Impact of Sorting and Catch Media on Porcine Sperm Motility, Capacitation, and Viability

BACKGROUND: Fluorescence-activated cell sorting (FACS) has emerged as a powerful tool for selecting spermatozoa of a desired population of fluorescent biomarkers, offering potential applications in reproductive biology and agriculture. However, concerns remain regarding sorting-induced physiological alterations that could compromise spermatozoa and/or their ability to capacitate. OBJECTIVES: Our objective was to determine whether catch media composition influences sperm recovery rate and physiological responses post-sort, before and after in vitro capacitation (IVC). MATERIALS AND METHODS: Five million spermatozoa per treatment were sorted and assessed immediately (0 h) and after 4 h of in vitro capacitation (4 h) using computer-aided sperm analysis (CASA) and Image-Based Flow Cytometry (IBFC). Treatments included six FACS-sorted groups across three media types with BSA or PVA supplementation, and one unsorted control. Functional parameters, including motility, zinc efflux, oxidative stress, membrane remodeling, and mitochondrial activity, were quantitatively assessed. RESULTS: We found that catch media without PVA or BSA had a near 10%-24% sperm recovery compared to 88%-92% with supplementation (p < 0.05). Immediately post-sort, spermatozoa exhibited minimal alterations relative to controls; however, after 4 h IVC, sorted groups demonstrated significant shifts in zinc efflux (p < 0.05), increased plasma membrane remodeling (p < 0.05), and reduced mitochondrial activity (p < 0.05). While LTX-based media preserved motility, differential responses in membrane integrity and zinc signature distribution were observed between treatments, suggesting that catch media can modulate capacitation outcomes. Notably, mitochondrial and membrane changes became more evident after capacitation incubation, indicating a delayed effect of sorting stress. CONCLUSION: These findings highlight the importance of optimizing catch media and incubation conditions to maintain post-sorting sperm functionality and inform the refinement of FACS protocols for agricultural and assisted reproductive technologies. By minimizing sorting-associated stress through media composition, sperm quality and fertilization potential may be better preserved.