Fresh-semen proAKAP4 concentration predicts sperm cryotolerance independently of conventional semen quality across three goat breeds

Abstract

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.