Introduction
Infertility is a global health issue that affects both men and women of reproductive age. It remains one of the major challenges in modern medicine. Male infertility, which accounts for approximately half of all infertility cases, is a disorder of the reproductive system. Male infertility may result from endocrine disorders, infections, physical damage, exposure to toxic substances, medication side effects, or idiopathic causes [1]. Additional contributing factors include sleep deprivation, environmental pollution, elevated scrotal temperature, and increased levels of reactive oxygen species (ROS) [2]. These factors negatively impact sperm quality, and many cases of male infertility are linked to sperm disorders [1]. Certain therapeutic drugs may impair spermatogenesis, as any agent that damages spermatogonia, Sertoli cells, or Leydig cells can disrupt this process and adversely affect fertility [3]. Quinolones—a group of antibiotics—are commonly prescribed by specialists to treat urogenital infections, particularly when high leukocyte counts are present in semen or as a precaution before in vitro fertilization procedures [4]. Prolonged quinolone use has been shown to impair spermatogenesis, compromise sperm quality, and alter hormone levels in experimental animal models [5,6]. Levofloxacin (LVFX), a member of the quinolone class, is used to treat urogenital tract infections. Numerous studies have shown that LVFX treatment alters rat testicular function and structure, affecting sperm quality, male sex hormone levels, spermatogenesis, and the morphology of Leydig and Sertoli cells [4,6,7]. Previous research indicates that LVFX induces the production of ROS, leading to oxidative stress and subsequent damage to testicular tissue. This oxidative stress is a key factor in the observed changes in sperm quality, hormone levels, and spermatogenic cells in rats [4,6]. When ROS accumulate in the testes, disrupting the balance between antioxidants and free radicals, oxidative stress ensues and damages testicular tissue [2]. LVFX-induced oxidative stress and a reduction in testicular antioxidants contribute to testicular damage [7]. Coenzyme Q10 (CoQ10) is an endogenous antioxidant present in all cells that protects against free radical damage, thereby preventing cell necrosis and apoptosis [8]. Previous studies suggest that CoQ10 improves sperm parameters and safeguards testicular tissue, significantly reducing infertility in male rats [9-14].
Although many effects of LVFX on rat testicular function have been documented, the quantitative evaluation of LVFX-induced structural changes in the testis remains underexplored. Thus, this study aimed to assess the effects of LVFX on sperm parameters, hormone levels, malondialdehyde (MDA) levels, and histological characteristics of the rat testis using stereological methods, as well as to investigate the potential protective effects of CoQ10 on these parameters.
Methods
1. Ethical approval
All experiments in this study 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.AEC.1401.024).
2. Chemicals
LVFX (LOGHMAN Pharmaceutical & Hygienic Company) and CoQ10 (JALINOUS Pharmaceutical Company) were used in this study. Both compounds were suspended in distilled water, and a fresh suspension was prepared for each experiment.
3. Animals
Eighty adult male Sprague-Dawley rats weighing 250 to 300 g were obtained from the Laboratory Animal Center of Shiraz University of Medical Sciences, Shiraz, Iran. The animals were housed in a controlled environment maintained at 22 to 25 °C, with a 12-hour light/12-hour dark cycle, 50% relative humidity, adequate ventilation, and free access to food and water.
4. Experimental design
After a 2-week acclimatization period, the animals were randomly assigned into eight groups (n=10). They received daily gavage treatments for 28 days. The LVFX doses were determined by a pharmacologist based on the latest human-to-animal dose conversion formulas for rats and relevant literature [15,16]. The groups were as follows: (1) control (1.5 mL of distilled water); (2) low dose LVFX, 25 mg/kg/day (LD-LVFX); (3) medium dose LVFX, 50 mg/kg/day (MD-LVFX); (4) high dose LVFX, 100 mg/kg/day (HD-LVFX); (5) LD-LVFX+Co Q10; (6) MD-LVFX+Co Q10; (7) HD-LVFX+Co Q10; and (8) CoQ10 (10 mg/kg/day) [9].
