Axiostar plus optical microscope

Cells were visualized using an Axiostar plus optical microscope (Carl Zeiss, Germany) in a phase contrast mode. Images were captured with the Pixera PRO 150ES camera (Pixera, USA) and analyzed using VideoTesT-Size 5.0 (Akond, Russia). DCF effects on cell morphology and surface relief were examined by a combined atomic-force and confocal laser scanning system consisting of a MFP-3D-BIO™ atomic force microscope (AFM, Asylum Research Inc., USA), and an Olympus Fluo View 1000 confocal laser microscope (CLSM, Olympus Corporation, Japan). To differentiate living and dead cells, the bacterial suspension was stained with LIVE/DEAD^®BacLight™ Bacterial Viability Kit (Invitrogen, USA). The root-mean-square roughness of the cell surface, cell length and width were determined, and the cell volume and surface area were calculated. The images were processed using the Igor Pro 6.22 A (WaveMetrics, USA).

Sperm plasma membrane biochemical functionality was evaluated by the hypo-osmotic swelling test (Figure 7), according to Pelufo et al. [73 (link)]. Briefly, 0.9 mL of hypo-osmotic solution (150 mOsm/L) and 0.1 mL of sperm were incubated at 37 °C for 30 min. After incubation, 20 μL of the sample was spread on a slide. Under light microscopy (AXIOSTAR Plus, Optical microscope, ZEISS, Germany; magnification ×400), 300 spermatozoa were counted and classified as HOST-positive (coiled or swollen tails) and HOST-negative (non-coiled or no-swollen tails). The results were expressed as the percentage (%) of HOST-positive spermatozoa.