120 Use of fixable dyes to analyze equine sperm membrane integrity and acrosome reaction after A23187 treatment

Conventional IVF is not successful in the horse, and current work is focused on factors affecting sperm capacitation in this species. Challenges arise in assessing equine sperm incubated in media containing capacitation promotors, as some of these factors cause sperm head-to-head binding (aggregation). Our preliminary microscopic findings showed that sperm aggregates are largely of viable sperm, whereas nonviable sperm individualize. Thus, data obtained using technologies that analyse only individual cells and gate out aggregates, such as flow cytometry, may not accurately represent the study population. We developed a fixable live/dead/acrosome staining protocol (LD-PSA) that minimizes sperm aggregation. The aim of this study was to evaluate the percentage of viable and acrosome-reacted equine sperm after A23187 treatment, using either LD-PSA or a standard staining protocol (PI-PSA). Sperm from 9 ejaculates were suspended in Hanks’ balanced salt solution (HBSS) and exposed for 10 min at 37°C to vehicle (V) or to 10 µM A23187 (C10). The sperm were washed, resuspended in HBSS medium with added lactate and pyruvate and containing 7 mg mL−1 of bovine serum albumin (BSA), and assessed immediately (0 h) or incubated at 37°C for 2 h (the period needed for equine sperm to respond to A23187). Motility was analysed using computer-assisted semen analysis. Each treatment was stained by PI-PSA: propidium iodide and fluorescein isothiocyanate-Pisum sativum agglutinin (PSA) in DPBS; and by LD-PSA: Live/Dead Fixable Red, paraformaldehyde 2%, Triton × 1%, and FITC-PSA in Dulbecco's phosphate-buffered saline with Accumax (Stem Cell Technologies), a commercial proprietary cell agglutination inhibitor, before flow cytometric analysis. Differences were analysed using repeated-measures two-way ANOVA. The % total motile sperm (TMOT) for V and C10 treatments were 76.3 ± 3.0 and 71.2 ± 4.7 at 0 h (P > 0.05), and 70.5 ± 14.8 and 2.4 ± 0.8 at 2 h (P < 0.05). On flow cytometry, the percentage of events outside the gate for V sperm (0 h and 2 h combined) was 31.9% in PI-PSA and 21.9% in LD-PSA samples (P < 0.01). Measured viability in V samples was significantly lower when stained with PI-PSA than with LD-PSA at 0 h (49.2 ± 4.6 vs. 67.1 ± 4.9) and tended to be lower (P = 0.07) at 2 h (44.0 ± 4.9 vs. 55.1 ± 2.8). Notably, the viability recorded in PI-PSA was 26 percentage points lower than was the TMOT at both 0h and 2h, indicating nonrepresentative results, as nonviable sperm should not be motile. By LD-PSA, this difference was 9 points at 0 h and 15 points at 2 h. Vehicle sperm showed significantly higher AR values in PI-PSA than in LD-PSA at 0 h (30.4 ± 4.0 vs. 17.7 ± 2.4) and 2 h (41.9 ± 4.5 vs. 24.0 ± 1.8), as did C10 sperm at 0 h (28.9 ± 2.7 vs. 18.0 ± 2.5). The lower values for viability than total motility likely reflect agglutination of viable sperm and thus their exclusion from analysis on flow cytometry. The anti-clumping measures employed in the LD-PSA protocol were associated with increased correspondence of measured viability with TMOT. Thus, LD-PSA may offer a more accurate technique to assess viability and acrosome status of equine sperm incubated in capacitating conditions.