Proteomic insights into the effects of cryopreservation on sperm of endangered Eld's deer (Rucervus eldii)

The success of fertilization largely depends on semen quality. However, semen cryopreservation can impair sperm function and reduce conception rates, thereby hindering the repopulation of threatened species, including Eld's deer (Rucervus eldii). Despite its importance, information on the effects of cryopreservation on sperm protein composition in Eld's deer remains limited. This study investigated the proteomic profiles of sperm from Siamese and Burmese Eld's deer at three stages-fresh, pre-freeze, and post-thaw-using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparative analysis revealed distinct protein expression patterns across stages. A total of 2294 proteins were identified, of which 16 and 14 were significantly differentially expressed between fresh and pre-freeze sperm and between fresh and cryopreserved sperm, respectively. Increased levels of coiled-coil domain-containing protein 136 (CCDC136) and signal transducer and activator of transcription 3 (STAT3) in pre-freeze sperm suggested cytoskeletal stabilization and immune activation during cooling. Post-thaw sperm showed decreased levels of nucleoside diphosphate kinase A (NME1) and zinc finger protein-like 1 (ZFPL1), indicating metabolic and chromatin disruption, whereas elevated cullin-4B (CUL4B) and small ubiquitin-like modifier 1 (SUMO1) reflected activation of DNA repair and protein turnover pathways. These molecular alterations were consistent with reduced post-thaw motility, viability, and membrane and acrosome integrity, highlighting key stress-response mechanisms. The identified proteins may serve as biomarkers for semen quality assessment and inform optimized cryopreservation strategies, thereby supporting conservation efforts for Eld's deer.