Quantifying Sperm Acrosome Integrity

For each data point, a minimum of 500 sperm per straw were assessed using previously described methods (Lieberman et al., 2016 (link)). Briefly, frozen semen straws were thawed in a water bath set at 35 °C for 30 s. Semen was then transferred to a 1.7-mL microcentrifuge tube and incubated at 35 °C for 1 h. Twenty microliters of semen solution was spread on a microscope slides and air-dried for 15 min. The slides were fixed by immersion in absolute methanol for 15 min. Slides were rinsed in PBS baths twice for 5 min each, transferred to a bath containing 25 μg/mL PNA-FITC (Sigma, St. Louise, MO) for 30 min, and rinsed in three phosphate buffered saline baths for 5 min each. Slides were gently dried using compressed air. One drop of Fluoroshield with DAPI Histology Mounting Medium (Sigma, St. Louise, MO) was then applied to each slide and acrosome integrity was measured using fluorescence microscopy.