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ProAKAP4, AKAP4 precursor, is a specific protein from spermatozoa flagella, essential for maintaining motility efficiency. In all mammals studied proAKAP4 levels have been shown to correlate with total and progressive motility. In stallions and donkeys, proAKAP4 serves as an indicator of sperm quality and sustained motility over the time once thawed. This study aimed to compare proAKAP4 concentration with cytometry parameters: acrosome integrity, ROS production, mitochondrial potential and lipidic disorder. Animal cohort consisted of eight Asturcon ponies. Three ejaculates of each pony (24 ejaculates) were collected and frozen by commercial protocol. Post-thawed semen was analyzed by CASA and flow cytometry (PNA, JC1, HE, Fluo3 and M540 stains) and the proAKAP4 amount was quantified by ELISA commercial kit, Horse 4MIDⓇ Kit. Results show a positive correlation between proAKAP4 concentration and flow cytometry dyes: PNA stain (r= 0.34, p=0.0178), indicating membrane integrity; a negative correlation with membrane lipid disorder, M540 stain (r= -0.327, p=0.0234); a positive correlation with ROS production, H2 stain intensity (r= 0.48, p=0.005); and a negative correlation with mitochondrial potential measured by JC-1 (r=-0.41, p=0.0042). These results suggest that a spermatozoon with an intact membrane possesses more proAKAP4. Additionally, a spermatozoon with a high proAKAP4 concentration and an intact membrane also implies higher motility, resulting in increased ROS production. Conversely, a spermatozoon exhibiting significant lipid disorder shows a lower proAKAP4 concentration, indicating lower quality and reduced likelihood of reaching the fertilization site. As observed in previous publications, mitochondrial activity is not associated with proAKAP4 concentration, suggesting that the energy expenditure of the spermatozoon is not linked to the quality of movement defined by proAKAP4 concentration. A quality threshold of 42.10 ng of proAKAP4/10M of spermatozoa was calculated using a cluster between viability and motility parameters. This concentration is consistent with the 37.77 ng/10M of spermatozoa identified in a previous study, it may serve as a criterion for determining the sperm quality in post-thaw conditions. A relationship with cytometry parameters is observed showing that proAKAP4 is positively correlated with intact acrosome membrane and production of ROS which it suggests that an intact and rapid spermatozoon will show more proAKAP4 concentration. On the contrary, proAKAP4 is negatively correlate with a lipid disorder of the membrane which it means that a less quality spermatozoa will show less proAKAP4 concentration. Even if correlations are not high, their significance is strong, further analysis with a bigger cohort are needed to confirm these results. [ABSTRACT FROM AUTHOR]