OPTIMIZED NO-ORIENTING NOZZLE FLOW CYTOMETRY GATING STRATEGY FOR THE IDENTIFICATION AND X/Y DISCRIMINATION OF BOAR SPERMATOZOA

Background & Objectives Flow cytometry, widely used for boar semen sexing, requires costly orienting-nozzle instruments and can be limited by debris and sperm agglutination which overlap with target signals and generate noise, constraining its applications in research and diagnostics. Here, we developed an optimized gating strategy for non-orienting flow cytometers to select the main sperm population and discriminate X and Y chromosome bearing spermatozoa through Hoechst 33342 fluorescence. Material & Methods Semen samples from eight Duroc boars were diluted to 5×106 sperm/mL, washed, stained with Hoechst 33342 at 7 μg/mL (previously determined by calibration curve) and incubated at 37°C for 10 minutes. Samples (200 μL/well) were analyzed in duplicate on a NovoCyte3000 (Agilent®) flow cytometer with low flow rate, recording 30,000 events in the main gate. Gating refinement was achieved using the Forward (FSC) and Side Scatter (SSC) parameters, by area (A) and height (H), wich describe particle size and complexity. Fluorescence was detected in the Pacific Blue channel (λex: 405 nm; λem: 445/45 nm). Statistical analysis was performed with RStudio (v2025.09.2+418). Results The first FSC-A/SSC-A gate enabled the initial exclusion of debris from the sample. A secondary gate in the FSC-A/FSC-H plot allowed the removal of aggregates, ensuring the selection of the target population that was finally observed in the FSC-A/Pacific Blue-A plot. Fluorescence detection revealed two distinct peaks corresponding to X and Y spermatozoa, respectively. Relative standard deviation (RSD) showed minimal variability both between replicates of each boar (RSD < 3%) and among different boars (RSD 10-15%), indicating intra- and inter-sample reproducibility. Statistical analysis confirmed no significant differences in X:Y proportion among ejaculates (p = 0.995). Discussion & Conclusion This gating strategy provides a reproducible method for the identification and discrimination of X/Y spermatozoa in a single analysis using non-orientation flow cytometers, supporting the development of accessible diagnostic and analytical methods for swine research and production.