• Journal of Veterinary Clinics
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• v.35 no.2
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• pp.39-45
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• 2018

We evaluated the effects of green tea extract (GTE) supplementation at different dilution steps on boar sperm freezing and in vitro fertilization. Sperm intracellular hydrogen peroxide ($H_2O_2$), motility, viability, acrosome integrity and morphology were determined. In addition, sperm IVF parameters (penetration and monospermy) and glutathione (GSH) levels of presumptive zygotes (PZs) were evaluated. Semen was diluted in lactose egg yolk (LEY) and cooled at $5^{\circ}C$ for 3 h (first dilution step) and then diluted in LEY with 9% glycerol and maintained at $5^{\circ}C$ for 30 min (second dilution step). Four experimental groups were compared: first and second dilution steps without GTE (control), first dilution step with GTE (Step 1), second dilution step with GTE (Step 2) and first and second dilution step with GTE (Step 1+2). The spermatozoa were frozen in nitrogen vapor. Higher sperm motility, viability and acrosome integrity after thawing were observed in Step 1, Step 2 and Step 1+2 groups compared with the control (P < 0.05). Lower $H_2O_2$ level was observed in Step 1+2 compared with control and Step 1 (P < 0.05). For IVF, matured oocytes were co-cultured with spermatozoa frozen according to the experimental groups. GSH levels of PZs were significantly higher in Step 2 and Step 1+2 than in control and Step 1 (P < 0.05) without a significant difference in IVF parameters. In conclusion, supplementation with GTE in both first and second dilution steps during the freezing process resulted in better boar sperm cryopreservation and might be beneficial for further embryo development.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.293-301
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• 1999

This study was to examine whether Hanwoo IVM/IVF/IVC blastocyst can be successfully survived in vitro/in vivo after vitrification and one-step dilution. For vitrification, blastocysts were serially exposed in glycerol (G) and ethylene glycol(EG) mixtures［10% (v Iv) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.］ which is diluted in 10% FBS added D-PBS. And then they were loaded in the straw, placed in cold nitrogen vapor for 3 min. and plunged into L$N_2$(－196$^{\circ}C$). One-step dilution within the straw was done in $25^{\circ}C$ and 36$^{\circ}C$ water for about 5 min. and 3 min., respectively. Recovered embryos after one-step dilution were cultured in cumulus cell mono-layered drop for 48 h or were transferred into recipient cows. When the embryo survival in vitro was assessed to re-expanded and hatched rates at 24 hand 48 h after one-step dilution, the results of vitrified group (85.4, 43.8%) was high, although these results were significantly lower than normal development (100.0, 63.3%) of control group, respectively (P＜0.001, P＜0.05). When in vitro survival of vitrified groups according to developmental stage was compared, the results of fast developed embryos (expanded blastocyst and early hatching blastocyst stage) were significantly higher than those of delayed developed one (early blastocyst stage) after one-step dilution (early hatching: 88.0, 48.0%: expanded: 81.1, 45.3%; early: 66.7, 14.3%) (P＜0.05). Also, in case of in vitro survival of vitrified groups according to embryo age (day 7, 8 and 9), when embryo age was younger, in vitro survival was significantly higher (day 7: 67.3, 34.5%; day 8: 76.9, 40.7%; day 9: 60.9, 23.9%)(P＜0.05). Finally, when in vivo development potential of vitrified and one-step diluted Hanwoo blastocysts was examined, 4 of 8 recipient (50%) cows became confirmed pregnant. These results demonstrated that our vitrification and one-step dilution technique can be applied easily and effectively on field trial without the equipment and embryological skills required for conservative dilution and transfer.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.77-83
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• 2011

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures [10%, (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.] which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into $LN_2$. One-step dilution in straw was done in $25^{\circ}C$ water for 1 min, by placing vertically in the state of plugged-end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

• Journal of Advanced Marine Engineering and Technology
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• v.33 no.5
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• pp.637-645
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• 2009

