2.3.4. Sperm motility, viability and morphology
Sperm motility analysis was performed according to Perobelli et al.
[28] and Favareto et al. [29]. Sperm were obtained from the right vas
deferens and diluted in 1 ml of modified HTF (Human Tubular Fluid,
Irvine Scientific™) pre-warmed at 34 °C. A 10 μl aliquot was placed in
a Makler chamber (Irvine) and analyzed under a phase-contrast microscope
(OSM-223287, Olympus) at 100× magnification. One hundred
sperms were evaluated per animal and were classified as motile or
immotile [28].
For sperm viability, the one step eosin–nigrosin staining technique
was used [30]. 50 μl of spermatozoa in HTF was mixed with eosin–
nigrosin and directly examined, and 100 sperms per animal were
evaluated and classified as viable or non-viable.
The evaluation of sperm morphology was performed according to
Fernandes et al. [27]. Sperm were recovered from the left vas deferens
by flushing with 1 ml of formol-saline (10%) and smears were prepared
on histological slides that were left to dry for 90 min. It was analyzed
200 spermatozoa per animal in a phase-contrast microscope (400×