Flow cytometry is an important tool to assess semen quality and sperm function, especially after cryopreservation, but the presence of egg yolk particles in many semen diluents can distort the results. The aims of the present study were to investigate the effect of the presence of egg yolk in the semen diluent on the outcome of flow cytometric analysis of dog spermatozoa and to explore the use of Laser Dye Styryl-751 (LDS-751), a permeant DNA stain with an emission wavelength of 712 nm, to exclude egg yolk particles from the analysis of plasma membrane fluidity (Merocyanine 540 and Yo-Pro-1) and intracellular Ca²⁺ (Fluo-3 and PI). Flow cytometric analyses were carried out on a Coulter Epics XL flow cytometer (Beckman-Coulter Inc., Fullerton, CA). Data were analyzed using EXPO32TM ADC software (Beckman-Coulter). A paired t-test was used to test whether gating out egg yolk particles using LDS-751 affected the analysis. Method agreement analysis was undertaken to assess the agreement between the 2 staining protocols (i.e. with or without LDS-751). The proportion of spermatozoa with high plasma membrane fluidity was significantly (P < 0.05) greater after gating out egg yolk particles; the measurements of high membrane fluidity spermatozoa were on average 7.7 percentage points greater than without gating out egg yolk as shown by the method agreement analysis (SD = 9.35). Similarly, the proportion of spermatozoa with high intercellular Ca²⁺ was significantly (P < 0.01) greater after gating out egg yolk particles; the proportions of spermatozoa with high intracellular Ca²⁺ were 5.95 percentage points greater when egg yolk particles were gated out compared with measurements in the presence of egg yolk particles (SD = 5.43). The method agreement shows that both methods show close agreement with and without egg yolk particles (i.e. results mostly fall within the mean of differences between pairs of repeated measurements ±2 SD). Therefore, the 2 methods are measuring the same phenomena. However, the change in measurement values with exclusion of egg yolk particles is significant. Therefore, accuracy is increased; that is, closer to the true value. In conclusion, using MC540/Yo-Pro-1 and Fluo-3/PI staining alone in the presence of egg yolk particles resulted in underestimation of the proportions of high plasma membrane fluidity and high intracellular Ca²⁺ spermatozoa, respectively. LDS-751 could be used with other fluorescent stains that target organelles such as mitochondria and acrosomes with multicolor flow cytometric analysis to exclude egg yolk particles.