Morphological analysis

The histological alterations induced by canine spontaneous CO condition were used to compare for resemblance with the histological pattern of human CO development.

The evaluation was performed by fixing testicular tissue samples in Karnovsky solution for 24 h, postfixed in 1% OsO₄, dehydrated in gradual ethanol solutions and embedded in EPON 812 (Ted Pella, INC. CA, USA). Semi-thin sections of 1 μm thickness were cut using an Ultracut UCT ultramicrotome (Leica, Vienna, Austria) and stained with 0.5% toluidine blue. Histological analysis of the seminiferous tubules was performed using a light microscope (BX 51, Olympus, Tokyo, Japan). From each tissue sample, a slide with 5 tissue sections was obtained. Therefore, 5 cross-sectioned tubules were evaluated, resulting in a total of 15 tubules per animal. The area of the seminiferous epithelium was determined by subtracting the internal area from the external area. The cellular area of at least 15 Leydig cells per animal was also determined using an image analysis system (Image-Pro Plus 6.2. Media Cybernetics, INC. MD, USA). Leydig cells were evaluated using the 100x objective lens and identified by their central location in the interstitial space, its polygonal shape of round nucleus, with an evident nucleolus. The maturation index (MI) or Johnsen index (31) and the histopathological index (HI) were determined (32).

To confirm the presence of gonocytes and cellular alterations, the testicular tissue was sectioned at 60–70 nm thickness. Sections were counterstained with uranyl acetate and lead citrate and analyzed with an electron microscope (JEM-1011, JEOL, Osaka, Japan). The presence of gonocytes was determined through their ultramorphological characteristics, among which are large round cells with the presence of cytoplasmic extensions in some cases, prominent nucleus and nucleolus, and mitochondria peripheral to the nucleus.