*2.2. Animals and Treatment*

Adult male Sprague Dawley rats (*n* = 24) were randomly distributed in t-BHP and control groups and were treated with 300 μmoles tert-BHP/kg b.w. or saline (control) once a day intraperitoneally for 15 days, respectively as done previously [18]. Treatment with tert-BHP showed to have no effects on the health of rats [18,24]. Animals were euthanized at 3, 6, and 9 weeks post-treatment. These end

points correspond to late, middle and early spermatogenesis, respectively [25]. At each given end time, reproductive organs were collected, weighted and kept at −80 ◦C until use. For sperm motility determination, cauda epididymes were cut one time at the base with a surgical blade and placed in phosphate-bu ffered saline (PBS; 1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) at 37 ◦C. Spermatozoa were allowed to swim-out for 10 min and were collected in clean tubes. All procedures with rats (handling, euthanasia, collections of tissues, etc.) were carried out following the regulations of the Canadian Council for Animal Care (CACC) and according the protocol #2009-5656 approved by the Facility Animal Care Committee (FACC) of the Research Institute, McGill University Health Centre.

### *2.3. Testes and Epididymes Homogenates Preparation*

Control and t-BHP treated adult male Sprague-Dawley rats' frozen testis, caput and cauda epididymis were thawed, weighed and homogenized in a glass potter in RIPA bu ffer containing protease inhibitors. The samples were then sonicated for 20 s at 30% amplitude twice with 20 s intervals with a Sonic Vibracell (Sonics and Materials, Inc., Newtown, CT, USA). The samples were centrifuged at 21,000× *g* for 20 min at 4 ◦C. The supernatant was extracted, aliquoted and stored at −80 ◦C.

### *2.4. Sperm Motility and DNA Oxidation Determinations*

Sperm motility was assessed by the same observer (CO) using the Olympus BH-2 microscope at 100 magnification with a thermal plate at 37 ◦C. At least 200 spermatozoa per duplicate were analyzed to determine percentage of total motility in each sample [18]. Sperm DNA oxidation was determined by immunohistochemistry using the anti-8-OHdG antibody as done previously [18]. Briefly, sperm samples were centrifuged at 2000× *g* for 5 min to remove the PBS medium and resuspended in 20 mM phosphate bu ffer (pH 6.0) with 1 mM EDTA for 5 min. Samples were then centrifuged and resuspended in 50 mM Tris-HCl (pH 7.4), 1% SDS and 40 mM dithiothreitol for 30 min. Final centrifugation of 5 min to replace the mixture with PBS was performed. The sperm PBS solution was smeared on Superfrost Plus slides (Fischer Scientific, Ottawa, ON, Canada) and they were fixed with 100% methanol at 20 ◦C for 30 min. Slides were incubated with 5% horse serum for 30 min at room temperature, then washed with PBS-T for 5 min and incubated with anti-8-OHdG antibody (1:100) (SMC-155D, StressMarq Biosciences Inc., Victoria, BC, Canada) diluted in 1% horse serum overnight at 4 ◦C. After a wash with PBS, the samples were incubated with biotinylated horse anti-mouse antibody in 1% BSA and PBS-T for 1 h, washed and finally incubated with Alexa Fluor 555-streptavidin (1:500 in PBS) for 45 min at 20 ◦C. ProLong Gold antifade with DAPI was added and smears sealed. Slides were analyzed with Zeiss Axiophot fluorescence microscopy (Carl Zeiss, Oberkochen, Germany). Two hundred spermatozoa per slide were counted in duplicate. A positive control was done by incubating spermatozoa with 2 mM H2O2 for 1 h at 37 ◦C. The specificity of the antibody was confirmed previously [18].

### *2.5. Testes Histological Analysis, and Sperm Count*

Testes were dissected, weighed, and fixed immediately with Bouin fixative for 24 h before processing and embedding in para ffin blocks, and tissues were sectioned (5 μm) and stained with hematoxylin-eosin as previously described [26]. Spermatozoa heads from testis homogenates were counted in a hematocytometer as previously described [27].
