Key Laboratory for Animal Genetics and Molecular Breeding of Jiangsu Province, Yangzhou University, Yangzhou, China.
Reprod Domest Anim. 2024 Oct;59(10):e14737. doi: 10.1111/rda.14737.
Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.
精子冷冻保存可以实现精子的长期保存。冰晶损伤以及氧化应激导致解冻后精子线粒体功能障碍和运动能力下降。然而，关于活性氧（ROS）和线粒体对绵羊精子冷冻保存的影响的信息有限。本研究的主要目的是研究 ROS 和线粒体与绵羊精子冷冻保存过程中精子质量的关系。本研究评估了精子运动能力、运动学特征、膜完整性、顶体完整性、线粒体膜电位（MMP）、三磷酸腺苷（ATP）水平、线粒体呼吸基因（NDUFV2、SDHA、CYC1 和 COXIV）的表达、ROS 水平、丙二醛（MDA）含量、磷脂酰丝氨酸外翻率、精子超微结构、mtDNA 拷贝数、凋亡相关基因（Bax、Caspase-3 和 Caspase-8）、细胞色素 C 和 Caspase-3 表达。结果表明，冷冻保存显著（p<0.05）降低了运动能力、动力学参数、膜完整性、顶体完整性、MMP、ATP、线粒体呼吸相关基因的 mRNA 表达水平，显著（p<0.05）增加了 ROS 水平、MDA 含量、磷脂酰丝氨酸外翻率、精子超微结构损伤、mtDNA 拷贝数、凋亡相关基因（Bax、Caspase-3 和 Caspase-8）、细胞色素 C 和 Caspase-3 表达的 mRNA 水平。综上所述，冷冻保存会导致线粒体损伤，从而增加 ROS 并引发随后的氧化应激。这一过程还会导致线粒体功能障碍，并干扰电子传递链，最终导致 MMP 和 ATP 生成减少。此外，细胞色素 C 的释放促使 Caspase-3 表达增加，随后发生精子凋亡，最终导致解冻后精子质量恶化。
