Effects of Fertilization Time and Culture Medium of Pig Oocytes Matured In Vitro by Liquid Boar Sperm Stored at 4℃

Park, C.S.,Yi, Y.J.,Kim, M.Y.,Chang, Y.J.,Lee, S.H.,Jin, D.I 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23℃) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800×g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0×10^(9) sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4℃. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10μg/ml insulin, 2μg/ml vitamin B_(12), 25 mM HEPES, 10μg/ml bovine apotransferrin, 150μM cysteamine, 10IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75μg/ml sodium penicillin G, 50μg/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5℃, 5% CO₂in air. Oocytes were inseminated with liquid boar sperm stored at 4℃ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 μl mTBM fertilization media with 1.0×10^(6) sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 μl NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4℃ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 μl TBM fertilization medium with 1×10^(6) sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

In vitro Fertilization and Development of Pig Oocytes Inseminated with Boar Sperm by Different Sperm Washing Media after Thawing of the Frozen Straws

Yi, Y.J.,Ko, H.J.,Lee, S.H.,Yang, C.B.,Son, D.S.,Kim, H.K.,Park, C.S. 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

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㎕ mTCM 199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to 24℃ over a 2 h period. Semen was centrifuged at 800g for 10 min and the seminal Plasma discarded. Sperm were resuspended in a lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent to contain 1×10^9 sperm/ml and cooled to 5℃ 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. Oocγtes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with 2×10^7 sperm concentration. Oocytes were coincubated for 6 h in 500㎕ mTBM fertilization. At 6 h after IVF, oocytes were transferred Into 500㎕ 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-PYA medium before in vitro fertilization of oocytes in mTBM medium.

In vitro Fertilization and Development of Pig Oocytes Inseminated with Boar Sperm by Different Sperm Washing Media after Thawing of the Frozen Straws

Yi, Y.J.,Ko, H.J.,Lee, S.H.,Yang, C.B.,Son, D.S.,Kim, H.K.,Park, C.S. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.2

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.

Systemic delivery and preclinical evaluation of Au nanoparticle containing β-lapachone for radiosensitization

Jeong, S.Y.,Park, S.J.,Yoon, S.M.,Jung, J.,Woo, H.N.,Yi, S.L.,Song, S.Y.,Park, H.J.,Kim, C.,Lee, J.S.,Lee, J.S.,Choi, E.K. Elsevier Science Publishers 2009 Journal of controlled release Vol.139 No.3

Effective delivery of radiosensitizer to target tumor cells, causing preferentially increased tumor cytotoxicity, while simultaneously minimizing damage to healthy cells around the tumor, is an ideal strategy for the improvement of radiotherapeutic efficacy against human cancer. We aimed to enhance radiotherapeutic efficacy by using biocompatible gold nanoparticles (AuNP) as a vehicle for systemic delivery of sz-lapachone (lap). Lap is a novel anticancer agent displaying potent cytotoxicity against cancer cells expressing NAD(P)H:quinone oxidoreductase-1 enzyme (NQO1). Although lap is expected to be a very promising radiosensitizer, its poor solubility and non-specific distribution obstruct preclinical evaluation and clinical application. In this study, the property of AuNPs carrying lap (AuNPs/lap) for active-targeting tumor cells and improving in vivo radiotherapeutic efficacy was evaluated. Murine monoclonal anti-EGFR antibody was conjugated to the AuNPs/lap as a ligand for active targeting. The active tumor-targeting property of AuNPs/lap conjugating anti-EGFR antibody was validated in vitro experiments using cell lines expressing EGFR at different levels. In mice bearing xenograft human tumors, the intravenous injection of AuNPs/lap exhibited highly enhanced radiotherapeutic efficacy. AuNPs/lap offers a new modality for improvement of radiotherapeutic efficacy and feasibility of further clinical application for human cancer treatment.

