**3. Results**

### *3.1. Low Sperm Motility and High DNA Oxidation Suggest Compromised Sperm Quality Due to Oxidative Stress*

Spermatozoa from cauda epididymis were collected and analyzed 3 weeks, 6 weeks and 9 weeks after the end of the t-BHP treatment to determine whether there were damages due to the treatment during the late, middle and early spermatogenesis, respectively. Spermatozoa from t-BHP-treated animals showed a significant reduction of their motility at all times compared to those sperm from the control group. Moreover, we observed significantly higher DNA oxidation levels in spermatozoa from treated rats compared to controls (Figure 1).

**Figure 1.** Impairment of sperm quality due to t-BHP treatment in male rats. (**A**) Sperm motility and (**B**) sperm DNA oxidation determined by 8-deoxyguanosine (8-OHdG) levels. The results are expressed as mean ± SEM. \* Means higher than the other group at the same time point (*p* ≤ 0.05; Mann–Whitney test, *n* = 4).

### *3.2. Lipid Peroxidation Increased in Caput and Cauda Epididymis of t-BHP Treated Male Rats*

We determined oxidative damage on lipids by detecting 4-hydroxynonenal (4-HNE), a known marker of lipid peroxidation. We observed multiple bands detected by the anti-4HNE antibody, suggesting that different proteins contain the 4-HNE adduct in the tissues analyzed (Figure 2). A significant increase of 4-HNE levels was observed in both the caput and cauda epididymis of treated rats at 3 weeks and 6 weeks after the end of the t-BHP treatment compared to controls. Interestingly, the levels of 4-HNE at 9 weeks after the end of treatment were similar in the caput epididymis when comparing treated and control rats but were significantly higher than those seen in the control group at 6 weeks. In cauda epididymis, the 4-HNE levels returned to control values at 9 weeks after treatment. Noteworthily, the levels of lipid peroxidation at 9 weeks was higher in caput compared to cauda epididymis, suggesting an early dysregulation of the antioxidant response in the caput epididymis.

**Figure 2.** Lipid peroxidation (determined by 4-HNE levels) increased in caput and cauda epididymis in t-BHP compared to control male rats. The results of relative intensities (upper panels) are expressed as mean ± SEM. The blots presented are representative of experiments with 4 different rats. Some lanes showing protein bands have been pasted but belong to the same blot and have the same film exposure. \* Means higher than the other group at the same time point (*p* ≤ 0.05; Two-way ANOVA and Bonferroni post-hoc test, *n* = 4).

### *3.3. PRDX1 and PRDX6 Are Differentially Upregulated in Caput and Cauda Epididymis at Different Time Points*

We assessed the expression levels of PRDX1 and PRDX6 in rat epididymis, and we observed a significant increase of PRDX6 in caput epididymis at 3 weeks and in cauda epididymis at 3 and 6 weeks post-treatment (Figure 3). We also found a trend of increase in PRDX1 expression levels in caput epididymis at week 3 and 9, and in cauda epididymis at week 9, but these increases were not significant (Figure 4).

**Figure 3.** Peroxiredoxin 6 (PRDX6) expression in caput and cauda epididymis of control and t-BPH-treated male rats. The results of relative intensities (upper panels) are expressed as mean ± SEM. The blots presented are representative of experiments with 4 different rats. Some lanes showing protein bands have been pasted but belong to the same blot and have the same film exposure. \* Means higher than the other group at the same time point (*p* ≤ 0.05; Two-way ANOVA, *n* = 4).

**Figure 4.** Peroxiredoxin 1 (PRDX1) expression in caput and cauda epididymis of control and *t*-BPH-treated male rats. The results of relative intensities (upper panels) are expressed as mean ± SEM, (*p* > 0.05; Two-way ANOVA and Bonferroni post-test, *n* = 4). The blots presented are representative of experiments with 4 different rats. Some lanes showing protein bands have been pasted but belong to the same blot and have the same film exposure.

