Revealing ROC-derived optimal thresholds of specific seminal plasma proteins as markers of bull semen freezability

This study examined differences in the concentrations of selected bovine seminal plasma proteins-osteopontin (OPN), clusterin (CLU), lactoferrin (LTF), and angiotensin-converting enzyme (ACE)-between ejaculates with good (GFE) and poor freezability (PFE). In addition, the potential of these proteins as indicators of semen freezability was assessed using receiver operating characteristic (ROC) curve analyses. A total of 72 ejaculates collected from six bulls were included in the study to enhance the biological robustness of the analysis, thereby reducing ejaculate-related variability. Using cluster analysis based on post-thaw sperm quality parameters (total motility and plasma membrane and acrosome integrity), ejaculates were classified as GFE (n = 41) or PFE (n = 31). As a result OPN, CLU, and LTF concentrations were higher in the GFE group compared to the PFE group (P < 0.05), while ACE concentrations did not differ between groups (P > 0.05). ROC curve analyses indicated that seminal plasma OPN, CLU, and LTF possess measurable diagnostic potential for predicting bull semen freezability, with optimal cut-off values identified for each protein. These associations may be related to the involvement of these proteins in maintaining sperm membrane integrity, modulating oxidative balance, and supporting cellular tolerance to cryopreservation-induced stress. Overall, seminal plasma OPN, CLU, and LTF may serve as practical indicators of semen freezability and could assist in optimizing semen processing strategies in artificial insemination centers. To our knowledge, this study is the first to define ROC-based predictive thresholds for seminal plasma proteins associated with bull semen freezability.