Bos taurus mongolian). The embryos of Korean Native Cow were produced by IVMFC or superovulation methods in Korea, cryopreserved by GMP vitrification, and subsequently trans-ported to Mongolia. The recipient cows were synchronized using a CIDR plus and prostaglandin $F_2\alpha$($PGF_2\alpha$) treatment. To produce in vivo embryos, seven cows were superovulated using FSH and PGF$_2$／sub $\alpha$／ treatment. A total of 64 blastocysts ( $9.1\pm2.94$ per cow) were collected. In vitro embryos were produced using a defined culture system which cleaved in 80.1％ ova (174／217), and developed to blastocyst stage embryos of 40.8％ (71／174). The post-thaw survival rate of in vivo blastocysts (93.7％; 45／48) was significantly higher than that of in vitro blastocysts (82.5％; 52／63, P＜0.05). Embryo transfer was carried out using 8 Mongolian recipient cows and 2 post-thaw blastocysts per recipient. Five of 8 recipients were found pregnant at Day 60 but one abortion occurred by Day 240. Two of offspring were produced from the Mongolian cows at 275 days after embryo transfer. These results indicated that a GMP vitrification method could be used as a cryopreservation technique for in vivo or in vitro bovine blastocysts and produced effectively a Korean Native Cow following embryo transfer into a Mongolian recipient cow.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.295-302
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• 2000

The objective of this study was to compare different superovulation treatments using PMSG or PG600$^{ }$ and to determine the optimal time of oocyte recovery after hCG administration. A total of 90 prepubertal Yorkshire x Landrace gilts crossed with Duroc, 6~7 months old and 100~120 kg of body weight, were used. PMSG (1,500 IU/head) or 5~7.5 ml of PG600$^{ }$(400 IU of PMSG and 200 IU of hCG) were administrated subcutaneously, and then 1,000 IU of hCG were administered intramuscularly at 72 hours after PMSG or PG600$^{ }$ injection. At carious time of 44, 46, 48 and 50 hours after hCG injection, superovulated gilts were slaughtered in a local abattoir. Ovaries together with oviducts were excised from the body immediately after slaughtered and transported to laboratory in 39$^{\circ}C$ saline. Ovaries were examined fur the number of corpus hemorrhagicum and unovulated follicles present in the surface of ovary. The unovulated follicles were categorized into small (1~3 mm in diameter) and large (4~8 mm) groups according to their diameter. Oocytes were recovered by flushing both oviducts with micropipette tip (1~100 $\mu$l) attached to a 10-ml disposable syringe. The number of CH on ovary and recovered oocytes at 46, 48 and 50 hr after hCG injection in PG600$^{ }$ treated groups were significantly higher than the other group. Group of phCG 50 hr among PMSG treated groups had a greater number of CH and recovered oocytes(P<0.05). The number of CH on ovary and recovered oocytes at 50 hr after hCG injection in 1$\frac{1}{2}$ vial(7.5 ml) of PG600$^{ }$ treated groups was significantly higher than 1 vial(5 ml) of PG600$^{ }$ treated group(P<0.05). In conclusions, considering a number of corpus hemorrhagicum and recovered oocytes after superovulation in gilts, effective time of oocyte recovery by treatment with PMSG and hCG was post-hCG 50 hr and with PG600$^{ }$ plus hCG was post-hCG 46, 48 and 50 hr. Also, admini-stration of 1$\frac{1}{2}$ vial(7.5 ml) of PG600$^{ }$ treated group had a great number of CH and recovered oocytes.covered oocytes.

• Applied Chemistry for Engineering
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• v.24 no.5
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• pp.443-455
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• 2013

Electrochemical studies on charge transfer reactions across the interface between two immiscible electrolyte solutions (ITIES) have greatly attracted researcher's attentions due to their wide applicability in research fields such as ion sensing and biosensing, modeling of biomembranes, pharmacokinetics, phase-transfer catalysis, fuel generation and solar energy conversion. In particular, there have been extensive efforts made on developing sensing platforms for ionic species and biomolecules via gelifying one of the liquid phases to improve mechanical stability in addition to creating microscale interfaces to reduce ohmic loss. In this review, we will mainly discuss on the basic principles, applications and future aspects of various sensing platforms utilizing ion transfer reactions across the ITIES. The ITIES is classified into four types : (i) a conventional liquid/liquid interface, (ii) a micropipette supported liquid/liquid interface, (iii) a single microhole or an array of microholes supported liquid/ liquid interface on a thin polymer film, and (iv) a microhole array liquid/liquid interface on a silicon membrane. Research efforts on developing ion selective sensors for water pollutants as well as biomolecule sensors will be highlighted based on the use of direct and assisted ion transfer reactions across these different ITIES configurations.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.40 no.1
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• pp.3-10
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• 2014

