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The aim of research is to conduct a comparative study of frozen-thawed semen from breeding bulls of dairy and combined breeds. Objectives: to evaluate the quality of cryopreserved semen at three time points: immediately after thawing, 1 hour after thawing, and 3 hours after thawing; to study the level of sperm DNA fragmentation in bulls of dairy and combined breeds. The object of the study was the semen of sires from breeding enterprises of the Russian Federation. Samples of cryopreserved semen (n = 87) from bulls of dairy breeds (Holstein and Yaroslavl), as well as combined breeds (Simmental and Kostroma) were selected for the study. Sperm motility and DNA structure integrity were studied immediately after thawing, 1 hour after thawing, and 3 hours after thawing. Immediately after thawing, the content of spermatozoa with rectilinear-progressive movement was (48.27 ± 1.896) % for dairy breed bulls and (46.04 ± 1.771) % for combined breeds. After 3 hours, the number of progressively motile sperm decreased to (16.05 ± 1.263) % for dairy breeds and to (12.66 ± 2.829) % for combined breeds. Higher DNA fragmentation was found in the semen of dairy bulls with the following parameters: Tail Moment – 0.291 ± 0.012 and Comet Length – 27.786 ± 0.042, while bulls of combined breeds had lower DNA damage values (Tail Moment – 0.091 ± 0.024, Comet Length – 25.663 ± 0.088). In the sample we studied, combined breed bulls were inferior to dairy breed bulls in sperm survival rate. Dairy bulls exhibited higher values for most parameters characterizing DNA damage compared to bulls of mixed breeds. These interbreed differences should be considered in selection and breeding work and the development of sperm cryopreservation technologies.