The results obtained by measuring the level of γH2AX fluorescence intensity in the paternal pronuclei show that the values differ between the individual groups of semen. The lowest values of fluorescence intensity, and thus the lowest DNA damage, were found in freshly collected semen (11.93 ± 8.06). Variability was also demonstrated between the experimental groups of frozen semen. The influence of different methods of semen collection and its freezing and the influence of the composition of the freezing medium were confirmed. The highest values of fluorescence intensity were found in the freezing medium supplemented with egg yolk (39.23 ± 8.06). This method of assessing the quality of cryopreserved is a relatively simple and inexpensive method that can replace previous sperm quality assessment techniques with the advantage of replacing hard-to-reach oocytes of endangered species with available mouse oocytes