Comet assay: Monitoring time-dependent DNA-fragmentation in two different types of cells, optimizing Flash-comet method for pig sperm analysis

Abstract: DNA-damage, caused by different factors such as oxidative stress in cells, is common in living organisms. The damage includes DNA-fragmentation, such as single- and double strands breaks. If the damage occurs in a sperm cell, spermatozoa, it can lead to fertility problems in male mammals, and the high levels of damage has been connected with lower fertilization rates and for the women, higher miscarriage rate. Comet assay is a method for assessing DNA-damage, based on electrophoresis. Flash-comet is a modified Comet assay, with improved speed and sensitivity. The aim of this project was to establish cell survival curves to monitor time-dependent DNA-fragmentation in two different types of cells, TK6-cells and pig sperm cells. The secondary purpose was to optimize the method Flash-comet for pig sperm analysis. Methods used for the experiments included Comet assay and Flash-comet. The results showed a clear time dependent decrease in overall amount of DNA in TK6-cells. Preliminary data indicated a possible lysis condition of the spermatozoa. The primary purpose was partially achieved, by determining cell survival curves to monitor time-dependent DNA-fragmentations in TK6-cells, but the curves were not achieved for the pig sperm cells. However, a major discovery was made, a new approach for measuring apoptotic cell death was discovered. The secondary purpose was partially achieved, by initiating the optimization and adjustment of the Flash-comet for pig sperm analysis, successfully optimizing the lysis step of the Flash-comet.