Poster Presentation IXth International Conference on Boar Semen Preservation 2019

Comparison of Hoechst 33342 and SYBR14 for the identification of boar spermatozoa during flow cytometric assessment of membrane and acrosome integrity (#6.5)

Lisa A Herickhoff 1 , Raul A Gonzalez-Castro 1
  1. Membrane Protective Technologies, Inc., Fort Collins, CO, United States

The integrity of the sperm membrane and acrosome are important components of sperm quality and fertilizing potential. Membrane integrity is commonly assessed with propidium iodide (PI) that binds to sperm DNA, but is unable to penetrate the membrane of the sperm cell so long as it remains intact and functional. To improve the accuracy of identification of sperm cells that have not taken up the PI, an additional stain is added which penetrates all cells regardless of their membrane status (eg. SYBR 14 or Hoechst 33342). A variety of fluorescent probes are used, often dependent on laser and filter capabilities of each flow cytometer. Here we compare the results of membrane and acrosome integrity assessment (PI and PNA-Alexa-647, respectively; Invitrogen, Eugene, OR, USA) by flow cytometry when either Hoechst 33342 (Invitrogen) or SYBR 14 (Invitrogen) is used to identify all sperm cells in the population. Cooled semen samples (n = 10 boars, five ejaculates/boar) were prepared with 25, 50, and 75% (v/v) of dead spermatozoa (previously snap frozen) for analysis of membrane and acrosome integrity with PI/PNA/SYBR14 or PI/PNA/Hoechst 33342. Membrane and acrosome-intact spermatozoa were considered as PI and PNA negative. Samples were analysed by flow cytometry (YETI™; Propel Labs, Fort Collins, CO, USA; 20000 events/sample). Unstained and single-stained samples were used to set quadrants, compensation and background fluorescence. Statistical analysis of results was by paired Student's t-test, Pearson’s correlation and Bland and Altman plots. The percentage of membrane and acrosome-intact spermatozoa did not differ when SYBR14 or Hoechst 33342 were used as the positive counterstain, with both methods highly correlated (r=0.86, P<0.0001). We conclude that SYBR14 or Hoechst 33342 are equally accurate in the identification of sperm events during PI/PNA analysis of boar sperm membrane and acrosome integrity.