Sources of variation in flow cytometric analysis of aquatic species sperm: The effect of cryoprotectants on flow cytometry scatter plots and subsequent population gating

Abstract

The use of fluorescent staining and flow cytometry to assess sperm quality in aquatic species has increased over the past decade, but comparisons among studies are difficult or impossible due to variation in application, analysis, and reporting of protocols and data.The goal of the present study was to determine the effect of exposure to two cryoprotectants commonly used for cryopreservation of sperm from aquatic species on the accuracy of flow cytometric assessment of sperm quality.Membrane integrity of zebrafish (Danio rerio) sperm exposed to 10% and 20%methanol and dimethyl sulfoxide (DMSO)in 300 mOsm kg(-1) Hanks' balanced salt solution (HBSS) or calcium-free HBSSwas determined using SYBR 14/propidium iodide staining. Both cryoprotectants significantly affected forward-scatter and side-scatter characteristics of sperm samples, resulting in significant changes in the number of total and gated events, and in the number and percentage of intact cells. These results indicate that it cannot be assumed that the approach to flow cytometric analysis of fresh sperm will be applicable to cryoprotectant-treated or cryopreserved sperm. In total, we document examples of five potentially interacting factors that produce errors of 5 to 50% each, resulting in underestimates and overestimates of total and intact sperm (actual numbers and percentages) in the presence of the two most commonly used cryoprotectants at the concentrations used most often for cryopreservation of sperm from aquatic species. This study provides methods to reduce or eliminate these errors and recommendations necessary for standardization and reporting.

Figure 1

Forward scatter vs. side scatter (FSC vs. SSC) and FL1 vs. FL3 (SYBR 14 vs. PI) scatter plots of zebrafish sperm exposed to treatments containing HBSS alone, and HBSS containing10% and 20% methanol (MeOH) ordimethyl sulfoxide (DMSO). The region designated as“P1” in the FSC vs. SSC plot is the gated sperm population, and the region designated as “P4” in the SYBR 14 vs. PI plot is the intact sperm population.

Figure 2

Forward scatter vs. side scatter (FSC vs. SSC) and FL1 vs. FL3 (SYBR 14vs. PI) scatter plots of zebrafish sperm exposed to treatments containing Calcium-free HBSS (C-F HBSS) alone, and C-F HBSS containing 10% and 20% methanol (MeOH) ordimethyl sulfoxide (DMSO). The region designated as“P1” in the FSC vs. SSC plot is the gated sperm population, and the region designated as “P4” in the SYBR 14 vs. PI plot is the intact sperm population.

Figure 3

Forward scatter vs. side scatter plots of solution debris in HBSS alone and containing 10% and 20% methanol (MeOH) ordimethyl sulfoxide (DMSO), before (first column) and after (second column) filtration through 0.45 μm syringe filters. Ultrapure water (dH₂O, third column) and C-F HBSS (fourth column) alone and containing 10% and 20% methanol (MeOH) ordimethyl sulfoxide (DMSO) are shown for comparison.