In this study, we obtained a total of 50 adult male mice from the Naval Medical Research Institute (NMRI). Each mouse had a body weight ranging from 25 to 30 g and was maintained under standard laboratory conditions. The Ethics Committee at Shahid Beheshti University of Medical Sciences (IR.SBMU.AEC.1402.12) conducted a thorough review and granted approval for the research protocol.

To induce hyperthermia in mice, we exposed them to a temperature of 43 °C for 20 minutes on alternating days over a period of 5 weeks. The mice were anesthetized with intraperitoneal injections of ketamine (100 mg/kg) and xylazine (5 mg/kg). We immersed their lower body, including the scrotum and hind legs, in a water bath. Subsequently, the mice were dried and returned to their cages. In the control group, the animals were anesthetized but maintained at room temperature for the duration of the experiment.

In the group subjected to hyperthermia, animals received 3 µg of vitamin B12 and 10 mL/kg of vitamin C based on their body weight. As both vitamins are water-soluble, they did not require a solvent for administration. The injections were administered intraperitoneally over a period of 35 days.

The animals were divided into four groups: (sham) control, hyperthermia, and two treatment groups. In the sham group (control), the animals were placed in water at a temperature of 35 °C five times and were injected intraperitoneally with saline for 35 days, with a 48-hour interval between each session, for a duration of 20 minutes each time. The same procedure was performed in the hyperthermia and treatment groups, but at a temperature of 43 °C. No additional interventions were performed in the hyperthermia group. In the group treated with vitamin B12, 3 µg of vitamin B12 was injected intraperitoneally after inducing hyperthermia. In the group treated with vitamin C, 10 mL/kg of vitamin C was injected intraperitoneally for 35 days following hyperthermia induction.

To collect sperm samples post-treatment, the tails of the epididymides were scraped and immersed in 1 mL of Ham's F-10 medium (Sigma-Aldrich Product No. N6635). This was followed by a 20-minute incubation at 37 °C. Subsequently, a 10 μL aliquot of the sample was placed on a microscope slide and sperm motility was assessed using an inverted microscope. In this study, approximately 100 sperm were counted in each chamber. The evaluation of sperm motility was based on the criteria set forth by the World Health Organization (1999 to 2010), which include (1) progressive motility, (2) non-progressive motility, and (3) immotility. A counting chamber was used to measure the sperm count [15,16].

After the conclusion of the experiment, blood samples were collected from the animals' hearts while they were deeply anesthetized to assess hormone levels. These samples were subsequently centrifuged at 6,000 ×g for 5 minutes at 4 °C and preserved at −80 °C for future analysis. To determine the serum testosterone levels in the blood, a mouse-specific enzyme-linked immunosorbent assay kit (catalog no. CSB-E11162r) was utilized.

After the experimental period ended and the animals were euthanized, their testicles were extracted and preserved in Bouin's fixative solution. Once the samples were adequately fixed, they underwent dehydration and embedding in a tissue processor. Following embedding in paraffin, sections of 5 and 10 microns were prepared from the samples and readied for hematoxylin and eosin staining.

The Cavalieri method was employed in the stereological analysis to determine the volume of testicular tissue. The calculation was performed using the formula [17,18]:

ΣP is the total number of points hitting the testis sections, a/p indicates the area associated with each sample point, and t represents the distance between tissue sections.

To quantify the total number of testicular cells, encompassing spermatogonia, primary spermatocytes, spermatids, as well as Sertoli and Leydig cells, researchers employ the optical dissector method. The cell count is determined using the formula provided below [17,18].

In the formula, ΣQ represents the overall count of cells, h denotes the thickness of the tissue under consideration, a/f represents the area per counting frame, and ΣP signifies the cumulative count of all the frames used for counting in all fields. Additionally, h corresponds to the height of the disector, t represents the actual section thickness measured using a microcator, and BA is the thickness of the tissue section.

This technique is capable of detecting apoptotic cells by analyzing DNA fragmentation in cell and tissue suspensions. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit was used to assess the level of apoptosis according to the provided instructions. After slide preparation, the samples were examined under a fluorescence microscope for counting. The number of TUNEL-positive cells across different groups was then compared.

The testicular cells were first separated using ethylene-diamine-tetraacetic acid (EDTA)/trypsin. Following this, the samples were centrifuged at 1,200 rpm and 4 °C for 5 minutes using phosphate-buffered saline. Afterward, 100 μL of dichlorodihydrofluorescein diacetate with a concentration of 20 μM was added to the sample and incubated in the dark at 37 °C for 45 minutes. Finally, the samples were analyzed using a spectrofluorometer with a wavelength of 488 nm [19].

To determine glutathione (GSH) levels, the spectrofluorometric method is employed. A suspension of testicular cells (0.5 mL) was stained with O-phthalaldehyde (OPA) and N-ethyl-maleimide (NEM). The cells were then centrifuged at 1,000 rpm for 1 minute, and the resulting cell sediment was dissolved in 2 mL of fresh culture medium. Subsequently, the cells were washed twice to eliminate the fluorescent dye from the medium. To measure each sample, a Shimadzu RF 5,000 U fluorescence spectrophotometer was utilized, with an excitation wavelength of 495 nm and an emission wavelength of 530 nm [19].

