Determination of Double- and Single-stranded DNA Breaks in Bovine Sperm is Predictive of Their Fertilizing Capacity

Background: The analysis of chromatin integrity has become an important determinant of sperm quality. In frozen-thawed bovine sperm, neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood. The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics, and to assess if this parameter can predict pregnancy rates in cattle.

Results: A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods (0-2 h and 2-4 h), analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays, chromatin deprotamination and decondensation, sperm motility, viability, acrosomal status, and intracellular levels of total ROS, superoxides and calcium. Insemination of 59,605 females was conducted using sperm from the same bulls, thus obtaining the non-return to estrus rates after 90 d (NRR). Results showed an increased rate of double-stranded breaks in the first period (0-2 h: 1.29 ± 1.01%/h vs. 2-4 h: 0.13 ± 1.37%/h; P <  0.01), whereas the rate of sperm with moderate + high single-stranded breaks was higher in the second period (0-2 h: 3.52 ± 7.77 %/h vs. 2-4h: 21.06 ± 11.69 %/h; P < 0.0001). Regarding sperm physiology, viability decrease rate was different between the two periods (0-2 h: - 4.49 ± 1.79%/h vs. 2-4 h: - 2.50 ± 3.39%/h; P = 0.032), but the progressive motility decrease rate was constant throughout post-thawing incubation (0-2 h: - 4.70 ± 3.42%/h vs. 2-4 h: - 1.89 ± 2.97%/h; P > 0.05). Finally, whereas no correlations between bull fertility and any dynamic parameter were found, there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet (Rs = - 0.563, P = 0.003), between NRR and basal progressive motility (Rs = 0.511, P = 0.009), and between NRR and sperm with high ROS at 4 h post-thaw (Rs = 0.564, P = 0.003).

Conclusion: The statistically significant correlations found between intracellular ROS, sperm viability, sperm motility, DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress, where viability and motility would be affected first and sperm chromatin would be altered at a later stage, thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw. Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.