Cryotolerance of brown bear (Ursus arctos) sperm depends on sperm origin: insights into sperm quality and proteomic profiles

Cryopreservation is a crucial tool for the ex situ conservation of endangered species, enabling the long-term storage of genetic resources. However, sperm from wild species often show variable cryoresistance, and the underlying molecular mechanisms remain poorly understood. This study evaluated how sperm origin (epididymal, pre-ejaculated, and ejaculated) influences the cryotolerance of brown bear (Ursus arctos) sperm by combining functional assessments (motility, viability, apoptosis, mitochondrial ROS production, and redox balance) with proteomic profiling before and after freezing. Epididymal sperm, despite superior initial motility, exhibited the greatest post-thaw decline in viability, apoptotic resistance, and mitochondrial ROS production. Ejaculated sperm showed intermediate pre-freeze values but retained the highest post-thaw quality, demonstrating better resilience to cryoinjury. Pre-ejaculated sperm, although starting with the lowest motility, displayed a more moderate decline, with intermediate post-thaw apoptosis and mitochondrial ROS levels. Redox analyses indicated higher basal oxidative stress in epididymal sperm and post-thaw reduction in antioxidant reserves in ejaculated sperm. Proteomic analyses identified 56, 33, and 53 downregulated proteins in epididymal, pre-ejaculated, and ejaculated sperm, respectively, with a core set of 22 proteins consistently affected across all sperm origins. Functional enrichment revealed that these shared proteins were mainly associated with energy and carbohydrate metabolism, highlighting core metabolic functions as common targets of cryodamage. In origin-specific analyses, epididymal sperm were the most affected, with disruptions in energy production, stress response, proteostasis, and fertilization processes. Pre-ejaculated sperm exhibited changes mainly in carbohydrate metabolism, while ejaculated sperm, although also affected in energy and stress processes, retained greater molecular stability, likely buffered by seminal plasma. These findings demonstrate that sperm origin strongly influences cryodamage patterns in the brown bear and identify a conserved metabolic signature of cryoinjury, supporting the value of proteomics for optimizing wildlife cryobanking strategies.