Proteomic and metabolomic profiling reveal alterations in freezing-thawing and fresh drake sperm.

Cryopreservation of semen is crucial for preserving genetic resources and maintaining biodiversity. However, the unique structural characteristics of avian sperm make them particularly susceptible to cryoinjury, often resulting in impaired motility following thawing. Currently, research on drake semen cryopreservation remains limited. In this study, an integrated proteomic and untargeted metabolomic analysis was employed to investigate the molecular mechanisms underlying cryodamage-induced impairment of drake sperm motility. The findings revealed that glycolytic enzymes and peroxisomes play essential roles in maintaining sperm function during cryopreservation. Proteomic and metabolomic profiling of fresh (FS) and frozen-thawed (FT) semen samples from drakes identified a total of 6,946 proteins and 1,568 metabolites. Comparative analysis showed that, relative to FS samples, the FT group exhibited an upregulation of 94 proteins and 80 metabolites, and a downregulation of 259 proteins and 138 metabolites. The findings of this indicate that several key antioxidant enzymes were significantly downregulated in the FT samples, suggesting a lack of capacity to eliminate excessive reactive oxygen species (ROS) generated during the freezing process. This imbalance likely contributes to oxidative stress and apoptosis in sperm cells. Additionally, suppressing the glycolytic pathway during freezing reduces ATP production, further compromising sperm motility. These results demonstrate that oxidative stress and energy metabolism disruption are major contributors to cryoinjury in drake sperm, providing potential targets for improving cryopreservation outcomes in drake species.