Relationships Between Metabolism of Cryopreserved Equine Sperm Determined by the Seahorse Analyzer and Sperm Characteristics Measured by Flow Cytometry and Computer-Assisted Analysis of Motility

Cryopreservation of stallion spermatozoa remains a challenge in equine reproduction, as it induces mitochondrial dysfunction that cannot be fully captured by conventional sperm quality assessment and requires further investigation. This study provides a detailed bioenergetic evaluation of cryopreserved stallion sperm using Seahorse XFp technology to measure basal oxygen consumption rate (OCR) and to perform a MitoStress Test. Three ejaculates from 20 Warmblood stallions (licensed by the Holsteiner Verband, Elmshorn, Germany) were analyzed at 15 min intervals from 15 to 120 min after thawing. CASA and multiparametric flow cytometry were used to assess motility, sperm viability, reactive oxygen species (ROS) synthesis, lipid peroxidation (LPO), and DNA fragmentation index (%DFI). Seahorse analysis revealed marked intra- and inter-individual variability in basal OCR among frozen–thawed samples. Functional mitochondrial parameters showed only partial correlations with motility and viability results, underlining the added diagnostic value of combined metabolic profiling. Over the 2 h measurement, viability and motility displayed an increasingly negative correlation with ROS synthesis, while no significant relation was found between OCR and ROS synthesis or LPO. These findings demonstrate that stallion sperm mitochondria are highly sensitive organelles whose functional state cannot be fully assessed by routine motility or viability testing alone. Integrating Seahorse-based profiling with CASA and flow cytometry can improve sperm quality assessment. Moreover, this novel approach has been proven to be a valuable tool, offering broad opportunities for further research in stallion reproduction.