Formation of the internal transport barrier in KSTAR

Chung, J.,Kim, H.S.,Jeon, Y.M.,Kim, J.,Choi, M.J.,Ko, J.,Lee, K.D.,Lee, H.H.,Yi, S.,Kwon, J.M.,Hahn, S.-H.,Ko, W.H.,Lee, J.H.,Yoon, S.W. International Atomic Energy Agency 2018 Nuclear fusion Vol.58 No.1

<P>One of key objectives of tokamak experiments is the exploration of enhanced confinement regimes, and the access of the internal transport barrier (ITB) formation is dealt with an important physics issue in the most of major tokamaks. Also, the advanced tokamak scenario with ITB is expected to lead to a continuous reactor with high fusion power density. From that point of view, the formation of the ITB in KSTAR which is designed for long pulse operation capability is very important although its heating and current drive systems are not fully equipped yet. We have therefore assumed that an early injection of the full NBI power (∼5.5 MW) during the current ramp-up would give a chance to form an internal barrier if the plasma could stay in the L-mode. To avoid the H-mode transition, we have produced inboard limited plasmas with detaching from the both upper and lower divertors. Using this approach, an ITB formation during L-mode has been observed which shows improved core confinement. Ion and electron temperature profiles show the barrier clearly in the temperature, and it was sustained for about 7 s in the dedicated experiment. This is the first stationary ITB observed in a full superconducting tokamak. This operation scenario with the ITB could be an alternative way to achieve a high performance regime in KSTAR, and the length of the ITB discharge could be extended even longer. In this paper, we present the formation of the ITB using measured and simulated characteristic profiles.</P>

유한요소법을 이용한 구보용 2024-T3 재료의 동적 물성치 평가

이재혁,김형삼,이동식,박세만 명지대학교 대학원 1999 대학원논문집 Vol.3 No.-

There are two types of beam theories: the first one is the traditional Euler-Bernoulli theory while the second one is Timoshenko beam theory that considers additional effects of shear deformations and rotary inertia. In this investigation, the both theories are utilized to evaluate elastic constants. leading to direct comparisons of the two theories. Also, for a numerical analysis the commercial FEM program. ANSYS 5.3 is employed to conduct modelling of test specimens for a modal analysis, enhancing the understanding of the nature of the FEM analysis and allowing comparisons with experimental results.

Comparison of Semen Characteristics, Frozen-Thawed Sperm Viability, Testosterone Concentration and Embryo Development between Yorkshire Boar A and B

Yi, Y.J.,Lee, S.H.,Park, C.S. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.5

This study was carried out to compare the semen characteristics, frozen-thawed sperm viability and testosterone concentration and in vitro fertilization (IVF) and development of in vitro matured pig oocytes between two Yorkshire boars. Semen and blood samples were collected once per week from October to November 2002 from two adult Yorkshire boars at 18 months of age with 170 kg body weight. Sperm were deep frozen in 5 ml maxi-straws with lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent and stored in liquid nitrogen. Blood samples were obtained at 10 a.m. by inserting a 21 gauge, hypodermic needle attached to 10 ml syringe into surface veins in the ear. The concentration of testosterone was determined by Competitive Enzyme Immunoassay. Ovaries were collected from prepubertal gilts at a local slaughter house. Cumulus oocyte complexes were aspirated from antral follicles (3 to 6 mm in diameter). The medium used for oocyte maturation was modified TCM 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at $38.5^{\circ}C$, 5% $CO_2$ in air. For IVF, one frozen 5 ml straw was thawed at $52^{\circ}C$in 40 sec and was diluted with 20 ml Beltsville thawing solution at room temperature. Sperm were washed 2 times in mTLP-PVA and inseminated without preincubation after thawing. Oocytes were inseminated with $2{\times}10^7$/ml sperm concentration. Oocytes were coincubated for 6 h in 500 ${\mu}$l mTBM fertilization medium. At 6 h after IVF, oocytes were transferred into 500 ${\mu}$l NCSU-23 culture medium for further culture of 48 and 144 h. There were no significant differences in the semen volume, motility, normal acrosome morphology and sperm concentration of raw semen between A and B of Yorkshire boar. However, motility and normal acrosome of boar A were higher than those of boar B at 0.5, 2, 3, 4, 5 and 6 h incubations of frozen-thawed sperm. Testosterone concentration (3.75 ng/ml) of boar A was higher than that (2.34 ng/ml) of boar B. The rate of blastocyst formation (15.1%) of boar A was higher than that (10.4%) of boar B. In conclusion, serum testosterone concentration of boar showed very important role for the frozen-thawed sperm viability and the blastocyst formation of pig oocytes matured in vitro.

