• Animal Bioscience
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• v.34 no.5
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• pp.844-854
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• 2021

Objective: The potential of extra virgin olive oil (EVOO), betaine (BET), and ginger (GIN), as natural antioxidants, in reducing negative effects of heat stress on physiological responses, antioxidant capacity, semen quality and fertility of bucks under heat stress were investigated. Methods: Forty adult Animal Production Research Institute line rabbit bucks were distributed randomly into four experimental treatments of ten rabbits each. The first treatment was fed the commercial pellet diet (CPD) without supplementation and served as a control. The other three treatments were fed CPD supplemented with EVOO (300 mg), BET (1,000 mg), and GIN (200 mg) per kg diet for 3 consecutive months during the summer season. Results: Supplementation of EVOO, BET, or GIN improved (p<0.05) the sexual desire, progressive motility, vitality, intact acrosome and membrane integrity, sperm cell concentration, sperm outputs and fertility. Seminal plasma total proteins, globulin, total antioxidant capacity, glutathione and glutathione S-transferase, and initial fructose increased (p<0.05), while total lipids, aspartate and alanine aminotransferases and malondialdehyde decreased (p<0.05) compared with the control. In comparing the natural antioxidants treatments, GIN evoked the largest improvement. Conclusion: The inclusion of GIN (200 mg/kg diet) appeared to improve the sexual desire, semen quality and oxidative stress of bucks. This may be a beneficial supplement for the management of rabbit bucks used in natural mating or artificial insemination.

• Clinical and Experimental Pediatrics
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• v.48 no.5
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• pp.545-550
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• 2005

Purpose : Newborn brain tissue has to be dissociated into a single cell suspension for flow cytometric analysis of cell death during hypoxia-ischemia. Thus the development of a method to dissociate cells from the brain tissue with least damage and maintenance of membrane and antigen integrity remains the challenge for the in vivo application of this technique. We evaluated the efficacy of mechanical or enzymatic (collagenase or tryspin) methods of brain tissue disaggregation. Methods : The extent of the damage to the plasma membrane and loss of the characteristics of the membrane induced with each dissociation method was determined by comparing the flow cytometric results labeled with both fluorescent annexin V and propidium iodide of the newborn rat pup brain tissue in the control group (n=10) and in the 48-hour after hypoxia-ischemia group (n=10). Results : In the control group, the cell percentage of damaged, apoptotic and necrotic cells of both hemispheres with the mechanical dissociation method was significantly increased compared to the trypsin or collagenase method. In the 48-hour after hypoxia-ischemia group, the cell percentage of apoptotic and necrotic cells of the right hemisphere with the collagenase method significantly increased, and live cells significantly decreased compared to the left hemisphere, control group. Although the same trend was observed, the extent of alterations made with the trypsin method was significantly less compared to the collagenase method. Conclusion : The dissociation of neonatal brain tissue for flow cytometric analysis with collagenase was most efficacious with the least cell damage and preservation of the plasma membrane characteristics.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.171-178
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• 2013

The purpose of this study was to evaluate the sperm viability, normal acrosome and mitochondrial activity in the frozen-thawed fowl semen by different cryoprotectants. The experiment was carried out on 10 sexually adult roosters of Ogye. The semen was collected twice a week and pooled semen was diluted 1:1 EK extender containing no cryoprotectant at $5^{\circ}C$. After equilibration for 30 minutes, diluted chicken semen was diluted 1:1 extender containing either 7% dimethylacetamide (DMA), 7% dimethylformamide (DMF) or 7.5% methylacetamide (MA) at final concentration and was put in 0.5 mL plastic straws and frozen for 30 minutes by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen. Frozen semen was thawed in water bath at $5^{\circ}C$ for 2 minutes. For cytometric analysis, the frozen-thawed semen was diluted with EK extender to a final concentration of 90 million spermatozoa per mL. Sperm membrane integrity was evaluated as SYBR-14 and propidium iodide (PI). Acrosome integrity was assessed with fluorescein isothiocyanate-labeled PSA and PI. The percentage of mitochondrial function was estimated by using Rhodamine123 (R123) and PI. In conclusion, freezing rooster semen by using 7% DMF as cryoprotectant was significantly highest in rates of survival and mitochondrial function while its rate of damage of acrosome was significantly lowest. As a result, DMF is the cryoprotectant that has the lowest influences on sperm membranes and acrosome integrity. Therefore it could be used for freezing method of animal genetic conservation method for poultry diversity.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.3
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• pp.189-199
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• 2006

