Ethics approval

The experimental procedures described in this study were performed in accordance with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and were approved by the Ethics Committee on Animal Use of the State University of Maringá (CEUA/UEM protocol no. 2910011021) and by the Ethics Committee on Animal Use of the State University of Londrina (OF CIRC CEUA/UEL protocol no. 144/2019).

Animals and experimental conditions and experimental design

Forty-eight male Wistar rats from different litters at PND 25 were retrieved from the Central Animal Facility of the State University of Maringa. Subsequently, they underwent a 5-d adaptation period in the Sectorial Vivarium of the Department of Biotechnology, Genetics, and Cell Biology. Animals were housed in polypropylene cages (40 × 34 × 17 cm) three animals per cage and remained under controlled temperature (22 ± 2°C) and photoperiod (07:00–19:00, light cycle) conditions, with ad libitum access to water and food.

Upon reaching PND 30, the animals were stratified into four groups. Two groups were subjected to an isocaloric low-protein palletized diet (4% protein, refer to Table 1; designated as LP60 and LP120), while the remaining two received a balanced commercial control diet (20.5% protein; Nuvital®, Curitiba/PR, Brazil; refer to Table 1; denoted as NP60 and NP120).

Table 1.

Composition of the isocaloric low-protein and normal protein diets

On PND 61, rats from NP60 and LP60 groups (n = 10 each) underwent euthanasia via decapitation using a guillotine without anesthesia. Rats from NP120 and LP120 groups (n = 14 each) were transported in an air-conditioned vehicle to the Sectorial Vivarium of Physiology Science at the State University of Londrina. They were subsequentially housed three per cage under the previously described experimental conditions. For the initial 2 weeks post-transportation, minimal contact with the animals was maintained to mitigate potential stressors.

From PND 61 to PND 120, all animals received the same commercial control diet (20.5% protein; Nuvital®, Curitiba/PR, Brazil; refer to Table 1). On PND 121, rats in NP120 and LP120 groups underwent anesthesia with thiopental (THIOPENTAX®, Itapira/SP, Brazil) followed by euthanasia through heart puncture.

Testis, epididymis, and sperm collection

On PND 61, rats (n = 10 per group) underwent euthanasia via decapitation. The testis and epididymis were excised, with the right counterparts undergoing fixation and dedicated to histological processing (n = 6). Simultaneously, the left counterparts were promptly frozen and stored in a –20°C freezer for subsequent spermatic count analysis (n = 10). The right vas deferens were retrieved and subjected to a formaldehyde wash (10%), with the contents preserved in the refrigerator for future analysis of sperm morphology (n = 10). From the left vas deferens, the content was washed with phosphate-buffered saline (PBS) buffer enriched with bovine serum albumin (BSA) and destined for spermatic motility analysis (n = 10).

Following the recovery period, at PND 121, rats (n = 14 per group) were euthanized by heart puncture after anesthesia induction with 8 units of thiopental (THIOPENTAX®, Itapira/SP, Brazil). The testis and epididymis were harvested, with the right counterparts destined for histological processing (n = 6) or swift freezing with liquid nitrogen and storage at –80°C freezer for subsequent evaluation of oxidative damage (n = 8). Concurrently, the left testis and epididymis were destined for future spermatic count analysis (n = 10). Sperm cells from the right vas deferens were destined for analysis of sperm morphology (n = 10). From the left vas deferens, the content destined to spermatic motility analysis (n = 10). Spermatozoa extracted from the epididymal tail were utilized for the assessment of oxidative damage (n = 8).

Daily sperm production per testis and transit time in the epididymis

The left testis and epididymis, decapsulated (n = 10), underwent weighing and homogenization following the methodology outlined by Robb et al., ^{Reference Robb, Amann and Killian23} with the adaptations previously described by Siervo et al. ^{Reference Siervo, Vieira and Ogo24} Post-homogenization, a small aliquot of the sample was transferred to the Neubauer chamber (five fields per animal) for the enumeration of homogenization-resistant spermatids (corresponding to stage XIX of spermatogenesis) in the testis and spermatozoa in the epididymis. To compute the daily sperm production (DSP), the concentration of homogenization-resistant spermatids per testis was divided by 6.1, which corresponds to the number of days during which mature spermatids remain in the seminiferous epithelium. Additionally, the sperm transit time through the epididymis was calculated by dividing the number of sperm in each epididymal portion by DSP.

