Sperm acrosome integrity was assessed using a fluorescent-labeled peanut agglutinin (FITC-PNA) (Sigma-Aldrich) as described previously [15 (link)]. Briefly, smears of sperm suspension were fixed in methanol for 15 min, air-dried, and stained with FITC-PNA (60 μg/ml in PBS) in the dark for 30 min. Slides were then washed with Milli-Q water to remove excessive staining. Two hundred spermatozoa were evaluated under Olympus Fluoview 3000 confocal microscope at 600× magnification and classified as intact or damaged acrosome based on the staining since FITC-PNA binds exclusively to the outer acrosomal membrane.
Sperm mitochondrial activity was assessed using a fluorescent dye Rhodamine 123 that is rapidly taken up by functional mitochondria. In brief, 5 μl of Rhodamine 123 (Sigma, U.S.A.) solution (1 mg/ml in DMSO) was added to 1 ml of diluted sperm suspension and incubated for 10 min at 25°C in the dark. Following incubation, the supernatant was removed by centrifugation (300× g, 10 min) and the sperm pellets were resuspended in 1 ml PBS. A drop of 10 μl of the suspension was placed on microscopic slides, covered with coverslips, and examined at 400× magnification under Olympus BX51 fluorescence microscope with Olympus Q5 camera. A total of 200 spermatozoa were examined per animal and expressed as percentage of mitochondrial activity.
Sperm mitochondrial activity was assessed using a fluorescent dye Rhodamine 123 that is rapidly taken up by functional mitochondria. In brief, 5 μl of Rhodamine 123 (Sigma, U.S.A.) solution (1 mg/ml in DMSO) was added to 1 ml of diluted sperm suspension and incubated for 10 min at 25°C in the dark. Following incubation, the supernatant was removed by centrifugation (300× g, 10 min) and the sperm pellets were resuspended in 1 ml PBS. A drop of 10 μl of the suspension was placed on microscopic slides, covered with coverslips, and examined at 400× magnification under Olympus BX51 fluorescence microscope with Olympus Q5 camera. A total of 200 spermatozoa were examined per animal and expressed as percentage of mitochondrial activity.
