The present study was conducted to examine the reactive oxygen species (ROS) generation levels and subsequent DNA damage in the bovine cultured somatic cells. Bovine ear skin cells were classified by serum starvation, confluence and cycling cells. Cells were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) or 10 μM hydroxyphenyl fluorescein (HPF) dye to measure the H2O2 or ˙OH radical levels. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each cell. H2O2 and ˙OH radical levels of cultured somatic cells were high in confluence group (7.1±0.7 and 8.4±0.4 pixels/cell, respectively) and significantly low in serum starvation group (4.9±0.4 and 7.0±0.4 pixels/cell, respectively, p<0.05). Comet tail lengths of serum starvation (148.3±5.7 μm) and confluence (151.1±5.0 μm) groups were found to be significantly (p<0.05) increased in comparison to that of cycling group (137.1±7.5 μm). These results suggest that the culture type of donor cells can affect the ROS generation, which leads the DNA fragmentation of the cells.

The objective of this study was to determine the potential hazardous effects of sorting process by flowcytometry on the quality of boar spermatozoa by flowcytometer. Freshly collected boar semen was diluted and divided into two groups; control none sorted and sorted. Sperms in sorted group were processed with flowcytometer for cell sorting with 100 uM nozzle under the 20 psi pressure. Measurements on each parameter were made at two time points, 0hr (right after sorting) and 24 hr post sorting. Although there was a tendency of lower viability in sorted group than none sorted control group, the percentage of live cells in control(75.83+-6.92 & 59.53+-10.34) was not significantly different from sorted (59.7+-7.34 & 43.97+-3.76) at both 0 and 24 hr post sorting. However, sorted sperm showed significantly lower mitochondrial function compared to the control at both 0 h (79.37+-3.22 vs. 63.50+-10.05) and 24hr(67.27+-3.22 vs. 46.97+-5.37) time points (p<0.007). Sperm DNA fragmentation rate was significantly lower in control (22.0+-7.04) than that of sorted (32.27+-7.49) at 24 hr time point (p<0.0002). Taken together, these data suggested thatsorting process by flowcytometer may have influenced sperm motility rather than viability. Also high speed sperm sorting by flowcytometer has significant effects on DNA fragmentation on elapsed time after sorting.

The changes in DNA damage were investigated during storage after irradiation. Potato, garlic were irradiated at 0.05, 0.07, 0.1 and 0.15 kGy and stored for 3 months. Ginger was irradiated at 0.01, 0.02, 0.03, 0.04 and 0.05 kGy and stored for 1 month. The comet assay was applied to the sample immediately after irradiation and at the end of storage. Samples were isolated, grounded and the suspended cells were embedded in an agarose layer. After lysis of the cells, they were electrophoresed for 1 min. and then stained. DNA fragmentation in seeds caused by irradiation was quantified as tail length and tail moment (tail length ×% DNA in tail) by comet image analyzing system. Right after irradiation, the differences in tail length between unirradiated and irradiated samples were significant(p〈0.05) in potato, garlic and ginger. With increasing the irradiation doses, statistically significant longer extension of the DNA from the nucleus toward anode was observed. The results represented as tail moment showed similar tendency to those of tail length. Similarly in the stored samples, even 1 or 3 months after irradiation, all the irradiated samples significantly showed longer tail length than the unirradiated controls. These results indicate that the comet assay could be one of the simple methods of detecting irradiated samples. Moreover, the method could detect DNA damage even after 1 or 3 months after irradiation.

The changes in DNA damage were investigated during storage after irradiation. Beef, pork and chicken were irradiated at 1.0, 3.0 and 5.0 kGy and stored for 6 months at -20℃. The comet assay was applied to the sample muscles at the beginning of irradiation and at the end of storage. Muscles were isolated, sliced, and the suspended cells were embedded in an agarose layer. After lysis of the cells, they were electrophoresed for 2 min. and then stained. DNA fragmentation in tissues caused by irradiation was quantified as tail length and tail moment (tail length × % DNA in tail) by comet image analyzing system. Right after irradiation, the differences in tail length between unirradiated and irradiated muscles were significant(p〈0.05) in beef, pork and chicken. With increasing the increasing doses, statistically significant longer extension of the DNA from the nucleus toward anode was observed. Similarly even 6 months after irradiation, all the irradiated muscles significantly showed longer tail length than the unirradiated controls. The results represented as tail moment showed similar tendency to those of tail length, but the latter parameter was more sensitive than the former. These results indicate that the comet assay could be one of the simple methods of detecting irradiated muscles. Moreover, this method suggest that using comet assay, we were able to detect DNA damage differences even after 6 months after irradiation.