The effect of different activation protocols on DNA integrity of porcine oocytes matured in vitro was analysed using the comet assay. The matured oocytes were then freed of cumulus cells and treated continuously or intermittently with a nitric oxide (NO) donor for 6 h. Standard activation with calcium ions (Ca2+) and culture without any treatment served as positive and negative controls, respectively. Exposure of oocytes to Ca2+ was associated with high activation efficiency, but decreased DNA integrity. The opposite was observed after continuous exposure to NO. Intermittent action of NO increased the activation rate, while the values of DNA damage remained at low levels. Our data suggest that an increased DNA instability could be the main reason compromising the further embryonic development of oocytes activated by the standard protocol. The intermittent treatment with NO thus represents a promising step to optimization of parthenogenetic activation of pig oocytes.