Effects of fluorescence-activated cell sorting on boar sperm motility subpopulations and in vitro embryo development

Authors: RODRIGUEZ, ISABEL - Iowa State University; WEIDE, TYLER - Iowa State University; GROTE, CALEIGH - Iowa State University; KELLER, ALEXANDRA - Iowa State University; BITTENCOURT, CATARINA - Iowa State University; SCHOELERMAN, EMILY - Iowa State University; SHOFNER, IAN - Iowa State University; KEATING, AILEEN - Iowa State University; Redel, Bethany; KERNS, KARL - Iowa State University

Submitted to: Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/26/2026
Publication Date: 6/7/2026
Citation: Rodriguez, I., Weide, T., Grote, C., Keller, A., Bittencourt, C., Schoelerman, E., Shofner, I., Keating, A., Redel, B.K., Kerns, K. 2026. Effects of fluorescence-activated cell sorting on boar sperm motility subpopulations and in vitro embryo development. Molecular Reproduction and Development. 93(6). https://doi.org/10.1002/mrd.70123.
DOI: https://doi.org/10.1002/mrd.70123

Interpretive Summary: Assisted reproductive technologies have become essential tools for improving reproductive efficiency and genetic progress in livestock species. Among the reproductive technologies currently used, fertilization in a petri dish allows us to evaluate both the sperm and egg, as well as, embryo development under controlled conditions. Despite significant advancements in porcine fertilization protocols, fertilization efficiency and embryo development rates remain highly variable, with overall reduced embryo viability compared with embryos that are naturally fertilized in the body. Fluorescence activated cell sorting is a tool that has been used to separate sperm that are better quality, however, there is limited data on how this technology influences sperm quality after sorting and subsequent embryo development. Here, we show that after cell sorting, sperm motility and fertilization potential is reduced. These findings indicate that sperm sorting can impair post-fertilization competence and represent the first report of sorting-associated cleavage defects in the pig. The observed developmental decline likely arises from subtle alterations induced during sorting that affect early embryo development.

Technical Abstract: 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.