After the treatment period, all animals were euthanized via intraperitoneal injection of a lethal dose of ketamine (240 mg/kg) combined with xylazine (15 mg/kg) [17]. Blood serum and the left testis were collected for serum testosterone measurement and biochemical analysis, respectively. The tail of the epididymis and the right testis were harvested for semen analysis and stereological evaluations, respectively.
5. Measurement of serum testosterone levels
Blood samples were centrifuged at 2,500 rpm for 15 minutes to separate the serum, which was then stored at −20 °C until further analysis. Serum testosterone levels were measured using a radioimmunoassay with a commercial kit (RIAKIT; IMMUNOTECH), following the manufacturer's instructions.
6. Measurement of oxidative stress marker in the testis (MDA)
A 100 mg sample of testicular tissue was weighed, finely chopped, and homogenized in cold phosphate buffered saline (PBS) (pH 7.4) to assess MDA levels, a biomarker of oxidative stress. The homogenate was centrifuged at 4,000 rpm for 15 minutes at 4 °C in the presence of 1 mM ethylene diamine tetra acetic acid. The supernatant was collected for MDA analysis using the thiobarbituric acid reactive substances method. In this assay, MDA, which is a byproduct of lipid peroxidation from oxidized unsaturated fatty acids, reacts with thiobarbituric acid to form a pink chromogen measurable at 532 to 535 nm [18].
8. Sperm analysis
2) Sperm motility
A pre-warmed microscope slide was used to assess sperm motility. Ten random microscopic fields at a final magnification of 400× were examined, evaluating an average of 150 to 300 sperm per animal. Sperm motility was classified into rapid progressive, slow progressive, non-progressive, and immotile categories based on previous studies. The percentage of motile sperm was calculated as (number of motile sperm×100)/total sperm count [19,20].
3) Sperm morphology
Semen was spread on a microscope slide and allowed to air-dry at room temperature before being stained with eosin Y. Sperm morphology was classified as normal or abnormal according to previous research. Normal sperm were defined by a falciform-shaped head and an elongated tail, whereas abnormal sperm exhibited head or tail defects. Ten random microscopic fields at a final magnification of 400× were evaluated, with an average of 150 to 300 sperm per animal. The percentage of normal or abnormal sperm was determined using the formula: (number of normal or abnormal sperm×100)/total sperm count [19,20].
4) Sperm viability
Semen was spread on a microscope slide, allowed to air-dry at room temperature, and then stained with eosin and nigrosin dyes (Sigma Aldrich). To prepare the staining solution, 0.67 g of eosin Y and 0.9 g of sodium chloride were dissolved in 100 mL of distilled water, and 10 g of nigrosin was added. Unstained sperm heads were considered viable, whereas stained sperm heads were classified as non-viable. Ten random microscopic fields at a final magnification of 400× were examined, with an average of 150 to 300 sperm evaluated per animal. Sperm viability was calculated as (number of live sperm×100)/total sperm count (live plus dead) [19,20].
9. Testis tissue collection and preparation
The right testicular weight and primary volume (V(testis)) were measured using a scale with 0.01 g precision and by immersion in normal saline, respectively. Uniform isotropic random sections of the testis were prepared using the orientator method as described in previous studies, as illustrated in Figure 1. A total of 8–12 slabs per testis were collected for histological processing, which included paraffin embedding, sectioning (at 4 and 25 µm thickness), and hematoxylin and eosin staining.
To determine the degree of tissue shrinkage (d(shr)), a circular section of the testis was obtained using a trocar. The areas of the circular sections after tissue processing (AA) and before processing (AB) were measured. The degree of shrinkage was calculated using the formula:
10. Estimation of the seminiferous tubules, epithelium, and interstitial tissue volumes
A stereological system comprising a Nikon E-200 microscope with a Plan Apo 60× oil objective lens (NA: 1.4) and a Samsung video camera (SCB-2000P; Hanwha Techwin) connected to a computer was used. Stereological probes were applied to live images of 4 µm-thick sections, and volume densities (Vv) of the seminiferous tubules, seminiferous epithelium, and interstitial tissue were evaluated using the point-counting technique with a stereology software system (Stereolite; SUMS). Volume density was calculated using the formula:
where ∑P(structures) is the number of points hitting the specific structure (seminiferous tubules, seminiferous epithelium, or interstitial tissue) and ∑P(total) is the total number of points hitting the entire testis tissue. The total volume of these structures was determined by multiplying their volume densities by the secondary testis volume [21,22].
