Holotomography and Multivariate Analysis Reveal Donor-Specific Responses to Antioxidant Supplementation During Stallion Sperm Cryopreservation

: Freeze-thaw procedures impair sperm morphology and function, affecting viability, motility, redox balance, and subcellular organization. Although antioxidants may mitigate these effects, their interaction with donor-specific variability remains unclear. We combined quantitative holotomography with conventional physiological assessments within a multivariate framework based on principal component analysis (PCA) and nested cross-validated Linear Discriminant Analysis (LDA) to evaluate donor-specific responses to antioxidant-supplemented cryopreservation. Spermatozoa from ten stallions was analyzed before and after freezing under five conditions: fresh semen; frozen semen with INRA Freeze, frozen semen with HF-20, and HF-20 supplemented with matcha, spirulina, horseradish, or quercetin. For each condition, sperm kinetics, mitochondrial activity, oxidative stress, DNA integrity, and three-dimensional volumetric measurements of whole-cell and subcellular compartments derived from holotomography were integrated into a single dataset. LDA achieved 0.734 cross-validated accuracy for stallion classification, revealing strong donor-specific signatures. In contrast, classification by antioxidant treatment was near chance (0.248). Fresh semen was clearly distinct from all cryopreserved groups. Holotomography showed reduced whole-cell and post-acrosomal/midpiece volumes after freezing, while nuclear volume was unchanged. Antioxidant supplementation produced minor, inconsistent effects, with partial midpiece preservation in some donors but no global pattern. Overall, inter-stallion variability dominates post-thaw sperm phenotype. Antioxidant effects were detectable but modest, supporting individualized strategies to optimize equine semen cryopreservation protocols.