ProAKAP4 as a predictor of semen freezability in Asturcon ponies

Abstract

BACKGROUND: Cryopreservation is a valuable tool in equine reproduction, although its success remains highly variable among stallions. The proAKAP4 protein has been proposed as a molecular marker of sperm quality and cryotolerance. AIMS/OBJECTIVES: This study aimed to evaluate the relationship between proAKAP4 concentration and sperm cryotolerance in ejaculates from Asturcon ponies, and to assess whether proAKAP4 levels in fresh semen can predict post-thaw sperm quality parameters before and after cryopreservation. METHODS: Twenty-four ejaculates from eight different stallions were used (three ejaculates per animal). Fresh and thawed ejaculates, as well as those frozen with BotuCRIO and FAO extender, were classified into two groups based on proAKAP4 concentration (low or high) using complete linkage hierarchical cluster analysis. Sperm quality was assessed using computer-assisted sperm analysis and flow cytometry, including evaluations of motility, viability, morphology, acrosome integrity, and plasma membrane lipid disorder. ProAKAP4 concentration was measured using a commercial ELISA kit. RESULTS: Fresh and frozen ejaculates with high proAKAP4 concentrations exhibited higher total motility, progressive motility, and sperm membrane integrity (P < 0.05) than ejaculates with lower proAKAP4 concentrations. Ejaculates frozen with BotuCRIO and FAO extender and classified as having high proAKAP4 concentrations also displayed improved (P < 0.05) total and progressive motility. BotuCRIO samples with high proAKAP4 levels showed higher (P < 0.05) plasma and acrosomal membrane integrity, and lower (P < 0.05) membrane lipid disorder compared to those with lower proAKAP4 levels. A pre-freeze ProAKAP4 concentration threshold of 70.33 ng/10(6) spermatozoa was identified as predictive of good freezability, and a post-thaw threshold of 40 ng/10(6) spermatozoa was associated with acceptable quality. CONCLUSION: These findings confirm that proAKAP4 is a promising biomarker for semen cryotolerance and support its integration into routine semen evaluation protocols for stallion selection.