Corrosion Behaviors of Coatings Fabricated Using Bulk Metallic Glass Powders with the Composition of Fe68.5C7.1Si3.3B5.5P8.7Cr2.3Mo2.5Al2.0

S. L. Wang,H. X. Li,S. Y. Hwang,S. D. Choi,S. Yi 대한금속·재료학회 2012 METALS AND MATERIALS International Vol.18 No.4

Two different types of coatings were prepared, by a high velocity oxy-fuel spraying method and a laser spraying method, respectively, using bulk metallic glass powders with the nominal composition of Fe68.5C7.1Si3.3B5.5P8.7Cr2.3Mo2.5Al2.0. The corrosion behaviors of the two coatings in 1M HCl, H2SO4, NaCl and NaOH solutions were investigated based upon the microstructural differences originating from the different coating methods. The amorphous coating layer formed by the high velocity oxy-fuel spraying method exhibited higher, excellent corrosion resistance in the 1M HCl solution. The coating layer formed by the laser spraying method exhibited a high pitting tendency attributed to the dendritic microstructure with various borides and carbides. Due to a great number of pores, the HVOF coating exhibits slightly lower corrosion resistance than the LS coating in alkaline solution.

Effects of a bio-assay guided fraction from Polygonum cuspidatum root on the viability, acid production and glucosyltranferase of mutans streptococci

Ban, S.H.,Kwon, Y.R.,Pandit, S.,Lee, Y.S.,Yi, H.K.,Jeon, J.G. Inverni Della Beffa S.p.A ; Elsevier Science 2010 Fitoterapia Vol.81 No.1

Polygonum cuspidatum root has been traditionally used for the treatment of dental diseases in Korea. The purpose of this study was to evaluate effects of P. cuspidatum root on the development of dental caries, especially its effects against bacterial viability and caries-inducing factors of Strptococcus mutans and Strptococcus sobrinus. Among methanol extract of P. cuspidatum root and its fraction tested, ethyl acetate fraction, composed of polydatin, resveratrol, anthraglycoside B, and emodin, showed inhibitory effects on glycolytic acid production and glucosyltransferase activity of S. mutans and S. sobrinus in addition to antibacterial activities.

Broadly neutralizing anti-HBV antibody binds to non-epitope regions of preS1

Chi, S.W.,Kim, J.,Yi, G.S.,Hong, H.J.,Ryu, S.E. North-Holland Pub ; Elsevier Science Ltd 2009 FEBS letters Vol.583 No.18

Broadly neutralizing anti-hepatitis B virus (HBV) antibody HzKR127 undergoes a fairly large conformational change of CDR H3 loop upon binding to HBV preS1 epitope peptide. In this study, we identified low-affinity antibody-binding sites in the largely unstructured preS1 region by nuclear magnetic resonance and biochemical studies, indicating that the antibody binds to the preS1 region outside the major immune epitope with low affinity. Surface plasma resonance experiments showed that the full-length preS1 has approximately three fold higher affinity for HzKR127 Fab than the preS1 epitope peptide, suggesting that the presence of low-affinity sites in the preS1 region increases the antibody-binding affinity. Therefore, the low-affinity binding of the antibody to non-epitope regions of preS1 may contribute to effective neutralization.