Spermatic quality

Sperm motility assessment followed the methods of Siervo et al. ^{Reference Siervo, Vieira and Ogo24} with adaptations. The left vas deferens (n = 10) underwent washing with 0.8 mL of PBS (137 mM NaCl, 2.68 mM KCl, 8.03 mM Na₂HPO₄ 1.47 mM, pH 7.2) enriched with 0.75% BSA at 34°–37°C to obtain spermatozoa. Subsequently, a Makler counting chamber (Sefi-Medical) at the same temperature was loaded with a 10 μL aliquot of the sperm solution. Sperm motility, assessed by visual estimation (100 spermatozoa/animal) under a light microscope (Motic®, Richmond, Canada) at 100× magnification, categorized spermatozoa as motile, when active movement in this cell’s tail was observed, and immotile, when no movement was seen.

Vas deferens content (n = 10) was removed via internal rinsing with 1.0 mL of 10% formol-saline using a needled syringe. Histological slides prepared from this solution underwent observation using an Opton photomicroscope (Berlin, Germany) at 400× magnification. One hundred spermatozoa were analyzed per animal, classifying morphology into three categories: normal morphology, head abnormalities (without characteristic curvature or isolated form, i.e., no tail attached), and tails abnormalities (broken, rolled into a spiral and isolated, i.e., no head attached, as per Fernandes et al.). ^{Reference Fernandes, Arena, Fernandez, Mercadante, Barbisan and Kempinas25}

Mitochondrial activity of the sperm (n = 6) was determined following the method described by Silva et al. (2014) with adaptations. Spermatozoa, obtained from the vas deferens by internal rinsing using a needled syringe filled with PBS enriched with 0,75% BSA, were added in microtubes containing 1 mg/mL of 3-30-diaminobenzidine (DAB) dissolved in PBS in a 1:3 (v/v) ratio and incubated at 37°C for 1 h in the dark. Smears were prepared under histological slides and fixed with 10% formaldehyde for 10 min and evaluated with a phase-contrast microscope. Cells were classified as DAB I (100% stained intermediate piece), DAB II (more than 50% staining in the intermediate part), and DAB III (less than 50% staining in the intermediate part) based on mitochondrial activity, indicating varying degrees of potential motility and fertilization capacity, from more to less capable.

Sperm acrosome status was assessed following the protocol by Silva et al. (2014). ^{Reference Silva, Vendramini, Restelli, Bertolla, Kempinas and Avellar26} Smears prepared onto microscope slides using fresh sperm suspension were fixed with methanol (n = 6). Slides were then stained with 40 μg/mL fluorescein-labeled peanut agglutinin (PNA; Sigma-Aldrich, St Louis, MO, USA) in PBS and covered with Fluoromont-G with DAPI (EMS, Hatfield, PA, USA). One hundred cells per slide were analyzed under a fluorescence Axio Zeiss microscope (Zeiss®, Oberkochen, GER). Cells had their acrosome classified as intact or damaged.

Histological analysis, testicular morphometry, and epididymal stereology

Testis and epididymis (n = 6) were fixed in methacarn solution (10% acetic acid, 60% methanol and 30% chloroform) before being immersed in alcohol 70%. Subsequent steps included Paraplast® wax imbibing, cutting into 5 μm sections, and staining with hematoxylin and eosin (H&E). Evaluation with a Motic microscope (Motic®, Richmond, Canada) at 100x and 400× magnification followed. For the testes, random seminiferous tubular sections (100/animal) in three nonconsecutive testis cross sections were analyzed for abnormalities, including immature germ cells in the lumen, acidophil cells, vacuoles, and tubular degeneration. In the epididymis, histological inspection was performed qualitatively in the caput-corpus and cauda of each animal as described by Favareto et al. ^{Reference Favareto, de Toledo and Kempinas27}

For evaluation seminiferous tubule diameter and epithelium height, 40 random testicular cross sections per animal (n = 6) in stage IX of the seminiferous epithelium cycle underwent examination. Seminiferous tubule diameters and epithelium heights were measured using an Opton photomicroscope (Berlin, Germany) (400× magnification) and Motic software (version 3.1 for MacOS). A mean of four measures for the diameters and heights was calculated and used in the statistical analysis, aligning with the protocol by Favareto et al. ^{Reference Favareto, de Toledo and Kempinas27} The IX stage was used given this stage is characterized by a well-defined and stable structure of the seminiferous epithelium, in which its height and the tubule’s diameter are representative of the overall structural integrity.

