Cryopreservation of bovine sperm causes single-strand DNA breaks that are localized in the toroidal regions of chromatin.

Background: Sperm cryopreservation is widely used in the cattle industry, as it allows for disassociating the localization of sires and the collection of semen from the timing of artificial insemination. While freeze-thawing is known to impair sperm DNA integrity, whether the damage induced consists of single- (SSB) or double-strand breaks (DSB) has not been determined. In addition, no previous study has addressed if DNA breaks preferentially reside in specific genome regions such as those forming the toroid linker regions, or are rather spread throughout the regions linked to protamines. The main aim of the present work, therefore, was to elucidate the type and localization of the DNA damage generated by cryopreservation and to evaluate its impact on artificial insemination outcomes in cattle.

Results: The incidence of SSB and DSB was evaluated in 12 ejaculates before and after cryopreservation with the Comet assay, and the localization of the DNA breaks was assessed using pulsed-field gel electrophoresis (PFGE). Before cryopreservation, the incidence of SSB was 10.99% ± 4.62% and involved 20.56% ± 3.04% of sperm cells, whereas these figures significantly (P < 0.0001) increased up to 34.11% ± 3.48% and 53.36% ± 11.00% in frozen-thawed sperm. In contrast, no significant differences in the incidence of DSB were observed (P > 0.990) before and after cryopreservation (before: incidence of 13.91% ± 1.75% of sperm DNA affecting 56.04% ± 12.49% of sperm cells; after: incidence of 13.55% ± 1.55% of sperm DNA involving 53.36% ± 11.00% of sperm cells). Moreover, PFGE revealed that the percentage of sperm DNA fragments whose length was shorter than a toroid (< 31.5 kb) was greater (P < 0.0001) after (27.00% ± 4.26%) than before freeze-thawing (15.57% ± 4.53%). These differences indicated that the DNA breaks induced by cryopreservation affect the regions condensed in protamines, which are structured in toroids. On the other hand, in vivo fertility rates were associated to the incidence of SSB and DSB in frozen-thawed sperm (P = 0.032 and P = 0.005), but not with the size of the DNA fragments resulting from these breaks (P > 0.05).

Conclusion: Cryopreservation of bovine sperm generates single-strand DNA breaks, which are mainly located in protamine-condensed toroidal regions. The incidence of DNA breaks in cryopreserved sperm has an impact on cattle fertility, regardless of the size of generated fragments.