Journal of equine veterinary science2026; 162; 105912; doi: 10.1016/j.jevs.2026.105912

ProAKAP4 as a predictor of semen freezability in Asturcon ponies.

Abstract: 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. Objective: 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 spermatozoa was identified as predictive of good freezability, and a post-thaw threshold of 40 ng/10 spermatozoa was associated with acceptable quality. Conclusions: These findings confirm that proAKAP4 is a promising biomarker for semen cryotolerance and support its integration into routine semen evaluation protocols for stallion selection.

Get Full Text

Publication Date: 2026-04-22 PubMed ID: 42031304DOI: 10.1016/j.jevs.2026.105912Google Scholar: Lookup

The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.

Facebook X LinkedIn Email Copy

Summary
Cite This
Publication
Affiliations
MeSH
Conflict of Interest
Citations

Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Research Overview

This study investigates whether the protein proAKAP4 can serve as a predictor of how well semen from Asturcon ponies withstands freezing and thawing processes (cryopreservation).
The research evaluates if proAKAP4 levels in fresh semen correlate with sperm quality after thawing, aiming to improve stallion semen evaluation and selection for breeding programs.

Introduction and Background

Cryopreservation in Equines: Freezing stallion semen is important for breeding management but yields inconsistent outcomes depending on individual stallions.
proAKAP4 Protein: A molecular marker located in spermatozoa, linked to sperm quality and resilience to cryopreservation stress.
Significance: Identifying reliable markers like proAKAP4 can help predict which stallions produce semen that remains viable after freezing, improving breeding success.

Objective

To evaluate the association between proAKAP4 protein concentration levels in Asturcon pony ejaculates and their sperm’s ability to tolerate freezing and thawing.
To determine if proAKAP4 levels measured in fresh semen can predict post-thaw sperm quality outcomes, before actual cryopreservation happens.

Methods

Sample Collection: 24 ejaculates collected from 8 stallions (3 ejaculates per stallion).
Extenders Used: Semen frozen using two extenders:
- BotuCRIO
- FAO
Grouping: Ejaculates were grouped into ‘high’ or ‘low’ proAKAP4 concentration clusters based on hierarchical cluster analysis.
Sperm Quality Assessment Techniques:
- Computer-assisted sperm analysis (CASA) for motility parameters.
- Flow cytometry for sperm viability, morphology, acrosome integrity, and membrane lipid disorder.
proAKAP4 Measurement: Commercial ELISA kit measured proAKAP4 levels, expressed in ng per 10 million spermatozoa.

Results

Fresh and Frozen Semen Quality: Samples with higher proAKAP4 levels showed statistically significant improvements in:
- Total sperm motility
- Progressive motility (forward movement quality)
- Sperm membrane integrity
Effect of Extenders: Both BotuCRIO and FAO groups with high proAKAP4 levels exhibited better motility after thawing.
BotuCRIO Specific Findings: Samples with high proAKAP4:
- Had greater plasma and acrosomal membrane integrity.
- Showed reduced membrane lipid disorder, indicating better membrane stability.
Thresholds for Prediction:
- Pre-freezing proAKAP4 level threshold: 70.33 ng per 10 million sperm predicted good freezability.
- Post-thaw proAKAP4 level threshold: ≥ 40 ng per 10 million sperm associated with acceptable semen quality.

Conclusions and Implications

ProAKAP4 as a Biomarker: The study confirms proAKAP4 protein concentration as a useful molecular marker to predict semen cryotolerance in Asturcon ponies.
Practical Use: proAKAP4 measurement could be integrated into routine stallion semen evaluations to improve selection criteria, minimizing variability in cryopreservation outcomes.
Benefits:
- More efficient breeding programs by selecting semen with higher freezing survival potential.
- Potential reduction in costs and time by avoiding freezing poor quality semen.
Future Directions: Research on proAKAP4 could extend to other horse breeds and possibly other species for broader cryopreservation applications.