Identification and validation of ram sperm proteins associated with cryoinjuries caused by the cryopreservation process

The change of sperm protein profile after the cryopreservation process may influence fertilization and early embryonic development. The purpose of the present study was to identify ram sperm proteomic modifications induced by the cryopreservation process using the isobaric tags for relative and absolute quantification labeling technology (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. Semen samples were collected from five Yunnan semi-fine wool rams using an electroejaculator. Sperm motility (CASA), plasma membrane (HOST test), and acrosome integrity (FITC-PSA) were evaluated after freeze-thawing. The total proteins of fresh and frozen-thawed sperm were extracted and purified, followed by identifying ram sperm proteomic modifications using the isobaric tags for relative and absolute quantification labeling technique (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. The results showed a significant reduction (P < 0.05) in all sperm parameters after thawing. 126 differentially abundant proteins (DAPs) were identified through comparison of the proteomes between fresh and frozen-thawed sperm. Among them, 90 proteins were down-regulated after the cryopreservation process. The remaining 36 proteins were up-regulated in frozen-thawed sperm. The results of functional annotation demonstrated the potential relationship of 10 DAPs with oxidoreductase activity. 18 and 15 DAPs may be involved in the stress and carbohydrate metabolic process, respectively. Furthermore, 8 DAPs may be functionally associated with reproduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results demonstrated the primary enrichment of these identified DAPs in metabolic activities, disease, and oxidative phosphorylation. In order to confirm the reliability of the iTRAQ results, the changing trends of 10 proteins analyzed by PRM were similar to those of the corresponding proteins identified by iTRAQ. In conclusion, the cryopreservation process modifies the proteome of ram sperm, possibly leading to compromised fertility of post-thaw sperm. Additionally, the identified DAPs in this study may function as potential biomarkers for assessing the post-thaw quality of ram semen.