The main purpose of this study lies on the improvement of micro dilution tunnel based on the typical porous tube type chamber. The characteristics of flow and temperature fields for steady state has been obtained by numerical analysis using FLUENT. Three different geometrical variations of the porous tube; a) increase of thickness at center, b) step increase of thickness at center and downstream, c) tapered increase of thickness, have been proposed. Accordingly results are obtained and compared in terms of penetration velocity and velocity ratio to therrmophoretic velocity for improvement against particulate deposition inside the tube. The penetration velocity and velocity ratio distributions in the upstream portion and portion of impinging of dilution air are apparently shown to be improved for the case of the step and tapered change of porous tube. The tapered change of tube thickness addition are shown to be the most effective among three geometrical changes. In addition, the considerable improvement against deposition are shown that its thickness should be at least 2mm.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.201-201
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• 2004

In this study we examined whether vitrified Hanwoo (Korean cattle) IVM/IVF/IVC blastocysts can survive in vitro/in vivo by a quick one-step dilution method and these embryos result in live births. Blastocysts produced in vitro were vitrified by serial exposure to glycerol (G) and/or ethylene glycol (EG) mixtures of 10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 see. (omitted)

• Analytical Science and Technology
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• v.8 no.4
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• pp.427-434
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• 1995

Inductively coupled plasma mass spectrometry combined with the isotope dilution method is used for the determination of lithium. The isotope dilution method is based on the addition of a known amount of enriched isotope (spike) to a sample. The analyte concentration is obtained by measuring the altered isotope ratio. The spike solution is calibrated through so called reverse isotope dilution with a primary standard. The spike calibration is an important step to minimize error in the determined concentration. It has been found essential to add spike to a sample and the primary standard so that the two isotope ratios should be as dose as possible. Since lithium is neither corrosive nor toxic, lithium is used as a chemical tracer in the nuclear power plants to measure feedwater flow rate. 99.9% $^7Li$ was injected into a feedwater line of an experimental system and sample were taken downstream to be spiked with 95% $^6Li$ for the isotope dilution measurements. Effects of uncertainties in the spike enrichment and isotope ratio measurement error at various spike-to-sample ratios are presented together with the flow rate measurement results in comparison with a vortex flow meter.

• Proceedings of the Korean Institute of Navigation and Port Research Conference
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• 2004.04a
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• pp.247-253
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• 2004

As a large scale port development in coastal waters proceeds step by step and populations in the vicinity of port are getting increased, the issue on "how to dispose the treated municipal water and wastewater in harbor" brings peoples′ concern. The submarine outfall system discharges the primary or secondary treated effluent at the coastline or in deep water, or between these two. The effluent, which has a density similar to that of fresh water, rises to the sea surface forming plume or jet, together with entraining the surrounding sea water and becomes very dilute. We intended in this paper to investigate the impact on dilution of effluent and the behavior of flume under the conditions of the seasonal and spatial temperature variations, which have not been noticeable in designing effective marine outfall system. To predict and analyze the behaviour and dilution characteristics of plume not just with the effluent temperature, but also with the seasonal variation of temperature of surround water and tidal changes, CORMIX(Cornell Mixing Zone Expert System)-GI have been applied. The results should be used with caution in evaluation the mixing zone characteristics of discharged water. We hope to help for the effective operation of outfall system, probable outfall design, protection of water quality, and warm water discharges from a power plant, etc.

