• Journal of Embryo Transfer
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• v.25 no.4
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

• Journal of the Society of Naval Architects of Korea
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• v.48 no.5
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• pp.451-456
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• 2011

To ensure structural integrity of membrane type LNG tank, the rational assessment of the sloshing impact responses of tank structures should be preceded. The sloshing impact pressures acting on the insulation system of LNG tank are typical irregular loads and the resulting structural responses show very complex behaviors accompanied with fluid structure interaction. So it is not easy to estimate them accurately and immense time consuming calculation process would be necessary. In this research, a simplified method to analyse the dynamic structural responses of LNG tank insulation system under pressure time histories obtained by sloshing model test or numerical analysis was studied. The proposed technique based on the concept of linear combination of the triangular response functions which are the transient responses of structures under the unit triangular impact pressure acting on structures. The validity of suggested method was verified through the example calculations and applied to the dynamic structural response analysis of a real Mark III membrane type insulation system using the sloshing impact pressure time histories obtained by model test.

• Journal of Veterinary Clinics
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• v.34 no.2
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• pp.156-160
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• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1797-1804
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• 2007

Lycopene, an acyclic carotenoid found in tomatoes (Lycopersicon esculentum) and a number off fruits, has shown various biological properties, but its antifungal effects remain poorly understood. The current study investigated the antifungal activity of lycopene and its mode of action. Lycopene showed potent antifungal effects toward pathogenic fungi, tested in an energy-independent manner, with low hemolytic effects against human erythrocytes. To confirm the antifungal effects of lycopene, its effects on the dimorphism of Candida albicans induced by fetal bovine serum (FBS), which plays a key role in the pathogenesis of a host invasion, were investigated. The results showed that lycopene exerted potent antifungal activity on the serum-induced mycelia of C. albicans. To understand the antifungal mode of action of lycopene, the action of lycopene against fungal cell membranes was examined by FACScan analysis and glucose and trehalose-release test. The results indicated that lycopene caused significant membrane damage and inhibited the normal budding process, resulting from the destruction of membrane integrity. The present study indicates that lycopene has considerable antifungal activity, deserving further investigation for clinical applications.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.213-217
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• 2013

The objective of this study was to investigate the effect of bacterial contamination on elapsed time after preservation on boar semen. Known numbers of Escherichia coli (E. coli) were inoculated to freshly ejaculated semen and sperm parameters such as viability, motility, agglutination, acrosome integrity and hypo-osmotic swelling test were performed during 7 days of liquid preservation. Semen samples were prepared using antibiotic free BTS extender and 4 different levels of E. coli were treated to semen with following concentrations; 3,000, 5,000, 7,000, 10,000 CFU/ml of sperms. Semen samples were preserved at $17^{\circ}C$ for 7 days in semen storage until analyzed. Aliquots were subjected to measure the sperm viability, motility and agglutination using computer assisted sperm analysis (CASA) system, acrosome integrity was performed using chlortetracycline (CTC) staining method and hypo-osmotic swelling test was performed using hypotonic solution from day 1 (day of semen collection) to 7. Detrimental effects on sperm motility and viability were observed 3 days after preservation at the level of 5,000 CFU/ml (p<0.05). Percentage of sperm abnormality was higher (p<0.05) in over 5,000 CFU/ml groups. Sperm agglutination rate was also significantly higher (p<0.05) in groups of 5,000 and 7,000 CFU/ml. The rate of acrosome reacted sperm was higher as preservation time goes in all the samples but the pattern was clearly higher among E. coli contaminated groups (p<0.05). The sperm membrane integrity in terms of hypo-osmotic test, E. coli affects little compared to other sperm parameters. The deleterious effects observed due to the bacterial contamination in semen suggest that importance of hygiene protocol to minimize the bacterial contamination during semen collection and processing.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.77-83
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• 2006

본 연구는 개 동결 정액 융해 시 straw 크기 및 융해 속도가 융해 정자의 질(quality)에 미치는 영향을 조사하고 최적의 융해 조건을 조사하는데 그 목적이 있다. 정상적인 번식능을 가진 비글 수컷 5마리에서 정액을 채취하여 원심 분리하여 정장을 버리고 남은 정자에 동결보호제인 glycerol이 첨가된 tris-glucose-egg yolk extender를 첨가하여 동결하고 액체질소에 보관한 후 융해하였다. 동결 융해 조건에 따른 효과를 알아보기 위해 straw는 0.25 ml과 0.5 ml크기를 사용하였고 융해 조건은 $75^{\circ}C$에 10초, $55^{\circ}C$에 12초 및 $37^{\circ}C$에서 120초로 하여 융해 후 정자의 활력도(vigor), 운동성(motility), Hypo-osmotic test(HOS test)를 이용한 생존성(viability) 및 $SperMac^{\circledR}$ 염색을 하여 정자의 membrane integrity를 비교 조사하였다. 조사 결과 0.5 ml 크기의 straw를 사용한 경우 $37^{\circ}C$ 융해가 $55^{\circ}C,\;75^{\circ}C$ 융해보다, 0.25 ml 크기의 straw를 사용한 경우에는 $37^{\circ}C,\;55^{\circ}C$ 융해가 $75^{\circ}C$ 융해보다 유의적으로 높은 활력 지수 및 생존성을 보였다(P<0.05). Straw크기에 따라 비교하였을 경우 0.5 ml 군에서 유의적으로 높은 활력도, 생존성 및 membrane integrity를 보였다(P<0.05). 결론적으로 개 정액이 동결 및 융해 시 0.5ml straw를 이용하여 동결한 후 $37^{\circ}C$에서 120초 동안 융해하는 것이 최적의 조건임이 사료된다.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.23 no.4
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• pp.388-396
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• 2020

