Eosin nigrosin

Sperm viability and morphology were assessed by one step Eosin-Nigrosin (Sigma Aldrich^®, Seelze, Germany) double stain method (Figure 6) [18 (link),36 ,49 (link)]. Spermatozoa (n = 300) were counted by means of an optical microscope (AXIOSTAR Plus, ZEISS, Germany; magnification ×1000). The recorded live and dead spermatozoa were expressed in a percentage ratio (%). Concerning morphology, the abnormal spermatozoa were classified as follows: (1) abnormal head (detached head, defects in size and shape); (2) midpiece defects (distal midpiece reflex, bowed midpiece, and others); (3) tail defects (bent tail, coiled tail, and others); and (4) presence of proximal cytoplasmic droplets [71 (link)]. If multiple abnormalities were identified on individual spermatozoa, all were recorded; thus, the actual frequency of each abnormality in the population was determined. In accordance with the major or minor impact of morphological abnormalities on male fertility [72 ], the total percentage (%) of abnormal spermatozoa was estimated by counting once those who had more than one abnormality, using the following priority: abnormal head, midpiece, tail, presence of droplets. Spermatozoa (n = 300) were scored at magnification ×1000, and the % ratio was calculated for each category.

After the mice had been sacrificed, we harvested the epididymides and testes immediately. The epididymis was dissected and spermatozoa were released in Krebs–Ringer medium^{75 (link)}. The spermatozoa suspension was collected for counting using the counting chamber. To analyze viability, the spermatozoa were collected for eosin–nigrosin staining (1% eosin and 10% nigrosin in normal saline, [2:1] by volume, Sigma-Aldrich, USA). The spermatozoa suspension was incubated for 15 min in two steps (swim-up and swim-out steps) at 37 °C. To evaluate the motility of the sperm, high-motility spermatozoa were collected from the uppermost 90% (by volume) of the spermatozoa suspension. We recorded videos of the spermatozoa suspensions for OpenCASA analysis, which assessed motility, VSL, VCL, VAP, linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and mean angular displacement (MAD)^{76 (link)}.