• Journal of Embryo Transfer
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• v.28 no.3
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from ${\alpha}$-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% $CO_2$ incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers ($CD45^-$, $29^+$, $90^+$ and $105^+$) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited $CD45^-$, $29^+$, $90^+$ and $105^+$ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at $1{\times}10^3$ cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.299-305
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• 2016

Although cryopreservation of sperm is routinely used for clinical requirement, it has some problems, such as high generation of reactive oxygen species (ROS) and cold-shock. To reduce the detrimental damage in sperm, anti-oxidants were added to cryoprotectant for sperm. Curcumin is one of anti-oxidants, which are added in cryoprotectants. However, recent studies have demonstrated that curcumin decreases sperm viability and motility. This study was performed to identify the effect of curcumin on hydrogen peroxide ($H_2O_2$)-exposed bovine sperm, which were cryopreserved-thawed. In $H_2O_2$-exposed bovine sperm, reactive oxygen species (ROS) were significantly reduced by treatment with curcumin in a dose-dependent manner (p < 0.05). Among tested concentrations of curcumin (1 to $50{\mu}M$), 30 and $50{\mu}M$ curcumin showed anti-oxidant effect on $H_2O_2$-induced ROS generation. On the other hand, combination of 30 or $50{\mu}M$ curcumin with anti-oxidant $H_2O_2$ increased the percentage of apoptotic sperm compared to only $H_2O_2$ treatment. Sperm viability was also decreased in the combination of 30 or $50{\mu}M$ curcumin with $H_2O_2$ as judged by FDA/PI staining. $H_2O_2$-induced decrease in sperm progressive motility was recovered by treatment with $1{\mu}M$ curcumin. These results show that high concentration of curcumin has anti-oxidant effect, but it has also cytotoxic effect on bovine sperm. Sperm viability and motility might be more affected by cytotoxic signals of curcumin compared to antioxidant signals.

• Journal of the Korean Society for Marine Environment & Energy
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• v.4 no.4
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• pp.12-20
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• 2001

The temporal variability in near-field mixing characteristics of discharging plumes in oceanic environment was investigated using the time series data of the buoyant jet parameters. Based on the currents and density profiles observed in Masan outfall site and effluent discharge flowrates for 63days of summer season, the temporal variabilities and those occurrence frequency were obtained by line plume equations. The results show that wide range of variability in Masan outfall's mixing characteristics was found due to the temporal changes of effluent flowrates and ambient oceanic conditions. The near-field dilution was in the range of 30~71 with the averaged dilution of 34, which was a good agreement with field measurements of salinity deficit. The length of mixing zone was in the range of 5.4~36.2 m with the average of 9.5 m, and the plume rise height was in the range of 8.1~10.2 m with the average of 8.9 m. However, only the 30~44% of the whole results are higher than the averages, which indicates the necessity of this frequency analysis with the continuously measured data for designing and managing the ocean outfall system.

• Journal of the Korean earth science society
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• v.41 no.5
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• pp.478-489
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• 2020

This study presents an analysis of infrasonic signals from two accidental explosions in Gwangyang city, Jeonnam Province, Korea, on December 24, 2019, recorded at 12 infrasound stations located 151-435 km away. Infrasound propagation refracted at an altitude of ~40 km owing to higher stratospheric wind in the NNW direction, resulting in favorable detection at stations in that direction. However, tropospheric phases were observed at stations located in the NE and E directions from the explosion site because of the strong west wind jet formed at ~10 km. The transmission losses on the propagation path were calculated using the effective sound velocity structure and parabolic equation modeling. Based on the losses, the observed signal amplitudes were corrected, and overpressures were estimated at the reference distance. From the overpressures, the source energy was evaluated through the overpressure-explosive charge relationship. The two explosions were found to have energies equivalent to 14 and 65 kg TNT, respectively. At the first explosion, a flying fragment forced by an explosive shock wave was observed in the air. The energy causing the flying fragment was estimated to be equivalent to 49 kg or less of TNT, obtained from the relationship between the fragment motion and overpressure. Our infrasound propagation modeling is available to constrain the source energy for remote explosions. To enhance the confidence in energy estimations, further studies are required to reflect the uncertainty of the atmospheric structure models on the estimations and to verify the relationships by various ground truth explosions.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.169-175
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• 2013

Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in vitro produced transgenic embryos has been a problem of the low productivity of transgenic animal because of absent of pre-estimate of pregnancy after transgenic embryos transferred into recipient. Therefore, this study is conducted to improve efficiency of transgenic cattle production by improving the non-surgical embryo transfer (ET) method. Transgenic bovine embryos were produced by injection of feline immunodeficiency virus enhanced green fluorescent protein (FIV-EGFP) lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization (IVF) was occured. Normal IVF and EGFP expressing blastocysts were transferred into recipients. Results indicated that 2 expanded blastocysts (34.7%) transferred group showed significantly (P<0.05) higher pregnancy rate than 1 expanded blastocyst (26.8%) transferred group. In case of parity of recipient, ET to heifer (34.9%) showed significantly (P<0.05) higher pregnancy rate than ET to multiparous recipient (21.2%). However, there are no significant differences of pregnancy rate between natural induced estrus and artificial induced estrus groups. Significantly (P<0.05) higher pregnancy rate was obtained from recipient group which have normal corpus luteum with crown group (34.8%) than normal corpus luteum without crown (13.6%). Additionally, treatment of $100{\mu}g$ Gn-RH injection to recipient group (38.6%) 1 day before ET significantly (P<0.05) increase pregnancy rate than non- Gn-RH injection to recipient group (38.6%). We also transferred 2 EGFP expressing expanded blastocysts to each 19 recipients, 7 recipients were pregnant and finally 5 EGFP transgenic cattle were produced under described ET condition. Therefore, our result suggested that transfer of 2 good-quality expanded blastocysts to $100{\mu}g$ of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.311-317
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• 2017

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen ($LN_2$). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in $50{\mu}l$ droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at $39^{\circ}C$ with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop ($47.1{\pm}6.9%$) than that of French mini straw ($15.9{\pm}12.5%$). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) ($84.5{\pm}14.2%$) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.287-295
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• 2017

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

• Journal of the Korean Society for Aeronautical & Space Sciences
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• v.38 no.12
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• pp.1240-1246
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• 2010

Enthalpy probe with the inner and outer diameters of 1.5 mm and 4.8 mm, respectively, is designed and used to measure the temperatures and velocities along the centerline of Ar arc plasma flow until the probe was destroyed. For this purpose, Ar arc plasma flow is generated by non-transferred type DC arc heater with the power level of 17 kW. From this experiment, it is shown that the designed enthalpy probe can measure the temperature and velocity of arc plasma flow up to 12,000 K and 600 m/s, respectively, without destroy of probe tip. In this extreme case, the arc plasma flow is calculated to transfer the heat flux of ${\sim}5{\times}10^7\;W/m^2$ to the probe based on the heat and thermal boundary equations near the forward stagnation point of a body immersed in arc plasma flow. Consequently, the designed enthalpy probe can measure the wide ranges of plasma temperatures, velocities and concentrations simultaneously, which are generated by various types of arc heaters within the heat flux ranges of $0{\sim}5{\times}10^7\;W/m^2$ on the probe tip.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.245-252
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• 2012

The purpose of the present study was to investigate the effect of IFN-${\tau}$ on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and concentration of progesterone (P4) in endometrial cells. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses of IFN-${\tau}$ (0, 0.02, 0.2 and 2 ug/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin $E_2$ and $F_{2{\alpha}}$ levels in the culture media were analyzed by enzyme immunoassays and total RNA was extracted from the cells for RT-PCR. P4 concentrations of blood samples were assayed by chemiluminescent immuno assays system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN-${\tau}$ (p<0.05), but it was not significantly different in all groups of stromal cells except for 2 ug/ml IFN-${\tau}$ group (p<0.05). Although IFN-${\tau}$ did not affect $PGE_2$ and $PGF_{2{\alpha}}$ production in epithelial cells, it decreased $PGE_2$ and $PGF_{2{\alpha}}$ production significantly in stromal cells (p<0.05). In vivo experiment, blood concentration of P4 was significantly increased after addition of IFN-${\tau}$ (1 ug/ml). The results indicate that PG production was mediated by COX-2 expression in stromal cells but it was not affected in epithelial cells and this suggest that treatment of IFN-${\tau}$ could improve the implantation environment of uterine by maintenance of high P4 concentration.

• Journal of Embryo Transfer
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• v.33 no.1
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• pp.23-30
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• 2018

The purpose of this study was to investigate the effects of the concentration of seminal plasma in aerobic and anaerobic conditions on the total motility(TM) and the progressive motility(PM) of spermatozoa in long term preservation of cooled equine semen. We also examine the pregnancy rates after artificial insemination using fresh, cooled or frozen semen, and different durations of cooled-preserved equine semen. In the aerobic state of cooled-preserved semen, As the increase of preserved duration to 24h, 48h, 72h, and 96h, TM tended to decrease in each of different concentrations of formalin-containing experimental group, TM tended to decrease regardless of the concentrations of SP. In different concentrations of SP, TM of without seminal plasma(SP W/O) group tended to be higher than that of SP 20%, SP 33% and SP 50%, especially TM of SP W/O group was significantly higher than other groups at 96 h (p<0.05). PM was higher in the groups of SP W/O and SP 20% than in the groups of SP 33% and SP 50% from 24 h to 72 h in cooled-preservation, especially PM of SP W/O group was significantly higher than other groups at 96 h (p<0.05). In the anaerobic condition of cooled-preserved semen, the results of TM and PM at different concentrations of SP were similar to the results in the aerobic condition although there was a difference in the ratio. The pregnancy rates of fresh-cooled, cooled-preserved and frozen semen were 66.3%, 60.7% and 34.5%, respectively, and the pregnancy rate of frozen semen was the lowest. We also found that it is possible to pregnancy after artificial insemination using 72 h cooled-preserved equine semen. There was similar of the pregnancy rates in the different month from April to August.