Purpose of study: Partial thickness skin graft is the golden standard regimen for full-thickness skin defect caused by burn or trauma. However, in case of extensive burns of more than 50% of total body surface area, the donor site is not sufficient to cover all defects. As a second choice, allograft, xenograft and synthetic materials have been used to treat skin defect. Among them the amniotic membrane(AM) was used as a biological dressing for centuries because of its potential for wound healing. In this study, quantification of EGF in AM and effect of AM-collagen complex on full thickness skin defects was examined. Materials & Methods: The concentration of EGF in fresh, deep frozen and freeze-dried AM was evaluated by ELISA. EGF-R immunostaining was performed in freeze-dried AM. SD rats weighing 250${\sim}$300g was used for wound healing experiment. Three full thickness skin defects(28mm diameter) were made on dorsal surface of SD rat. The control group was covered by Vaselin gauze and AM-collagen complex and $Terudermis^{(R)}$. was grafted in two other defects. Healing area, Cinamon's score were evaluated before biopsy. Grafted sites were retrieved at 3 days, 1 week, 2 weeks and 4 weeks after operation. H & E and Factor VIII immunohistochemical stain was performed to evaluate the microscopic adhesion and structural integrity and microvessel formation. Results: 1. EGF concentration of fresh, deep frozen and freeze-dried AM showed similar level and EGF-R was stained in epithelial layer of freeze-dried AM. 2. At 4 weeks after grafting, the healing area of AM-collagen and Terudermis group was 99.29${\pm}$0.71% and 99.19${\pm}$0.77 of original size. However, that of control group was 24.88${\pm}$2.90. 3. The Cinamon's score of AM-Collagen and $Terudermis^{(R)}$. group at 4 weeks was 15.6${\pm}$1.26 and 14.6${\pm}$3.13 and that of control group was 3.7${\pm}$0.95. Significant difference was observed among control and experimental groups(p<0.05). 4. Histologic examination revealed that AM protected leukocyte infiltration and epithelial migration was nearly completed at 4 weeks. $Terudermis^{(R)}$. group showed mild neutrophil infiltration until 2 weeks and completion of epithelization at 4 weeks. Control group showed massive leukocyte infiltration until 4 weeks. 5. Microvessels were increased sharply at 1 week and control group at 1 and 4 week showed significant differences with $Terudermis^{(R)}$. group of same interval(p<0.05) but no differences were found with AM group(p<0.05). Conclusion: EGF and EGF-R were well preserved in freeze-dried AM. AM attached to collagen acted as excellent biologic dressing which had similar effect with $Terudermis^{(R)}$. AM showed anti-inflammatory action and healing was completed at 4 weeks after full-thickness skin defect.

• Tuberculosis and Respiratory Diseases
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• v.40 no.3
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• pp.236-249
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• 1993

Background: Among the injurious agents to which the lung airspaces are constantly exposed are reactive species of oxygen. It has been widely believed that reactive oxygen species may be implicated in the etiology of lung injuries. In order to elucidated how this oxidant causes lung cell injury, we investigated the effects of exogenous $H_2O_2$ on alveolar epithelial barrier characteristics. Methods: Rat type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with HEPES-buffered Ringer solution. The changes in short-circuit current (Isc) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against $H_2O_2$ injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ, an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results: These monolayers have a high transepithelial resistance (>2000 ohm-$cm^2$) and actively transport $Na^+$ from apical fluid. $H_2O_2$(0-100 mM) was then delivered to either apical or basolateral fluid. Resulting indicated that $H_2O_2$ decreased Isc and R gradually in dose-dependent manner. The effective concentration of apical $H_2O_2$ at which Isc (or R) was decreased by 50% at one hour ($ED_{50}$) was about 4 mM. However, basolateral $H_2O_2$ exposure led to $ED_{50}$ for Isc (and R) of about 0.04 mM. Inhibition of cellular catalase yielded $ED_{50}$ for Isc (and R) of about 0.4 mM when $H_2O_2$ was given apically, while $ED_{50}$ for basolateral exposure to $H_2O_2$ did not change in the presence of ATAZ. The rate of $H_2O_2$ consumption in apical and basolateral bathing fluids was the same, while cellualr catalase activity rose gradually with time in culture. Conclusion: Our data suggest that basolateral $H_2O_2$ may affect directly membrane component (e.g., $Na^+,\;K^+$-ATPase) located on the basolateral cell surface. Apical $H_2O_2$, on the other hand, may be largely degraded by catalase as it passes through the cells before reaching these membrane components. We conclude that alveolar epithelial barrier integrity as measured by Isc and R are compromised by $H_2O_2$ being relatively sensitive to basolateral (and insensitive to apical) $H_2O_2$.