### *3.4. Catalase Expression Shows Trends of Increase, with Significant Individual Variation in the Epididymis*

Catalase expression levels were determined in rat epididymis from control and treated rats. Although not significant, we observed a trend of increase in treated rats compared to control at 3 and 6 weeks in cauda epididymis (Figure 5). Caput epididymis did not show upregulation of catalase at any time point.

**Figure 5.** Catalase expression in caput and cauda epididymis of control and t-BPH-treated male rats. The results of relative intensities (upper panels) are expressed as mean ± SEM, (*p* > 0.05; Two-way ANOVA and Bonferroni post-test, *n* = 4). The blots presented are representative of experiments with 4 different rats. Some lanes showing protein bands have been pasted but belong to the same blot and have the same film exposure.

No significant difference was observed between the control and treated groups at any time points for PRDX1, PRDX6, catalase or TRX-1 (Figures 6 and 7).

**Figure 6.** Peroxiredoxin 1 (PRDX1) and 6 (PRDX6) expression in testis of control and t-BPH-treated male rats. The results of relative intensities (upper panels) are expressed as mean ± SEM, (*p* > 0.05; Two-way ANOVA and Bonferroni post-test, *n* = 4). The blots presented are representative of experiments with 4 different rats. Some lanes showing protein bands have been pasted but belong to the same blot and have the same film exposure.

**Figure 7.** Catalase ( **A**) and thioredoxin (**B**) expression in testis of control and t-BPH-treated male rats. The results of relative intensities (upper panels) are expressed as mean ± SEM, (*p* > 0.05; Two-way ANOVA and Bonferroni post-test, *n* = 4). The blots presented are representative of experiments with 4 di fferent rats. Some lanes showing protein bands have been pasted but belong to the same blot and have the same film exposure.

The levels of 4-HNE in testis were similar in control and treated rats, suggesting that the oxidative stress generated by the treatment was well tolerated by the testis (Figure 8). Noteworthy, the levels of 4-HNE increased in testis from both control and treated rats at 6 and 9 weeks, suggesting that there is time dependency in the levels of lipid peroxidation in this organ.

**Figure 8.** Lipid peroxidation in the testis of control and t-BPH-treated male rats. The results of relative intensities (upper panels) are expressed as mean ± SEM. The blots presented are representative of experiments with 4 di fferent rats. Lanes showing protein bands have been pasted but belong to the same blot and have the same film exposure. # Means lower than all other groups, (*p* ≤ 0.05; Two-way ANOVA and Bonferroni post-test, *n* = 4).

### *3.6. Reproductive Organs Weight, Spermatogenesis, and Sperm Production Were Not Affected by the t-BHP Treatment*

To determine whether the damages observed in epididymal spermatozoa may have originated due to impairment of spermatogenesis by t-BHP treatment, we analyzed testis sections from control and treated groups to identify the di fferent stages of spermatogenesis, compared the reproductive organs weight and sperm production in the experimental groups. We did not observe di fferences in body and organ weights between the treated and control rats at any time point (Figure 9 and Supplementary Materials Table S1).

**Figure 9.** Testis and epididymis weight (A and B) and sperm production (C). (*n* = 4, Two-way ANOVA, *p* > 0.05).

The analysis of testis sections revealed that spermatogenesis proceeded normally in both control and treated rats during the 9 weeks after the end of treatment (Figure 10) that spermatogonia are transformed into spermatozoa [25]. We identified all the stages of the spermatogenesis in the rat, including the stages VII and VIII that contained elongating spermatids and fully formed spermatozoa, respectively in the luminal edge of the seminiferous epithelium, indicating active sperm production by the testes. As shown in Figure 10, some seminiferous tubules contain spermatozoa in the lumen (stage VIII), be ready to be spermiated to enter the epididymis. In addition, Sertoli and Leydig cells were morphologically normal.

**Figure 10.** Histological analysis of testes from control and t-BHP-treated male rats. Testis sections were stained with hematoxylin and eosin to evaluate spermatogenesis. All testis sections displayed normal spermatogenesis (Stages VII and VIII showing elongating spermatids and spermatozoa in the lumen of the seminiferous tubules, respectively), *n* = 4. Bar = 300 μm.
