Chenglei Song, Qiufei Jiang, Chunmao Zhu, An Shi, Guoxiong Nan, Wenjuan Shen, Jiamin Zhou, Zhuo Yang, Zhengjun Tuo, Xiaohong Han, Jinzhong Tao
Sperm cryodamage is a key factor affecting its post-thaw quality. This study aims to systematically elucidate the molecular response mechanisms of bovine sperm cryodamage by integrating transcriptomic and proteomic analyses. By utilizing the swim-up technique to isolate sperm subpopulations with mild and severe cryodamage, thereby minimizing interference from individual genetic variation. Results demonstrated that mildly damaged sperm exhibited superior motility, structural integrity, and antioxidant capacity, along with less reactive oxygen species accumulation and more active Wnt, mTOR, and Hippo signaling pathways at the transcriptional level. Severely damaged sperm exhibited activation of pathways related to oxidative phosphorylation, thermogenesis, peroxisome, glutathione metabolism, phagocytosis, and DNA repair at the protein level, with ATP5MG, GPX1, XRCC1, and AP1M2 serving as core regulatory factors in these pathways. Additionally, integrated analysis identified eight potential biomarker proteins, including apoptosis-related protein ARL6IP5, anti-apoptotic protein GRINA, adhesion factors CDHR1/LGALS1, DNA repair proteins CUL4B/POLR2E, and core factors RPL14/A1BG in the protein-protein interaction network. Notably, the protein levels of ARL6IP5, CDHR1, and LGALS1 were inconsistent with their gene expression levels. These candidate molecules showed significant correlations with post-thaw sperm quality and were quantitatively validated. The results indicate that mitochondrial energy metabolism, antioxidant and anti-apoptotic defense systems, as well as self-repair and clearance responses, including nucleic acid repair and phagocytosis, represent the molecular response characteristics of sperm cryodamage. These findings complement the molecular mechanisms and regulatory networks of sperm cryodamage, and provide potential targets for evaluating the cryopreservation potential of bull semen and optimizing cryoprotectants.