Abstract / Summary
Abstract Cryopreservation compromises sperm quality, yet conventional semen analysis poorly predicts which ejaculates will withstand freezing. proAKAP4, a sperm-specific structural protein, is a candidate freezability biomarker, but is usually measured after thawing. We tested whether proAKAP4 quantified in fresh semen predicts post-thaw sperm quality in bucks across breeds. Five ejaculates from each of 23 bucks (Boer, n = 10; Anglo-Nubian, n = 8; Murcia-Granada, n = 5) were cryopreserved and assessed for computer-assisted motility and flow-cytometric plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, viability and DNA fragmentation; fresh-semen proAKAP4 was measured by ELISA. Fresh-semen proAKAP4 was repeatable (average-measure ICC = 0.84) and correlated strongly with post-thaw membrane integrity, mitochondrial membrane potential, viability, motility and velocity, and inversely with DNA fragmentation. Notably, it was unrelated to any fresh-semen parameter, indicating that it captures cryotolerance-specific rather than baseline quality. Using a composite cryotolerance score, proAKAP4 discriminated high- from low-cryotolerance bucks with high accuracy (AUC = 0.985; exploratory cut-off 36.81 ng/10 6 sperm). Although both proAKAP4 and cryotolerance differed among breeds, partial correlation ( r = 0.86) and a linear mixed model ( p < 0.001) indicated that, under the cryopreservation conditions applied, proAKAP4 predicted cryotolerance consistently across the three breeds examined. These findings identify fresh-semen proAKAP4 as a promising, non-invasive candidate biomarker for selecting bucks with superior freezability, warranting validation in larger, independent cohorts and under different cryopreservation protocols.