11. Estimation of the length of the seminiferous tubules
Furthermore, 4 µm-thick sections and an unbiased counting frame were used to estimate the length density (LV) of the seminiferous tubules. Each counting frame contained two exclusion lines (on the left and lower sides and their extensions) and two inclusion lines (on the right and upper sides). Seminiferous tubules that were entirely or partially within the frame and did not touch the exclusion lines were counted. LV was calculated using the formula:
12. Estimation of the diameter of the seminiferous tubules
The diameter was measured on tubules sampled using the unbiased counting frame. The maximum diameter perpendicular to the longest axis, approximating the tubule’s center, was recorded as the tubule diameter.
13. Stereological estimation of the cell number
The optical dissector technique was applied to thick histological sections (25 µm) to evaluate the numerical density (Nv) and total number of spermatogenic, Sertoli, and Leydig cells. The optical dissector system comprised a Nikon Eclipse E200 Microscope (Nikon) with a 40× oil immersion objective (Nikon Plan Fluor 40×, NA 1.3) and a Heidenhain MT12 microcator (243602-01; Heidenhain, ND 280, ID 636280-01) connected to a video camera. The unbiased counting frame was superimposed on the live image. Guard zones were established at the upper and lower surfaces of the histological section to avoid tissue-processing artifacts, and cells within these zones were excluded. The distance between the guard zones defined the dissector height.
Each testicular cell was identified and examined in focus within the sampling frame, ensuring it was entirely or partially within the frame without touching the exclusion lines. The Nv of spermatogenic, Sertoli, and Leydig cells was calculated using the formula:
where ΣQ– represents the number of cells that come into focus within the dissector height, ΣP is the total number of frames analyzed, h is the dissector height, and a/f is the area of the counting frame. The total number of each cell type was obtained by multiplying the numerical density by the secondary testis volume.
14. Statistical analysis
Data were analyzed using GraphPad Prism software ver. 8.0.0 for Windows (GraphPad Software). Sperm quality parameters (count, motility, normal morphology, and viability) and stereological parameters (volume, cell number, and tubule length) were compared using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant.
Results
1. Sperm count, motility, morphology, and viability
As shown in Table 1, rats treated with LD-LVFX exhibited significant reductions in sperm count and motility (p=0.01 and p=0.02, respectively), and similar reductions were observed in the MD-LVFX group (p=0.0009 and p=0.0013, respectively) compared to the control group.
The parameters improved significantly in animals treated with LD-LVFX+CoQ10 (p=0.02 and p=0.04, respectively) and MD-LVFX+CoQ10 (p=0.018 and p=0.0019, respectively) when compared to their corresponding LVFX-treated groups (Table 1). The HD-LVFX group showed significant decreases in sperm count, motility, normal morphology, and viability relative to controls (p=0.0002, p<0.0001, p<0.0013, and p=0.009, respectively). However, in HD-LVFX rats receiving CoQ10, these parameters were significantly improved compared to the HD-LVFX group (p<0.03, p<0.0003, p<0.04, and p<0.04, respectively) (Table 1). The results revealed negligible changes in normal morphology and viability in the LD-LVFX and MD-LVFX groups regardless of CoQ10 administration (Table 1).
2. Serum testosterone levels
Serum testosterone levels were significantly reduced in the MD-LVFX and HD-LVFX groups compared to the control group (p=0.0002 and p=0.0001, respectively). In addition, testosterone levels increased significantly in rats treated with MD-LVFX+CoQ10 and HD-LVFX+CoQ10 compared to the corresponding LVFX-treated groups (p=0.0006 and p=0.006, respectively) (Table 2).
3. Malondialdehyde level
MDA levels were significantly elevated in the MD-LVFX and HD-LVFX groups relative to the control group (p=0.03 and p=0.009, respectively). Furthermore, rats treated with MD-LVFX+CoQ10 and HD-LVFX+CoQ10 showed significant improvements in MDA levels compared to the LVFX-only groups (p=0.0015 and p=0.001, respectively) (Table 3).
