Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, S17003, Girona, Spain.
Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, S17003, Girona, Spain.
Biol Res. 2022 Apr 1;55(1):15. doi: 10.1186/s40659-022-00386-2.
The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays.
The results showed that global DNA damage negatively correlates (P < 0.05) with normal sperm morphology (R = - 0.460) and progressive motility (R = - 0.419), and positively with the percentage of non-viable sperm (R = 0.507). Multiple regression analyses showed that non-viable sperm were related to SSB (β = - 0.754). In addition, while fertilisation did not seem to be affected by sperm DNA integrity, global DNA damage, DSB and SSB were found to be correlated to embryo development outcomes. Specifically, whereas global DNA damage and DSB negatively affected (P < 0.05) the later preimplantation embryo stages (percentage of early blastocyst/blastocyst D6: for global DNA damage, R = - 0.458, and for DSB, R = - 0.551; and percentage of hatching/hatched blastocyst D6: for global DNA damage, R = - 0.505, and for DSB, R = - 0.447), global DNA damage and SSB had a negative impact (P < 0.05) on the developmental competency of fertilised embryos (R = - 0.532 and R = - 0.515, respectively). Remarkably, multiple regression analyses supported the associations found in correlation analyses. Finally, the present work also found that the inclusion of Comet assays to the conventional sperm quality tests improves the prediction of blastocyst formation (AUC = 0.9021, P < 0.05), but not fertilisation rates (P > 0.05).
Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility.
精子 DNA 完整性的评估已被提议作为传统哺乳动物精液分析的补充测试。从这个意义上说，已经报道了精子 DNA 碎片化 (SDF) 的两种类型，即单链 (SSB) 和双链 (DSB) DNA 断裂，它们具有不同的病因，并与牛和人类的不同生育结果相关。考虑到在猪中没有研究表明 SDF 如何影响精子质量和生育结果，本研究旨在确定总 DNA 损伤、SSB 和 DSB 对精子质量和体外受精能力的影响。为此，将 24 个精液样本（每个公猪一份）分为三份：第一份用于通过计算机辅助精子分析 (CASA) 系统和流式细胞术评估精子质量参数；第二份用于进行体外受精，第三份用于使用碱性和中性彗星试验评估精子 DNA 完整性。
结果表明，总 DNA 损伤与正常精子形态呈负相关（P<0.05）（R=-0.460）和前向运动（R=-0.419），与非存活精子的百分比呈正相关（R=0.507）。多元回归分析表明，非存活精子与 SSB 有关（β=-0.754）。此外，虽然受精似乎不受精子 DNA 完整性的影响，但总 DNA 损伤、DSB 和 SSB 与胚胎发育结果相关。具体而言，虽然总 DNA 损伤和 DSB 对后期植入前胚胎阶段（早期囊胚/囊胚 D6 的百分比：总 DNA 损伤，R=-0.458，DSB，R=-0.551；孵化/孵化囊胚 D6 的百分比：总 DNA 损伤，R=-0.505，DSB，R=-0.447）有负面影响（P<0.05），但总 DNA 损伤和 SSB 对受精卵的发育能力有负面影响（R=-0.532 和 R=-0.515）。值得注意的是，多元回归分析支持相关性分析中发现的关联。最后，本研究还发现，将彗星试验纳入传统精子质量检测可提高囊胚形成的预测能力（AUC=0.9021，P<0.05），但不能提高受精率（P>0.05）。
考虑到所有这些发现，本研究建立了一个有用的模型来研究 SDF 如何对生育能力产生负面影响。
