Effects of Fluorescence-Activated Cell Sorting on Boar Sperm Motility Subpopulations and In Vitro Embryo Development

Flow cytometric sorting can be used to select sperm subpopulations with defined physiological or molecular traits; however, the impact of sorting on sperm function and subsequent embryo development in the porcine model remains unresolved. The objective of this study was to evaluate the effect of fluorescence-activated cell sorting (FACS) on boar sperm motility and developmental competence following in vitro fertilization (IVF). Pooled semen from boars was divided into sorted and unsorted (control) treatments and analyzed for motility and kinematic parameters using computer-aided sperm analysis (CASA) with both population-level (conventional, population-averaged) metrics and single-cell track data collected before and after sorting. Oocytes were fertilized with either treatment, and cleavage (≥ 2-cell stage) and blastocyst development were evaluated. Cleavage and blastocyst percentages were reduced in the sorted (48.8% ± 9.9% and 8.5% ± 4.2%) compared with control (92.2% ± 1.5% and 28.4% ± 2.8%, respectively; p < 0.001) group. Population-level CASA revealed statistically significant but biologically modest changes in motility and kinematic parameters following sperm sorting. In contrast, single-cell CASA coupled with k-means clustering identified four distinct motility subpopulations and demonstrated that sorting redistributed existing motility states, characterized by depletion of fast, progressive sperm and enrichment of less energetically demanding populations. These findings suggest that FACS alters the composition of sperm motility subpopulations, alongside a marked reduction in post-fertilization developmental competence in porcine IVF.