4. Volume of the testis
Testis volume showed negligible changes across the groups receiving LD-LVFX, MD-LVFX, and HD-LVFX, with or without CoQ10 (Figure 1).
5. Volumes of the seminiferous tubules
The seminiferous tubule volume decreased by 35% in the MD-LVFX and 37% in the HD-LVFX treated groups compared to the control groups (p=0.0028 and p=0.0013, respectively). However, CoQ10 supplementation significantly improved tubule volumes in MD-LVFX and HD-LVFX treated rats compared to the LVFX-only groups (p=0.006 and p=0.0023, respectively) (Figure 2).
6. Volumes of the seminiferous epithelium
The volume of the seminiferous epithelium decreased by 39% and 56% in the MD-LVFX and HD-LVFX treated groups, respectively, compared to controls (p=0.02 and p=0.0003, respectively). CoQ10 supplementation significantly restored epithelial volumes in MD-LVFX and HD-LVFX treated rats compared to the LVFX-only groups (p=0.017 and p=0.0093, respectively) (Figure 2).
7. Volumes of the interstitial connective tissue
The interstitial tissue volume increased by 44%, 48%, and 53% in the LD-LVFX, MD-LVFX, and HD-LVFX groups, respectively, compared to control animals (p=0.0169, p=0.0026, and p=0.0001, respectively). Additionally, CoQ10 supplementation significantly improved interstitial tissue volumes in rats treated with LD-LVFX, MD-LVFX, and HD-LVFX (p=0.0044, p=0.0238, and p=0.0039, respectively) (Figure 2).
8. Length of the seminiferous tubules
The length of the seminiferous tubules decreased by 25% in the MD-LVFX group and 32% in the HD-LVFX group compared to controls (p=0.0042 and p=0.0001, respectively). CoQ10 supplementation significantly improved tubule length in rats treated with MD-LVFX and HD-LVFX (p=0.0027 and p=0.0005, respectively) (Figure 3).
9. Diameter of the seminiferous tubules
The tubule diameter increased by 6% in the MD-LVFX group and by 8% in the HD-LVFX group relative to controls (p=0.0357 and p=0.002, respectively). CoQ10 supplementation significantly restored tubule diameter in rats treated with MD-LVFX and HD-LVFX (p=0.0129 and p=0.0091, respectively) (Figure 3).
10. Cell number
The numbers of spermatogonia (reduced by 24% and 40%), spermatocytes (29% and 33%), round spermatids (51% and 50%), long spermatids (32% and 39%), Sertoli cells (40% and 74%), and Leydig cells (51% and 76%) were significantly decreased in the MD-LVFX (p=0.0149, p=0.0019, p=0.0025, p=0.0055, p=0.0011, and p=0.0011, respectively) and HD-LVFX groups (p=0.0004, p=0.0003, p=0.0025, p=0.0003, p=0.0001, and p=0.0001, respectively) compared to the control group.
Conversely, cell numbers increased significantly in animals treated with MD-LVFX+CoQ10 (p=0.0293, p=0.0379, p=0.0004, p=0.0386, p=0.02966, and p=0.0306, respectively) and HD-LVFX+CoQ10 (p=0.0023, p=0.0089, p=0.0011, p=0.0085, p=0.0054, and p=0.0384, respectively). In contrast, stereological parameters showed negligible changes in the LD-LVFX groups, irrespective of CoQ10 supplementation.
11. Qualitative changes
Figure 6 illustrates the qualitative assessment of the testis. Histological analysis of rats treated with HD-LVFX revealed structural alterations, including a reduced number of seminiferous tubules and evidence of atrophy. However, co-administration of CoQ10 with HD-LVFX alleviated these adverse effects.
Discussion
The present study demonstrates, for the first time, the beneficial effects of CoQ10 on stereological parameters in the rat testis following administration of various LVFX doses. The protective effect of CoQ10 was evident against the adverse outcomes observed with both low and high doses of LVFX, including diminished semen quality, lowered serum testosterone, elevated MDA levels, and alterations in testicular stereological parameters. A key strength of this study is the use of unbiased stereological methods, which provide accurate estimates and are less biased than alternative techniques [23]. The results indicate that treatment with LVFX for 28 days causes structural and functional alterations in rat testes, with high doses producing the most pronounced disruption.
