All experiments in this research were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Medical and Research Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (Ethic code: IR.SUMS.REC.1399.1183).

CPFX (Tehran Darou Pharmaceutical Company) and CoQ10 (DarmanYab Darou) were utilized in the current study. Both CPFX and CoQ10 were suspended in distilled water and freshly prepared for the experiment.

In the current study, 60 adult male Sprague-Dawley rats, each weighing between 245 and 255 g and aged 8 weeks, were sourced from the laboratory animal center at Shiraz University of Medical Sciences in Shiraz, Iran. These rats were maintained in an environment with a room temperature of 22 to 24 ºC, a 12-hour light/12-hour dark cycle, 50% humidity, standard ventilation, and unrestricted access to water and food.

The rats were given at least 2 weeks to acclimate to their new housing environment before being randomly assigned to one of six groups (n=10). Each day for 45 days, the animals were administered treatments orally through an intragastric tube [10]: (1) Control (1.5 mL distilled water); (2) Low-dose CPFX, 103 mg/kg/day (LD-CPFX) [10]; (3) High-dose CPFX, 206 mg/kg/day (HD-CPFX) [10]; (4) LD-CPFX+CoQ10 (10 mg/kg/day); (5) HD-CPFX+CoQ10; and (6) CoQ10 (10 mg/kg/day) [17].

The dose of 206 mg/kg/day was selected based on the human daily therapeutic dose [21]. At the conclusion of the experimental period, all rats were euthanized using a lethal intraperitoneal injection of ketamine (240 mg/kg) combined with xylazine (15 mg/kg) [22]. A blood sample was immediately collected via cardiac puncture during a laparotomy; additionally, the tail of the epididymis and the testis were quickly excised for sperm analysis and stereological study, respectively.

The blood samples from the rats were centrifuged for 15 minutes at 2,500 rpm, and the serum was separated from the blood cells. The serum samples were then immediately stored at –20 °C until further analysis. The serum testosterone level was measured using a radio-immunoassay with a commercial kit (RIAKIT; Immunotech), following the manufacturer's instructions.

The tail of the epididymis was dissected, removed, cleared of fat, and promptly placed in a small petri dish containing 3 mL of phosphate-buffered saline. It was then incubated at 37 °C for 30 minutes to allow the semen to diffuse. The diluted semen was gently shaken to achieve a uniform suspension of the spermatozoa [23].

In total, 3 mL (3,000 mm³) of diluted semen was taken from the sperm sample collection and spread into the counting chamber between the cover glass and the Neubauer hemocytometer. The chamber was completely filled (each Neubauer counting chamber has a volume of 0.1 mm³). Sperm heads were manually counted using an optical microscope in the central part of the counting chamber. Approximately 150 to 300 sperm heads were counted at a final magnification of ×400 in a 0.1 mm³ volume of the counting chamber. Subsequently, the total number of sperm in the 3 mL (3,000 mm³) volume of diluted semen was calculated. The results were expressed as the total number of sperm per milliliter [23].

The diluted semen was placed on a pre-warmed microscopic slide. Ten randomly selected microscopic fields were examined at a final magnification of ×400, assessing 150 to 300 sperm per rat. Sperm motility was categorized as follows: (1) rapid progressive, where sperm move quickly in a straight line; (2) slow progressive or non-linear progressive, where sperm move forward in a curved or crooked path; (3) non-progressive, where sperm exhibit tail movement but do not move forward; and (4) immotile, where sperm do not move at all. The percentage of motile spermatozoa was calculated by multiplying the number of motile sperm by 100 and then dividing by the total number of sperm [23].

The smears were prepared using diluted sperm samples and were allowed to dry at room temperature. Subsequently, the slides were stained with 1% eosin Y for 5 to 10 minutes. The sperm morphology was categorized as either normal or abnormal. Abnormal morphology included a range of atypical head and tail shapes, such as blunt hooks, banana-shaped heads, amorphous forms, pin-heads, double heads, double tails, small heads, and bent tails. To evaluate the morphology, we examined the smears under a microscope at a final magnification of ×400, selecting 10 random fields and assessing 150 to 300 sperm per rat. Finally, the percentage of sperm with normal or abnormal morphology was calculated using the following formula:

percentage of normal sperm=the number of normal sperm×100/total number of sperm [23].

To evaluate sperm viability, we stained the sperm smears using a combination of eosin Y (CAS No: 15086-03-9; Sigma-Aldrich) and nigrosin dyes (CAS No: 8005-03-6; Sigma-Aldrich). To prepare the eosin and nigrosin solution, we dissolved 0.67 g of eosin Y and 0.9 g of sodium chloride in 100 mL of distilled water. Subsequently, 10 g of nigrosin was added to the mixture. Sperm heads that remained unstained were considered alive, while those that took up the stain were counted as dead. To estimate the percentage of live sperm, we examined 10 randomly selected microscopic fields at a final magnification of ×400, counting between 150 and 300 stained and unstained sperm per rat. The percentage of live sperm was then calculated using the formula: (number of live sperm×100)/total number of live and dead sperm [24].

