Reactive oxygen species (ROS) production and F-actin cytoskeleton dynamics play important roles in the survival rate of blastocysts after the vitrifiedwarming process. However, the protective effects of Mito-TEMPO against cryo-injury and viability through F-actin aggregation and mitochondrial-specific ROS production in vitrificated-warmed bovine embryos have not been investigated. The aims of the present study were to: (1) determine the effects of Mito-TEMPO on embryonic developmental competence and quality by F-actin stabilization during in vitro culturing (IVC), and (2) confirm the effects of Mito-TEMPO through F-actin structure on the cryotolerance of vitrification-warming in Mito-TEMPO exposed in vitro production (IVP) of bovine blastocysts. Bovine zygotes were cultured with 0.1 μM Mito-TEMPO treatment for 2 days of IVC. Mito-TEMPO (0.1 μM) exposed bovine embryos slightly improved in blastocyst developmental rates compared to the non-treated group. Moreover, the viability of vitrified-warmed blastocysts from Mito-TEMPO treated embryos significantly increased (p < 0.05, non-treated group: 66.7 ± 3.2% vs Mito-TEMPO treated group: 79.2 ± 5.9%; re-expanded at 24 hours). Mito-TEMPO exposed embryos strengthened the F-actin structure and arrangement in the blastocyst after vitrification-warming. Furthermore, the addition of Mito-TEMPO into the IVC medium enhanced embryonic survival and quality through F-actin stabilization after the vitrification-warming procedure. Overall, our results suggest that supplementing the culture with 0.1 μM Mito- TEMPO improves the embryonic quality and cryo-survival of IVP bovine blastocysts.

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito- TEMPO (0.1 μM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozenthawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

Cryopreservation is used for blastocyst preservation of most mammalian embryos and is an important technique for breeding. We aimed to compare the efficiency of the cryopreservation method using the standard Cryotop device and the ReproCarrier device, a domestic product manufactured in Korea. The efficacy of the two devices was analyzed based on the survival rate, intracellular levels of reactive oxygen species (ROS), and apoptosis of the vitrified bovine blastocysts. The survival rates of the vitrified-warmed blastocysts were similar between the ReproCarrier group (58.4 ± 17.7%) and Cryotop group (59.9 ± 14.1%). Intracellular ROS levels and apoptotic index were determined by DCFDA staining and TUNEL assay. Changes in intracellular ROS levels, number of total nuclei, and cellular apoptosis of vitrified blastocysts after cryopreservation were not significantly different between the two groups. These results indicate that the ReproCarrier device method is as effective as the standard Cryotop method for vitrification of bovine blastocysts in vitro.

This paper is to investigate the employees' corresponding types and casual analysis. It proposes the legal and practical measures for improvement of Industrial Accident Compensation Insurance's usability. The results from the empirical analysis indicate that (1) 91.4 percent of the respondents feel the necessity of Industrial Accident Compensation Insurance, (2) 67.4 percent of the respondents perceive that Industrial Accident Compensation Insurance is useful, (3) employers’ perceptions of the specific items of Industrial Accident Compensation Insurance appears to be low. (4) 35.9 percent of the respondents deal with industrial accidents through other ways such as health insurance and car insurance. The study ends with discussion of the findings and provides several theoretical and managerial implications and recommendations for future research and applications.

The numerical experiments using a particle tracking model have been performed for predicting the change of water quality and shoreline. In present study, comparison of the numerical model results with the analytic solution shows that the point of the maximum concentration and the distribution pattern is very similar. The reflection effect from the boundary was newly introduced for making clear the effect of the closed boundary which set limits to application of a particle tracking model. The present model seems to reappear physical phenomenon well. This model shows well qualitative appearance of pollutant diffusion in Kwangan beach. Therefore, this model is regarded as a useful means for predicting diffusion of pollutant, movement of suspended sand, and change of water quality.