Objectives: Although nerve growth factor (NGF) could promote the functional regeneration of an injured peripheral nerve, it is very difficult for NGF to sustain the therapeutic dose in the defect due to its short half-life. In this study, we loaded the NGF-bound heparin-conjugated fibrin (HCF) gel in the NGF-delivering implants and analyzed the time-dependent release of NGF and its bioactivity to evaluate the clinical effectiveness. Materials and Methods: NGF solution was made of 1.0 mg of NGF and 1.0 mL of phosphate buffered saline (PBS). Experimental group A consisted of three implants, in which $0.25{\mu}L$ of NGF solution, $0.75{\mu}L$ of HCF, $1.0{\mu}L$ of fibrinogen and $2.0{\mu}L$ of thrombin was injected via apex hole with micropipette and gelated, were put into the centrifuge tube. Three implants of experimental group B were prepared with the mixture of $0.5{\mu}L$ of NGF solution, $0.5{\mu}L$ HCF, $1.0{\mu}L$ of fibrinogen and $2.0{\mu}L$ of thrombin. These six centrifuge tubes were filled with 1.0 mL of PBS and stirred in the water-filled beaker at 50 rpm. At 1, 3, 5, 7, 10, and 14 days, 1.0 mL of solution in each tubes was collected and preserved at $-20^{\circ}C$ with adding same amount of fresh PBS. Enzyme-linked immunosorbent assay (ELISA) was done to determine in vitro release profile of NGF and its bioactivity was evaluated with neural differentiation of pheochromocytoma (PC12) cells. Results: The average concentration of released NGF in the group A and B increased for the first 5 days and then gradually decreased. Almost all of NGF was released during 10 days. Released NGF from two groups could promote neural differentiation and neurite outgrowth of PC12 cells and these bioactivity was maintained over 14 days. Conclusion: Controlled release system using NGF-HCF gel via NGF-delivering implant could be an another vehicle of delivering NGF to promote the nerve regeneration of dental implant related nerve damage.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.3
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• pp.155-168
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• 2004

Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of $3{\sim}4$ weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at $37^{circ}C$, 5% $CO_2$ and 21% $O_2$ (95% air) or 5% $CO_2$, 5% $O_2$ and 90% $N_2$. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of $0.0001{\mu}M$, $0.01{\mu}M$ or $1.0{\mu}M$. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of $0.01{\mu}M$ melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with $0.0001{\mu}M$ or $1.0{\mu}M$ melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of $0.01{\mu}M$ melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.122-128
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• 2018

Objective: To determine whether fragment removal on in vitro fertilization (IVF) day 2 improved the subsequent development and pregnancy outcomes of fragmented embryos compared to similar-grade embryos without fragment removal. Methods: This study was a retrospective analysis involving 191 IVF cycles in which all embryos had over 10% fragmentation (grade 3 or 4) on day 2 of the IVF-embryo transfer cycle from March 2015 to December 2017. IVF cycles were divided into the fragment removal group (n = 87) and the no fragment removal group (n = 104) as a control cohort. Before fragment removal, embryos with fragmentation on day 2 were incubated in $Ca^{2+}$- and $Mg^{2+}$-free biopsy medium under paraffin oil for 30 minutes. Microsurgical fragment removal was performed with later-assisted hatching and a handmade suction micropipette that had an outer diameter of $30{\mu}m$. Results: There were no significant differences in the characteristics of the patients between the control and the fragment removal groups. After fragment removal and subsequent in vitro culture for 24 hours, the number of blastomeres ($7.1{\pm}1.7$ vs. $6.9{\pm}1.6$) was comparable between the transferred embryos in the two groups, but the morphological grade of the embryos in the fragment removal group ($1.9{\pm}0.7$) was significantly higher than that of the control group ($3.1{\pm}0.5$, p< 0.01). The clinical pregnancy (43.7%) and implantation rates (25.8%) in the fragment removal group were significantly higher than those in the control group (28.8% and 14.0%, respectively; p< 0.05). Conclusion: Early fragment removal on day 2 significantly improved the subsequent development and pregnancy outcomes of fragmented embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.235-243
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• 2002

This study was designed to examine the ability of the bovine (MII) oocytes cytoplasm to support several mitotic cell cycles under the direction of differentiated somatic cell nuclei of bovine, porcine, mouse and human. Bovine GV oocytes were matured in TCM-199 supplemented with 10% FBS. At 20h after IVM, recipient oocytes were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst and their 1st polar body (PB) and MII plate were removed by enucleation micropipette under UV filter. Ear skin samples were obtained by biopsy from an adult bovine, porcine, mouse and human and cultured in 10% FBS added DMEM. Individual fibroblast was anlaysed chromosome number to confirm the specificity of species. Nuclear transferred (NT) units were produced by electrofusion of enucleated bovine oocytes with individual fibroblast. The reconstructed embryos were activated in 5 $\mu$M ionomycin for 5 min followed by 1.9 mM 6-dimethylaminopurine (DMAP) in CR1aa for 3 h. And cleaved NT embryos were cultured in CR1aa medium containing 10% FBS on monolayer of bovine cumulus cell for 8 days. Also NT embryo of 4~8 cell stage was analysed chromosome number to confirm the origin of nuclear transferred somatic cell. The rates of fusion between bovine recipient oocytes and bovine, porcine, mouse and human somatic cells were 70.2%, 70.2%, 72.4% and 63.0%, respectively. Also, their cleavage rates were 60.6%, 63.7%, 54.1% and 62.7%, respectively, there were no differences among them. in vitro development rates into morula and blastocyst were 17.5% and 4.3% in NT embryos from bovine and human fibroblasts, respectively. But NT embryos from porcine and mouse fibroblasts were blocked at 16~32-cell stage. The chromosome number in NT embryos from individual fibroblast was the same as chromosome number of individual species. These results show that bovine MII oocytes cytoplasm has the ability to support several mitotic cell cycles directed by newly introduced nuclear DNA.