After weighing the testis, its primary volume "V (testis)" was assessed through immersion in normal saline. To evaluate stereological parameters such as the length of the seminiferous tubules, isotropic uniform random sections were prepared. The testis was then sectioned according to the "orientator method." Briefly, the testis was placed on a circle divided into 10 equal segments. A random number between 0 and 10 was chosen, and the testis was bisected along that axis. Each half of the testis was then positioned in the 0–0 direction of a second circle, which was divided into 10 unequal sinus-weighted segments, and a second cut was made (Figure 1A, 1B). Subsequently, the testis was sectioned into slices parallel to the direction of the second cut, spaced 1 mm apart, yielding 8 to 12 slabs (Figure 1B, 1C). These slabs were preserved in 10% neutral buffered formalin for tissue processing [25,26]. Following this, the slabs were processed, embedded in paraffin blocks, sectioned (4 and 25 µm thickness), and stained with hematoxylin and eosin.

In general, tissue processing results in the shrinkage of the testis. A straightforward method to assess the extent of this shrinkage, referred to as "d(shr)," involves punching a circular sample from the section slabs of the testis using a trocar (Figure 1B).

Subsequently, the areas of the circle before and after tissue processing were calculated. The degree of tissue shrinkage, denoted as "d(shr)," was assessed using the following formula:

d(shr)=1–(AA/AB)^1.5,

where AA and AB were the areas of circular parts of the testis after and before tissue processing, respectively. Consequently, the secondary volume of the testis was estimated by multiplying the primary volume by the degree of shrinkage.

Sections with a thickness of 4 µm are prepared from paraffin blocks using a microtome to estimate the volume of the seminiferous tubules, seminiferous epithelium, and interstitial tissue. The stereological analysis utilizes a microscope (Nikon Eclipse E200; Nikon) equipped with an oil objective lens (Plan Apo 60×, n/a: 1.4) and a Samsung video camera (SCB-2000P; Hanwha Techwin) connected to a computer. Stereological probes are applied to live images, and the aforementioned parameters are assessed using the "point-counting method" facilitated by a stereology software system (Stereolite; Shiraz University of Medical Sciences) [25,26]. The volume densities of various testicular structures (seminiferous tubules, seminiferous epithelium, and interstitial tissue) were also evaluated. Specifically, the parameter “Vv (testis structures/testis sections)” of the testis was evaluated by the point-counting method (Figure 1D, 1E) and the following formula:

V (testis structures)=[∑P (testis structures)/∑P (testis section)]×V (secondary testis volume),

where ∑P (testis structures) is the number of points hitting the seminiferous tubules, seminiferous epithelium, and interstitial tissue and ∑P (testis section) is the number of points hitting the section of the testis.

The length density (LV) of the seminiferous tubules was measured using sections that were 4 µm thick, employing an unbiased counting frame. This frame is defined by the left and bottom lines (referred to as “forbidden lines”), along with their extensions, and the right and top lines referred to as “acceptance lines”). The frame was randomly positioned on the live monitor image of the testis section at a final magnification of ×160. Only the seminiferous tubules that were completely or partially within the counting frame and touched the right and top acceptance lines were counted. Any tubules that touched the forbidden lines were excluded from the count (Figure 1F). The LV of the seminiferous tubules was evaluated by the following formula:

LV (seminiferous tubules/testis section)=2 ∑Q/(∑P×a/f),

where ∑Q is the total number of the seminiferous tubules sampled by the frame and counted, ∑P is the total number of counting frame-associated points hitting the testis section, and (a/f) is the area of the counting frame. The total length of the seminiferous tubules was assessed by multiplying the LV by the secondary volume of the testis [25,26].

The optical disector is a stereological technique used for counting cells in thick histological sections (25 μm). This method was employed to assess the numerical density (Nv) and to evaluate the total number of spermatogenic, Sertoli, and Leydig cells in the testis. Following the principles of systematic uniform random sampling, microscopic fields were selected by moving the stage uniformly in the x and y directions at consistent intervals.

An optical disector consists of a microscope supplied with a 40× oil immersion objective lens (Nikon Plan Fluor 40× oil immersion microscope objective with 1.3 NA) with a high numerical aperture. It is also fitted with a microcator (Heidenhain MT12 243602-01; Nd 280, ID 636280-01; Heidenhain), which is connected to a video camera. This setup transmits live microscopic images to a computer monitor. Additionally, the microcator assesses the movement in the Z-direction with a precision of 0.5 μm [25,26]. One of the stereological probes used is an unbiased counting frame, which is employed to count the number of optic cells. This counting frame is placed over the live image (Figure 1G). Guard zones are located at the upper and lower parts of the histological section surfaces. These zones help prevent the introduction of tissue artifacts that can occur during the processing of these sections.

Cells located within the guard spaces were not included in the count. The term “height of disector” refers to the distance between the upper and lower guard spaces. All cells were identified, and each cell that appeared in high focus within the subsequent focal sampling frame, and was either wholly or partially within the counting frame without touching the exclusion lines, was counted (Figure 1G).