• Journal of Veterinary Clinics
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• v.24 no.4
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• pp.486-492
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• 2007

Seminal plasma(SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing. The purpose of this study was to determine the effect of SP on sperm survival by adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from four healthy dogs(1-4 years old) of various breeds were pooled, centrifuged at $300{\times}g$ for 10 min at $25^{\circ}C$, and the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris(EYT) buffer. The study comprised two experiments: [Exp 1] Sperm were suspended in EYT extender containing either 0, 20, 40, 80 or 100% SP and were slowly cooled to $4^{\circ}C$ for 2h or held at $25^{\circ}C$ as controls. Sperm concentration was adjusted to $2{\times}10^8/ml$. [Exp II] Sperm samples, each of which contained $1{\times}10^8/ml$, were assigned to nine groups to be frozen. In the first four groups, sperm in EYT containing either 20, 40, 80 or 100% SP were cooled to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol and then were frozen. The final concentrations of SP were 10, 20, 40 or 50%. In the other four groups, sperm in EYT alone were first cooled slowly to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol plus 10, 20, 40 or 50% SP and then were frozen. Spermatozoa, which chilled in EYT alone and diluted to contain final concentrations of EYT+0.6M glycerol without seminal plasma, and then frozen, was regarded as control. Spermatozoa were frozen at $25^{\circ}C/min$ of cooling rate in plastic straws that were suspended above liquid nitrogen and thawed in water at $38^{\circ}C$ for 1 min. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at $200{\times}$ magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that adding SP did not improve motility of spermatozoa compared to those incubated without SP regardless of temperature. The results of the second experiment showed that spermatozoa suspended in EYT+0.6M glycerol containing SP exhibited the higher progressive motility before being frozen(P<0.05). However, frozen-thawed spermatozoa that had suspended in EYT+0.6M glycerol containing SP showed the similar or lower viability(P<0.05). In summary, although seminal plasma did not affect spermatozoa that were chilled in EYT without cryoprotectant(CPA), addition of seminal plasma to EYT containing CPA did significantly improved progressive motility of canine spermatozoa that were chilled.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.155-159
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• 2013

This study was carried out to investigate synthetic extender for semen cryopreservation of Jeju Native Black Bull. The semen was collected using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by Tris-Egg yolk extender and contrifuged in 1,500 rpm for 15 minutes. The supernatant was removed. The pellect was diluted to final sperm concentration of $2{\times}10^8/ml$ by doubling in every 30 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 4 hours at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm for 10 minutes. The height and duration affect the freezing speed by temperature. The frozen straw was plunged to $LN_2$. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. Frozen-thawed sperm were evaluated sperm viability, membrane integrity and acrosome integrity. Post-thawed sperm viability has been significantly higher (p<0.05) in fresh sperm ($93.27{\pm}1.62%$) than frozen-thawed sperm ($73.34{\pm}3.27%$). However, there were no significant differences between fresh and frozen-thawed dead cell rate ($7.35{\pm}2.63$ vs, $13.71{\pm}2.85$). In sperm motility, between Triladyl and AndroMed Extender, there was no significant different ($72.86{\pm}2.83$ vs, $81.47{\pm}2.48$), similarly, the dead cell rates was similar ($18.41{\pm}3.42%$ and $17.26{\pm}4.25$). The results of our study suggest that AndroMed to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Jeju Native Black bull semen.

• Journal of Animal Science and Technology
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• v.54 no.2
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• pp.95-101
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• 2012

