Regular verification of the quality of cryopreserved semen derived from native cattle is one of the tasks performed at the bank as recommended by the Food and Agriculture Organization. The purpose of the present study was to evaluate the quality of semen from PR bulls stored for 40, 50 and 60 years in the BMB using standard evaluation parameters such as sperm motility as well as structural-functional parameters such as plasma membrane integrity, transmembrane mitochondrial potential and sperm chromatin damage. Semen pellets from 27 PR bulls (3 ejaculates/bull) were tested. The data were analysed by one-way and two-way ANOVA, and the significance of the difference (P≤ 0.01) between the means was determined using Duncan’s test. Our study results revealed that the long-term storage of semen had no effect on sperm characteristics after thawing. However, statistically significant differences (P≤0.01) in sperm plasma membrane integrity and transmembrane mitochondrial potential, following storage in liquid nitrogen were noted between bulls at all time points. However, there were no significant differences (P>0.01) in sperm chromatin damage between breeds or between different storage times, and the degree of DNA fragmentation ranged from 0.4 to 0.8%.