Table 1_Comparative proteomic analysis of bovine spermatozoa before and after cryopreservation.xlsx

Semen cryopreservation has transformed the livestock industry by enabling the long-term storage and global distribution of genetic material from high-indexing sires. However, despite its benefits, cryopreservation of bull semen is known to impair sperm quality, as seen by declines in motility, membrane viability, and fertilization potential. The molecular mechanisms behind these impairments remain poorly understood, prompting the need for proteomic profiling to uncover biomarkers associated with cryosurvival and reproductive function. This study employed a bottom-up proteomic approach to characterize proteomic changes in bovine sperm before and after cryopreservation. Semen samples from five sexually mature bulls with proven fertility were divided into fresh group and cryopreserved groups and analyzed by LC-MS/MS. A total of 1,373 proteins were identified across all samples after filtering was applied. Principal component and hierarchical clustering analyses revealed 84 proteins with significant differential abundance (greater than log2FC of 2 and p < 0.05); 59 were more abundant in fresh sperm and 25 more abundant in cryopreserved sperm. Functional enrichment analysis indicated that proteins increased after cryopreservation were primarily associated with metabolic processes and keratinization, while proteins more abundant before cryopreservation were primarily involved in reproductive processing and cytoskeletal organization. Image-based flow cytometry further demonstrated cryo-induced redistribution cytochrome c (CYCT), dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48kDa subunit (DDOST), and 40s ribosomal protein S3a (RPS3A) across distinct sperm cell regions. These results implicate mitochondrial integrity, redox homeostatic, and cytoskeletal structure as pathways disrupted during semen cryopreservation, consistent with apoptosis-like and capacitation-like changes. This work establishes a framework for future evaluation of protein biomarkers involved in critical sperm functions associated with cryoinjury and detecting susceptible sperm populations.