The objective of this study was to examine the effect of amides as a cryoprotectant for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing 5% dimethyl acetamide (DMA), 5% dimethyl formamide (DMF), 5% methyl formamide (MF) or 7% glycerol. Post-thawed sperm were evaluated for sperm motility, viability, acrosome integrity and membrane integrity. Post-thawed sperm motility was significantly higher (p<0.05) in glycerol and DMF ($64.00%{\pm}9.62$ and $59.00%{\pm}5.48$, respectively) than DMA and MF ($50.00%{\pm}3.24$ and $44.00%{\pm}4.18$, respectively). Sperm viability wassignificantly higher (p<0.05) in glycerol and DMF ($58.25%{\pm}7.35$ and $53.05%{\pm}3.77$, respectively) than others. However, for sperm motility and viability, there were no differences among glycerol and DMF. Also, swelling sperm ratio by hypo-osmetic selling test (HOST) was significantly increased (p<0.05) in glycerol and DMF treatments ($45.12%{\pm}25.08$ and $44.95%{\pm}8.58$, respectively). The percentage of capacitated sperm assessed by CTC staining, F pattern was lower (p<0.05) in DMF than others. B pattern was increased (p<0.05) in DMA, DMF and MF when compared with glycerol. AR pattern ratio was decreased (p<0.05) in glycerol and DMF when compared with DMA and MF. These results suggested that amides performed better and could be used as a cryoprotectant for semen freezing of Korean Jeju Black Bull.

• Journal of Chest Surgery
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• v.40 no.1 s.270
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• pp.8-16
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• 2007

Background: We performed a prospective clinical study to evaluate the ultrastructural integrity of the myocardium after using Histidine-Tryptophan-Ketoglutarate (HTK) solution in comparison with blood cardioplegic solution during congenital heart surgery. Material and Method: Twenty two patients with acyanotic heart disease, who were scheduled for elective open heart surgery, were randomized into two groups. The HTK Group (n=11) received HTK cardioplegic solution; the blood group (n=11) received conventional blood cardioplegic solution during surgery. The preoperative diagnoses included ventricular septal defect (n=9) and atrial septal defect (n=2) in each group. A small biopsy specimen was taken from the right ventricle's myocardium, and this was processed for ultrastructural examination at the end of 30 minutes of reperfusion. Semiquantitative electron microscopy was carried out 'blindly' in 4 areas per specimen and in 5 test fields per area by 'random systematic sampling' and 'point and intersection counting'. The morphology of the mitochondrial membrane and cristae were then scored. The interstitial edema of the myocardium was also graded. Result: The semiquantitative score of the mitochondrial morphology was $19.65{\pm}4.75$ in the blood group and $25.25{\pm}5.85$ in the HTK group (p=0.03). 6 patients (54.5%) in the blood group and 3 patients (27.3%) in the HTK group were grade 3 or more for the interstitial edema of the myocardium. Conclusion: The ultrastructural integrity was preserved even better with HTK solution than with conventional blood cardioplegic solution.

• Journal of Veterinary Clinics
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• v.24 no.4
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• pp.577-583
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• 2007

It is important to obtain semen with good quality for efficient fertilization and pregnancy. To obtain these semen, various methods have been developed but most of these methods are time consuming and require costly equipment. Therefore, the objective of this research is to investigate the usability of column filtration system as quick and simple method to get sperm with better quality. Ejaculates were obtained from 5 dogs and analyzed with basic quality parameters before each filtration. Sperm concentration was adjusted to $5{\times}10^7/ml$ after dilution. The experimental groups were divided into non-filtered group(control) and filtered groups(glass wool, Sephadex 5% and Sephadex 20%). Ejaculates were filtered through each filter system and assessed by recovery rate of sperm, motility, normal morphology, CFDA/PI stain and plasma membrane integrity(hypo-osmotic swelling test, HOST). The lowest recovery rate of spermatozoa was recorded in glass wool filtration group, followed by 20% Sephadex filtration group(p<0.05). There was no significant difference between control(non-filtered) and 5% Sephadex filtration poop. Also, there was no significant difference of sperm motility assessed under light microscope among experimental groups. Morphological normality of canine spermatozoa was the highest in the glass wool filtration group and the lowest in the 5% Sephadex filtration group with no significant differences versus 20% Sephadex filtration and control group, respectively(p<0.05). Viability of canine sperm assessed by CFCA/PI staining was the highest in the glass wool filtration poop with no significant difference versus the control group, and the lowest in the 20% Sephadex filtration group with no significant difference versus 5% Sephadex filtration group, respectively(p<0.05). HOS values of canine sperm was the highest in the 20% Sephadex filtration group with no significant difference versus 5% Sephadex filtration group, and the lowest in the control poop with no significant difference versus glass wool filtration group, respectively(p<0.05). Therefore, these results indicated that filtration treatment for extended canine sperm would be useful method to get sperm with better quality by trapping the damaged sperm, consequently filter would be physical barrier against injured or immotile sperm.