The first part of the study revealed significant decreases in sperm count, motility, normal morphology, viability, and serum testosterone levels in the HD-LVFX group. Zaki et al. [6] reported similar decreases in sperm parameters in animals exposed to 40 mg/kg body weight (BW) of LVFX for 2 months. Likewise, El-Demerdash et al. [4] found that daily administration of LVFX (300 mg/kg BW) for 2 weeks reduced sperm quality and serum testosterone, whereas Ahmadi et al. [7] reported no significant changes in serum testosterone with lower LVFX doses (0.03, 0.06, and 0.08 mg/kg BW) over 60 days.
The second part of the study indicated that LVFX treatment significantly increased testicular MDA levels, consistent with the findings of El-Demerdash et al. [4], who attributed this to increased lipid peroxidation in the testes.
The third part of the study demonstrated significant reductions in the volumes of the seminiferous tubules and epithelium, along with a decrease in tubule length; conversely, tubule diameter and interstitial tissue volume increased significantly. These findings align with the qualitative histological observations by Zaki et al. [6], which noted that LVFX induces dilation, damage, edema, and congestion in seminiferous tubules and interstitial tissue.
The fourth part of the study revealed a significant reduction in the numbers of spermatogenic, Sertoli, and Leydig cells following LVFX treatment, corroborating earlier reports that LVFX alters germinal epithelium cell numbers [6,7].
The fifth part of the study showed that CoQ10 markedly mitigated LVFX-induced alterations in stereological parameters of the semen and testis. This improvement, which implies that CoQ10 is an acceptable treatment for testicular damage, is likely due to the antioxidant properties of CoQ10, as evidenced by reduced MDA levels in CoQ10-treated rats. Previous studies have shown that CoQ10 enhances sperm count, motility, viability, and serum testosterone levels [9-11,14]. Recent reports by Arafa et al. [14], Behairy et al. [24], Akbel et al. [12], Eid et al. [13], and Iftikhar et al. [10] further indicate that CoQ10 prevents testicular tissue alterations in rats exposed to methotrexate, cadmium chloride, cyclophosphamide, and bisphenol A. Based on our findings, CoQ10 prevented histological changes in the seminiferous tubules, interstitial tissue, and cell numbers in LVFX-treated rat testes, most likely due to its ability to mitigate oxidative stress.
These results are consistent with those reported by other researchers, who showed that CoQ10 could mitigate the unfavorable changes induced in rat testis tissue after exposure to free radicals by inhibiting oxidative stress [10-14].
The current study confirmed that treating rats with MD-LVFX (50 mg/kg/day) and HD-LVFX (100 mg/kg/day) for 4 weeks induced testicular toxicity. The reduction in sperm parameters appears to result from a decline in the germinal epithelium volume following LVFX exposure. The rat testis is composed primarily of seminiferous tubules separated by interstitial tissue containing blood vessels and Leydig cells. Damage to the seminiferous tubules leads to reductions in their length, diameter, and volume, while degeneration of the seminiferous epithelium reduces spermatogenic and Sertoli cell numbers—a finding consistent with reports by Ahmadi et al. [7] and Zaki et al. [6]. Sertoli cells support spermatogenic cells and facilitate spermatogenesis; thus, their reduction likely contributes to the decline in spermatogenic cell numbers. Furthermore, an increase in connective tissue may explain the observed reduction in Leydig cells, as LVFX has been shown to cause Leydig cell damage and interstitial edema [6,7]. Oxidative stress, evidenced by elevated MDA levels, may also contribute to these effects [10-14].
In conclusion, LVFX treatment disrupts testicular tissue and function in rats, whereas CoQ10 ameliorates LVFX-induced impairments in sperm parameters, serum testosterone, MDA levels, and testicular histology. Therefore, CoQ10 may be a suitable dietary supplement to prevent testicular disorders and reduce fertility problems associated with LVFX treatment in animals. Further preclinical and clinical studies in humans are needed to determine whether CoQ10 supplementation can protect testicular tissue in patients receiving LVFX.
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