Comprehensive proteomic characterization of Eld's deer (Rucervus eldii) seminal plasma during cryopreservation identifies proteins associated with cryostress responses.

In the conservation of endangered wildlife such as Eld's deer (Rucervus eldii), assisted reproductive technologies including artificial insemination play an important role in enhancing reproductive success and maintaining genetic diversity. However, semen cryopreservation induces molecular alterations that may impair post-thaw sperm function. This study aimed to characterize changes in the seminal plasma proteome of Eld's deer across fresh, pre-freeze, and post-thaw stages of cryopreservation, using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cryopreservation resulted in a consistent reduction of fertility-associated and immune-related proteins, including binder of sperm 1 (BSP1), spermadhesin-1 (SPADH1), polymeric immunoglobulin receptor (PIGR), and beta-defensin 1 (DEFB1). In contrast, proteins associated with oxidative stress defense and ubiquitin-mediated protein regulation, including glutathione S-transferase theta-4 (GSTT4), ubiquitin thioesterase OTUB1 (OTUB1), and ubiquitin carboxyl-terminal hydrolase 1 (USP1), were increased following cryopreservation. These findings indicate a functional shift in the seminal plasma proteome from fertility-related components toward stress-response and cellular maintenance pathways during cryopreservation. Notably, GSTT4, OTUB1, and USP1 were identified as candidate proteins associated with cryopreservation-related responses. Further validation is required to determine their potential utility as biomarkers of sperm cryotolerance and their relevance to improved semen preservation strategies for endangered Eld's deer.