Purpose: Impaired intestinal mucosal integrity may affect the gastrointestinal function, especially in relation to nutrition, absorption, and barrier function. The purpose of this study was to measure the prevalence of impaired intestinal mucosal integrity in presumed healthy children aged 1-3 years and assess the effects of zinc, glutamine, fiber, and prebiotic supplementation in them. Methods: A cross-sectional study was conducted in 200 children aged 1-3 years in Pasar Minggu, South Jakarta, Indonesia. A randomized double-blind parallel group method clinical trial was then performed to assess the effects of zinc, glutamine, fiber, and prebiotic supplementation. Results: Elevated calprotectin was found in 91/200 subjects (45.5%) at the onset of the study. After 10 months, 144 subjects completed the study: 72 subjects received the trial formula, whereas the other 72 received the standard formula. A transitory decrease in fecal calprotectin (FC) was observed after 6 months in the subgroup with normal FC levels, who were fed the test formula (p=0.012). Conclusion: The prevalence of impaired intestinal mucosal integrity in this group of Indonesian children aged 1-3 years was high. Supplementation with zinc, glutamine, fiber, and prebiotics during 6 months reduced FC only in those who had low levels at baseline but not in those with impaired integrity.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.265-271
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• 2011

This study was undertaken to find out the effect of methyl-beta-cyclodextrin (MBCD) on cold shock and membrane cholesterol quantity of sperm during the freezing process in miniature pigs. For this study, semen ejaculated from PWG M-type miniature pig was diluted that freezing solution (with egg yolk group) and m-Modena B (without egg yolk group) treated with 0, 1, 5, 10 and 20 mM MBCD before freezing process. The diluted semen was monitored sperm ability at room temperature, after cooled until $5^{\circ}C$ and after forzen-thawed for cold shock test of spermatozoa. Also, membrane cholesterol of sperm was extracted by folch solution at the same time sperm ability was assessed for viability and acrosomal status. The membrane cholesterol quantity was measured by thin-layer chromatography (TLC) method. The result, viability and acrosome integrity in semen diluted without egg yolk groups were decreased at all temperature range by increasing of MBCD concentration. In particular, sperm of egg yolk group was showed that significantly higher viability and lower acrosome damage when treated with 5 mM MBCD (p<0.05). The results of TLC experiment, cholesterol amounts were increased with MBCD cocentration in egg yolk, and decreased with MBCD concentration in m-Modena B. In cryopreservation efficiency, there was no significant difference at viability, and acrosomal state of sperm in 5 mM MBCD concentration was significantly lower in acrosome damage than other groups (p<0.05). Therefore, the addition MBCD in egg yolk was protected spermatozoa from cold shock injury. This protective effect of MBCD may be due to addition of sperm membrane cholesterol.

• Journal of Periodontal and Implant Science
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• v.31 no.2
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• pp.299-315
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• 2001

Application of membranes for guided tissue regeneration(GTR) have been confined to the subgingival barrier functions; however, many studies have provided evidence that some drugs, including tetracycline, initially can promote the growth of periodontal ligament or alveolar bone in peridontal therapy. Osseous regeneration in periodontal defects is increased by local administration of tetracycline due to its anti-collagenolytic effect, which enhances bone-forming ability via osteoblast cell chemotaxis and reduced bone resorption. The aim of this study was to evaluate effects of tetracycline loaded poly-L-lactide(PLLA) barrier membranes for guided bone regenerative potential. Tetracycline was incorporated into the PLLA membrane with the ratio 10% to PLLA by weight. Ability to guided bone regeneration of the membranes were tested by measuring new bone in the tibial defects($7{\times}10{\times}5\;mm^3$) of the beagle dog for 4,5, and 6 weeks. In control, drug-unloaded PLLA membranes were used in same size of defect. In histologic finding of the defect area, a few inflammatory cells were observed in both groups. These membrane were not perforated by connective tissue and maintained their mechanical integrity for the barrier function for 4-6 weeks. New bone formation was greater in defects covered by tetracycline-loaded membrane than in defects covered by drug- unloaded membranes. In bone regeneration guiding potential test, tetracycline-loaded membrane was more effective than drug- unloaded membranes(p<0.05). These results suggest that tetracycline-loaded PLLA membranes potentially enhance guided bone regenerative efficacy and might be a useful barrier for GTR in periodontal treatment.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.