# Integrated Proteomics and Metabolomics Reveal Spermine Enhances Sperm Freezability via Antioxidant Pathways

## TL;DR

This study finds that spermine improves sperm survival after freezing by boosting antioxidant activity and enhancing sperm quality in bulls.

## Contribution

The novel contribution is identifying spermine as a key factor in improving sperm freezability through antioxidant pathways.

## Key findings

- Spermine levels are positively correlated with post-thaw sperm motility and antioxidant enzyme activity.
- Spermine supplementation during cryopreservation improves sperm kinematic parameters and membrane integrity.
- Spermine enhances antioxidant defenses and reduces oxidative stress markers in frozen-thawed sperm.

## Abstract

Sperm freezability exhibits marked individual variability, yet the mechanisms remain unclear. Using bulls as the experimental model, we integrated proteomic (sperm) and metabolomic (seminal plasma) analyses of high-freezability (HF) and control (CF) bulls to identify key biomarkers associated with sperm freezability. Post-thaw motility and membrane integrity were significantly higher in HF bulls (p < 0.05). Sperm proteome analysis revealed upregulated antioxidant proteins (PRDX2, GSTM4), heat shock proteins (HSP70, HSP90), and key enzymes in arginine and proline metabolism (PRODH, LAP3). Seminal plasma metabolomics revealed elevated spermine in HF bulls. Meanwhile, we found that spermine abundance was positively correlated with post-thaw motility, as well as with the expression levels of both PRODH and LAP3 (r > 0.6, p < 0.05). Functional validation demonstrated that 200 μM spermine supplementation in cryopreservation extenders enhanced post-thaw motility, kinematic parameters (VAP, VSL, VCL), membrane integrity, and acrosome integrity (p < 0.05). Concurrently, spermine enhanced antioxidant enzyme (SOD, CAT, GSH-Px) activity and reduced ROS and MDA levels (p < 0.05). Our study reveals a spermine-driven antioxidant network coordinating sperm–seminal plasma synergy during cryopreservation, offering novel strategies for semen freezing optimization.