Technological stress factors and sperm DNA integrity: The impact of cryopreservation on bull fertility

This study focuses on the dynamics of nuclear DNA fragmentation in Holstein bull sperm under technological stress, including freezing-thawing and incubation. The relevance of this research is due to the high incidence of infertility in livestock (up to 25% in bulls) and the economic losses associated with the use of low-quality semen in artificial insemination. Particular attention is given to the assessment of DNA integrity using alkaline comet assay and the Argus-CASA computer system, as well as analysis of the correlation between DNA fragmentation and sperm motility. Results showed that freezing-thawing followed by 3-h incubation leads to progressive DNA damage. The DNA content in the sperm head (Head DNA) decreased from 154.59 ± 0.56 to 150.34 ± 0.56 pixels (p < 0.01), while the proportion of DNA in the comet tail (Tail DNA) increased from 1.74 ± 0.01% to 2.02 ± 0.01%. The integral parameters Tail Moment and Olive Tail Moment increased by 33% and 22%, respectively, confirming increased fragmentation. At the same time, a sharp decrease was observed in the proportion of progressively motile sperm (PR%) from 48.12 ± 1.20% to 12.50 ± 0.12% after 3 h. Correlation analysis revealed a strong negative relationship between motility and DNA damage level (r = −0.92). The results demonstrate a pronounced detrimental effect of cryopreservation on bull sperm DNA integrity and motility, underscoring the need to optimize cryopreservation protocols and develop improved biomarkers for sperm quality assessment.