• 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.

• Korean Journal Plant Pathology
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• v.3 no.3
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• pp.180-186
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• 1987

Cell suspensions of two isolates of Pseudomonas syringae. PS8401 from sweet persimon and PS8402 from tea plant, were active in ice nucleation at -2.5 and $-3.8^{\circ}C$, respectively. Ice nucleation at those temperature was, using micropipette method, detected in suspensions ($10^8$ olony forming unit/ml of distilled water) of cells that had been grown on nutrient agar supplemented with $2.5\%$ glycerol. Using the same method, on the other hand, the freezing temperature of distilled water only was approx. $-21.8^{\circ}C$, and those of various plant saps including corn were lower than $-11.6^{\circ}C$. Corn seedlings sprayed with cell suspensions $(10^8\;cfu/ml)$ of nutrient broth) of PS8401 began to be damaged at $-2^{\circ}C$ and were almost completely damaged at $-4^{\circ}C$, whereas seedlings sprayed with nutrient broth only were not injured until the temperature down to $-9^{\circ}C$. Amounts of frost damage measured 48 hr after application of PS8401 suspensions increased as applied bacterial cell densities were increased. Ice-nucleation activity of the cell suspensions in vitro increased with increasing the number of cells in suspension. The activity also affected by growth-medium composition or growth-temperature. Ice nucleation thus occured at -4.0, -4.4 and $-7.2^{\circ}C$ in suspensions $(10^2\;cfu/ml)$ of PS8401 that had been grown at$25\%$ nutrient agar with $2.5\%$ glycerol, nutrient agar with $2.5\%$ glucose and nutrient agar only, respectively, and occured at -4.0 and $-7.6^{\circ}C$ in suspensions $(10^2\;cfu/ml)$ of PS8401 hat had been grown on nutrient agar with $2.5\%$ glycerol at $15\~25^{\circ}C$ and $30^{\circ}C$, respectively.

• The Korean Journal of Nuclear Medicine Technology
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• v.18 no.1
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• pp.76-81
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• 2014

Purpose: Diversity and the lachrymal duct deformities and the passage inside the nasal cavity except for anterior image such as epiphora happens during the test were able to express more precisely during the dacryoscintigraphy. Also, we thought about the necessity of a method to classify the passage into the naso-lachrymal duct from epiphora. Therefore, we are to find the validity of the method to obtain both oblique views except for anterior views. Materials and Methods: The targets of this research are 78 patients with epiphora due to the blockage at the lachrymal duct from January 2013 to August 2013. Average age was $56.96{\pm}13.36$. By using a micropipette, we dropped 1-2 drops of $^{99m}TcO4^-$ of 3.7 MBq (0.1 mCi) with $10{\mu}L$ of each drop into the inferior conjunctival fold, then we performed dynamic check for 20 minutes with 20 frames of each minute. In case of we checked the passage from both eyes to nasal cavity immediately after the dynamic check, we obtained oblique view immediately. If we didn't see the passage in either side of the orbit, we obtained oblique views of the orbit after checking the frontal film in 40 minutes. The instrument we used was Pin-hole Collimator with Gamma Camera(Siemens Orbiter, Hoffman Estates, IL, USA). Results: Among the 78 patients with dacryoscintigraphy, 35 patients were confirmed with passage into the nasal cavity from the anterior view. Among those 35 patients, 15 patients were confirmed with passage into the nasal cavity on both eyes, and it was able to observe better passage patterns through oblique view with a result of 8 on both eyes, 2 on left eye, and 1 on right eye. 20 patients had passage in left eye or right eye, among those patients 10 patients showed clear passage compared to the anterior view. 13 patients had possible passage, and 30 patients had no proof of motion of the tracer. To sum up, 21 patients (60%) among 35 patients showed clear pattern of passage with additional oblique views compared to anterior view. People responded obtaining oblique views though 5 points scale about the utility of passage identification helps make diagnoses the passage, passage delayed, and blockage of naso-lachrymal duct by showing the well-seen portions from anterior view. Also, when classifying passage to naso-lachrymal duct and flow to the skin, oblique views has higher chance of classification in case of epiphora (anterior:$4.14{\pm}0.3$, oblique:$4.55{\pm}0.4$). Conclusion: It is considered that if you obtain oblique views of the bilateral orbits in addition to anterior view during the dacryoscintigraphy, the ability of diagnose for reading will become higher because you will be able to see the areas that you could not observe from the anterior view so that you can see if it emitted after the naso-lachrymal duct and the flow of epiphora on the skin.