This method involved observing polymerase chain reaction (PCR) in real-time by measuring the amount of fluorescence emitted and recording it in a detector. We used this method to semi-quantitatively study the expression of c-kit, proliferating cell nuclear antigen (Pcna), and stimulated by retinoic acid gene 8 (Stra8) genes.

First, we extracted whole RNA samples and added DNase to eliminate any remaining genomic DNA. Then, following the instructions provided with the kit, we synthesized cDNA in a 20-μL volume at a temperature of 42 °C for 60 minutes. We used SYBR Green Ι dye for labeling. This dye binds to and replaces the DNA minor groove during the annealing and DNA replication stage. As the amount of double-stranded DNA increases, more fluorescent light is emitted and measured by the device.

To investigate the genes, we used a pair of forward and reverse primers designed with Primer 3 Plus software for the exon-exon junction. These primers allowed us to distinguish cDNA from genomic DNA. Before conducting the tests, we confirmed the PCR primers using the Primer-Blast method available at www.ncbi.nlm.nih.gov/tools/primer-blast (Table 1).

The mean±standard deviation of the quantitative data was obtained from five separate samples (n=5). Statistical analyses were conducted using SPSS version 20 (IBM Corp.). Significance was determined using one-way analysis of variance (with the Tukey test). The significance level was set at p<0.05.

The hyperthermia group exhibited a significantly lower total sperm count than both the hyperthermia+vitamins and control groups (p<0.0001). Additionally, the total sperm count in the groups treated with vitamins demonstrated a higher recovery rate than that of the hyperthermia group, along with a statistically significant difference from the control group. Additionally, sperm motility was significantly reduced in the hyperthermia group in comparison to the hyperthermia+vitamins and control groups (p<0.0001). Furthermore, the groups receiving treatment showed a greater improvement in sperm motility than the hyperthermia group (p<0.0001). The research also indicated that vitamin C was more effective in improving these parameters than vitamin B12 (p<0.001). Despite the lack of a significant difference between the two vitamins in these parameters, vitamin C showed superior restorative effects (Figure 1).

Based on a stereological assessment of the histological studies, the hyperthermia group exhibited a significant decrease in testis volume compared to both the hyperthermia+vitamins and control groups (p<0.0001). There was no significant difference in effectiveness between vitamin C and vitamin B12 (p<0.0001). When comparing the hyperthermia group to the hyperthermia+vitamins and control groups, the hyperthermia group demonstrated a significant reduction in the number of spermatogonia, primary spermatocytes, and round spermatids. However, vitamin C treatment significantly increased the counts of spermatogonia, primary spermatocytes, and round spermatids following sustained scrotal hyperthermia (p<0.0001). In contrast, the number of Sertoli cells did not differ significantly between the vitamin-treated groups. The number of Leydig cells decreased significantly in the hyperthermia group compared to the hyperthermia+vitamins and control groups. Notably, there was a significant increase in Leydig cells in the hyperthermia+vitamins group, with vitamin C showing the most pronounced effect (p<0.0001) (Figure 2).

The serum testosterone level was significantly lower in the hyperthermia group than in both the hyperthermia+vitamins group and the control group. Our findings indicate that vitamin C treatment effectively elevated serum testosterone levels. No significant difference was observed in serum testosterone levels between the hyperthermia group and the group treated with vitamin B12. However, vitamin C supplementation resulted in serum testosterone levels that more closely approximated those of the control group (p<0.0001) (Figure 3).

ROS are small, unstable, and highly reactive molecules that have the potential to oxidize proteins, lipids, and DNA. ROS are generated through the incomplete reduction of oxygen involving an electron. A marked increase was observed in ROS levels in the hyperthermia group compared to the control group. Conversely, ROS levels were significantly reduced in the hyperthermia + vitamins group. There was no significant difference between the two hyperthermia+vitamin groups (p<0.0001) (Figure 3). In contrast the level of GSH in the hyperthermia group decreased compared to the control group. The GSH level increased in the hyperthermia+vitamins group, and the increase in GSH levels was higher in the hyperthermia+vitamin C group (p<0.001, p<0.0001) (Figure 3).

The results of the TUNEL assay revealed a significant increase in apoptotic cells in the hyperthermia group compared to both the hyperthermia+vitamins and control groups. Moreover, vitamin C was found to be more effective in reducing apoptotic cells than vitamin B12. In other words, vitamin C led to a greater decrease in apoptotic cells, approaching the levels observed in the control group (p<0.0001) (Figure 4).

The expression of all three genes (c-kit, Stra8, and Pcna) was decreased in the hyperthermia group compared to the control group. However, the expression of all three genes increased in the hyperthermia+vitamin C and hyperthermia+vitamin B12 groups and was close to the control group. Nonetheless, the increase in the expression of genes in the hyperthermia+vitamin C group was slightly greater than that in the hyperthermia+vitamin B12 group, although the difference was not significant (p<0.001, p<0.01) (Figure 5).