• Transactions of the Korean Society of Mechanical Engineers B
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• v.27 no.9
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• pp.1209-1219
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• 2003

The dynamic behaviors of the single vortex interacting with $CH_4-Air$ jet diffusion flame are investigated numerically. The numerical method is based on a predict-corrector scheme for a low Mach number flow. A two-step global reaction mechanism is adopted as a combustion model. Studies are conducted in fixed initial velocities for the three cases according as where $CO_2$ is added; (1) without dilution, (2) dilution in fuel stream and (3) dilution in oxidizer stream. A single vortex is generated by an axisymmetric jet, which is made by an impulse of a cold fuel when a flame is developed entirely in a computational domain. The simulation shows that $CO_2$ dilution in fuel stream results in somewhat larger vortex radius, and greater amount of entrainment of surrounding fluid than in other cases. Thus, the dilution of $CO_2$ in fuel stream enhances the mixing in single vortex and increases the stretching of the flame surface. The budgets of the vorticity transport equation are examined to reveal the mechanism of vortex formation when $CO_2$ is added. It is found that, in the case of $CO_2$ dilution in fuel stream, the vortex destruction due to volumetric expansion and the vortex production due to baroclinic torque are more dominant than in other cases.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.73-73
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• 2001

This study was to examine whether the vitrified, one-step diluted and direct transferred Hanwoo IVM/IVF/IVC blastocysts can be successfully survived in vivo and they were succeeded into the live birth. For vitrification, blastocysts were serially exposed in glycerol (G) or/and ethylene glycol (EG) mixtures [10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 sect] which is diluted in 10% FBS added D-PBS. Thawing of straw was carried out in air for 10 sec and then in water bath of $25^{\circ}C$ for 20 sec. One-step dilution within the straw was done in water bath of $25^{\circ}C$ for 1 min. Vitrified and one-step diluted embryos were directly transferred into 36 (natural or hormone induced synchronized) recipient cows in 6 areas of Kyungsang Buk-Do. Pregnancies were confirmed at first when recipient cows did not return to the subsequent estrus cycle, and later by manual palpation per rectum on day 45, 90 and then living calves were derived into parturition. Overall pregnancy was 33.3%(12/36), However, higher pregnancy was obtained when the recipients exhibited estrus one day earlier than the age of transferred embryos (53.3 vs 25.0-27.3%), irrespective of synchronization methods. Also, parous recipients became pregnant higher than nulliparous heifers, And, there were not different in pregnancy rates by the aspect of corpus luteum (CL) quality of recipients (good, 29.4; fair, 37.5; poor, 33.3%). One hundred eight of frozen-thawed Hanwoo blastocysts were directly transferred into 36 recipient cows. In 12 of pregnant cows, 3 cows were aborted and 9 cows were calved [single, 66.7% (6/9): twin, 33.3% (3/9)]. Total embryo implantation rate was 11.1% (12/108). However, 9 Hanwoo calves were lived. Therefore, these results demonstrate that direct transfer technique of vitrified and one-step diluted bovine blastocysts can be applied easily and effectively with the higher pregnancy rate on field trial without the equipment and embryological skills.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.124-129
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• 2016

Endotoxin, also known as lipopolysaccharide (LPS) produced by the cell wall of gram negative bacteria can be present in any liquid or on any biomaterial. Endotoxin in blood can cause fever and inflammation. In this study, we compared bacterial endotoxin using Escherichia coli O157:H7, Klebsiella oxytoca, Salmonella Typhi, Shigella sonnei and Morganella morganii. Bacteria were cultured for use in the experiment, and diluted to $1.5{\times}10^8CFU/mL$. A check marked sensitivity confirmatory test of the Limulus amebocyte lysate (LAL) reagent was performed to examine the validity. The end point reaction to each bacteria sample was confirmed with 10 fold dilution and then the final reaction end point was confirmed by 2 fold dilution between the dilution step and the upper dilution step. According to the results, in detection of endotoxins in more than 0.015 EU/mL, E. coli O157 was 75~37.5 CFU/mL, K. oxytoca 37.5~18.75 CFU/mL, M. morganii and S. Typhi 3.75~1.875 CFU/mL, and S. sonnei 7.5~3.75 CFU/mL. The resulting value was finally ensured by a confirmation test for the inhibitory factor. Based on this study, conduct of further research on bacterial endotoxin is encouraged.