The Nv of the mentioned cells was assessed using the following formula:

Nv (cells/tissue testis)=ΣQ–/[Σp×(a/f)×h],

where ΣQ- is the number of the cells coming into the height of the disector, ΣP is the total counting frames in the whole microscopic fields, h is the height of the disector, and (a/f) is the area of the frame. The total number of the cells was assessed by multiplying the Nv by V (secondary volume of the testis) [25,26].

GraphPad Prism ver. 8.0.0 for Windows (GraphPad Software) was used to analyze the data. Sperm quality (number, motility, normal morphology, and viability) and stereological parameters (volume, number of cells, and tubule lengths) were compared using one-way analysis of variance and the Tukey post hoc tests. A p-value less than 0.05 was considered statistically significant. The data are presented as mean±standard deviation.

As shown in Table 1, there was a significant decrease in sperm count, the percentage of motile spermatozoa, sperm with normal morphology, and sperm viability in the animals treated with LD-CPFX and HD-CPFX (p=0.0001 for all parameters in both groups) compared to the control group. However, the sperm parameters in the rats treated with LD-CPFX+CoQ10 (p=0.01, p=0.01, p=0.03, and p=0.003, respectively) and HD-CPFX+CoQ10 (p=0.05, p=0.004, p=0.04, and p=0.01, respectively) showed improvement compared to their respective CPFX-treated groups (Table 1).

The serum testosterone levels were lower in the LD-CPFX and HD-CPFX administered groups compared to the control group (p=0.0003 and p=0.0001, respectively). Additionally, testosterone levels increased in the rats treated with LD-CPFX+CoQ10 and HD-CPFX+CoQ10 compared to the corresponding CPFX-treated groups (p=0.05 and p=0.01, respectively) (Table 2).

The results showed a negligible change in testis volume in the LD-CPFX or HD-CPFX groups, with or without CoQ10 (Figure 2).

The volume of the seminiferous tubules decreased by 10% and 14% in rats treated with LD-CPFX and HD-CPFX, respectively, compared to the control groups (p=0.01 and p=0.006, respectively). However, administration of CoQ10 significantly restored the seminiferous tubule volume in the CPFX-treated animals compared to the CPFX-only groups (p=0.04 and p=0.01, respectively) (Figure 2).

The total volume of the seminiferous tubule epithelium decreased by 35% and 36% in the rats that received LD-CPFX and HD-CPFX, respectively, compared to the control rats (p=0.0001 and p=0.001, respectively). However, these levels improved after the rats were treated with CoQ10, compared to the CPFX-treated groups (p=0.007 and p=0.02, respectively) (Figure 3).

The volume of interstitial connective tissue increased by 25% and 28% in the rats that received LD-CPFX and HD-CPFX, respectively, compared to the control rats (p=0.03 and p=0.008, respectively). Additionally, CoQ10 mitigated the effects on interstitial connective tissue in the animals treated with LD-CPFX and HD-CPFX (p=0.01 and p=0.07, respectively) (Figure 2).

The results showed a 19% and 31% reduction in the length of the seminiferous tubules in animals treated with LD-CPFX and HD-CPFX, respectively, compared to matched controls (p=0.006 and p=0.0001, respectively). Furthermore, the length of the seminiferous tubules recovered in rats that received LD-CPFX+CoQ10 and HD-CPFX+CoQ10, compared to the CPFX-treated group (p=0.02 and p=0.0002, respectively) (Figure 3).

The numbers of spermatogonia (52%, 55%), spermatocytes (41%, 48%), round spermatids (36%, 40%), long spermatids (43%, 38%), Sertoli cells (42%, 48%), and Leydig cells (68%, 75%) were decreased in the LD-CPFX (p=0.0007, p=0.0001, p=0.04, p=0.0001, p=0.01, and p=0.0001, respectively) and HD-CPFX (p=0.003, p=0.0001, p=0.01, p=0.0001, p=0.002, and p=0.0001, respectively) groups compared to the matched control group. The number of cells increased significantly in animals treated with LD-CPFX+CoQ10 (p=0.01, p=0.0009, p=0.02, p=0.01, p=0.01, and p=0.0001, respectively) and HD-CPFX (p=0.03, p=0.002, p=0.04, p=0.0009, p=0.01, and p=0.01, respectively). These increases were observed when compared to the corresponding LD-CPFX and HD-CPFX groups (Figures 4 and 5).

Histological assessment revealed the normal features of seminiferous tubules, including all spermatogenic cell lineages, as well as Sertoli and Leydig cells (Figure 6). In the LD-CPFX and HD-CPFX groups, destruction of the seminiferous tubule epithelium was observed, and the number of spermatogenic cells was reduced. This qualitative evaluation confirms the stereological data. A decrease in Leydig cells was also observed in the interstitial tissues. The degenerative impact of both doses of CPFX was reversed by CoQ10 administration, as the density of spermatogenic and Leydig cells in the testis was restored to levels observed in the control group (Figure 6).