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. 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). 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.

中文翻译：

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牛精子的冷冻保存会导致单链 DNA 断裂，这些断裂位于染色质的环形区域


精子冷冻保存广泛用于养牛业，因为它可以将公牛的定位和精液的收集与人工授精的时间分开。虽然已知冻融会损害精子 DNA 的完整性，但尚未确定诱导的损伤是单链断裂 （SSB） 还是双链断裂 （DSB）。此外，以前的研究没有解决 DNA 断裂是否优先驻留在特定的基因组区域，例如形成环形接头区域的基因组区域，或者更确切地说是分布在与鱼精蛋白相连的整个区域。因此，本研究的主要目的是阐明冷冻保存产生的 DNA 损伤的类型和定位，并评估其对牛人工授精结果的影响。用 Comet 法评估冷冻保存前后 12 例射精的 SSB 和 DSB 发生率，并使用脉冲场凝胶电泳 （PFGE） 评估 DNA 断裂的定位。冻融精子中 SSB 的发生率分别为 10.99% ± 4.62%，涉及 20.56% ± 3.04% 的精子细胞，而冻融精子的 SSB 发生率显著增加至 34.11% ± 3.48% 和 53.36% ± 11.00%。相比之下，冷冻保存前后 DSB 的发生率没有显著差异 （P > 0.990） （之前：13.91% ± 1.75% 的精子 DNA 发生率影响 56.04% ± 12.49% 的精子细胞;之后：13.55% ± 1.55% 的精子 DNA 发生率，涉及 53.36% ± 11.00% 的精子细胞）。此外，PFGE 显示冻融后 （27.00% ± 4.26%） 后 （15.57% ± 4.53%） 长度短于环形体的精子 DNA 片段百分比 （% 3C 0.0001） （P < 0.0001）。 这些差异表明，冷冻保存诱导的 DNA 断裂会影响鱼精蛋白中浓缩的区域，这些鱼精蛋白以环形结构构建。另一方面，体内生育率与冻融精子中 SSB 和 DSB 的发生率相关 （P = 0.032 和 P = 0.005），但与这些断裂导致的 DNA 片段的大小无关 （P > 0.05）。牛精子的冷冻保存会产生单链 DNA 断裂，这些断裂主要位于鱼精蛋白浓缩的环形区域。冷冻保存的精子中 DNA 断裂的发生率对牛的生育能力有影响，无论产生的片段大小如何。