The number of Sertoli cells with observable nuclei was determined in 20 cross sections of the seminiferous tubules per testis (n = 6) in each rat, employing a light microscope at 400× magnification, following the procedure by Nassr et al. ^{Reference Nassr, Arena and Toledo28} The tubules were evaluated at stages VII–VIII of spermatogenesis, given the crucial role of Sertolli cells in spermiation and acrosomal development, which occurs during these stages.

The number of Leydig cell nuclei was determined in 10 images of the interstitium per testis (n = 6) for each rat, utilizing an Opton photomicroscope (Berlin, Germany) at 400× magnification.

Stereological analysis of the epididymis involved capturing and analyzing 10 random epididymal cross sections per animal. Weibel’s multipurpose graticulate, with 168 points superimposed on the images, permitted the counting of overlapping dots in the seminiferous cord or interstitial compartment. This enabled the establishment of respective proportions of each of these components in the epididymis for each experimental group, adhering to the method by Favareto et al. ^{Reference Favareto, de Toledo and Kempinas27}

Staging of the spermatogenic cycle

Random seminiferous tubular sections (100/animal; n = 6) in three nonconsecutive testis cross sections were classified into four stages: I–VI, VII–VIII, IX–XIII, and XIV of the seminiferous epithelial cycle. This classification followed the criteria established by Leblond and Clermont, ^{Reference Leblond and Clermont29} using a photomicroscope at 400× magnification. This analysis allowed for the assessment of the proportion of staging of the seminiferous tubules.

Testosterone assay

Blood samples were collected in heparinized tubes and centrifuged at 2400 g for 20 min (4°C). The plasma was separated and stored at −20°C until the assay. Total plasmatic testosterone concentration was measured using chemiluminescence (Architect System, Wiesbaden, Germany) according to the manufacturer’s protocol. All samples were included in the same assay to avoid inter-assay errors.

Oxidative stress of the testis, epididymis, and sperm cells

Sperm collected from the epididymis tail was homogenized with and Ultraturrax homogenizer (Marconi, Piracicaba, Brazil) in 1 mL of PBS (pH 7.2) and centrifuged at 9500 g for 10 min at 4°C, as described by Erthal et al. (2022). Tissue samples from the testis and epididymis head and tail were homogenized in 500 mL of PBS (pH 7.2) and centrifuged at 9500 g for 10 min at 4°C. Protein quantification of tissular and sperm samples was determined by the Bradford method, using BSA as a standard. ^{Reference Bradford30} Samples were then normalized to 1 mg of protein per mL of solution, following the methodology described by Erthal et al. (2022), and utilized for the following analyses: lipid peroxidation (TBARS), reduced glutathione (GSH), catalase (CAT) activity, glutathione S-transferase (GST) activity, and superoxide dismutase (SOD) activity.

Lipid peroxidation

Lipid peroxidation was measured through lipid hydroperoxide, a step of fatty acids oxidation. ^{Reference Aviram and Eias32,Reference Parfitt, Desomeaux, Bolton and Hartog33} Hydroperoxide was determined through ferric oxidation (Fe²⁺; ammonious ferric sulfate). The Fe³⁺ ion that is formed reacts with xylenol orange, forming a colorful compound detected through its absorbance at 560 nm. ^{Reference Jiang, Woollard and Wolff34} Results were expressed as nmol/protein mg.

Reduced glutathione concentration

The concentration of GSH was determined as described by Rahman et al., ^{Reference Rahman, Kode and Biswas35} with adaptations made by Erthal et al. ^{Reference Erthal, de Siervo G.E.M., Frigoli, Zaninelli, Verri and Fernandes31} GSH levels were measured by adding 5,5,-dithiobis-2-nitrobenzoidc acid into the normalized samples, resulting in a yellow solution, with its absorbance read at 412 nm. The results are expressed as micromoles/mg of protein. Results were expressed as μg/protein mg.

Catalase activity

The enzymatic activity of CAT was determined by degradation of hydrogen peroxide into water and oxygen. Using the normalized solution (1 mg/mL), 297 μL of the reaction medium was added in a UV4 microplate, and the absorbance was read at 240 nm for 60 s. ^{Reference Aebi36} Results were expressed as mM/min/protein mg.

Superoxide dismutase activity

The enzymatic activity of SOD was determined as described by Senthikulmar et al. ^{Reference Senthikulmar, Amaresan and Sankaranaranarayanan37} with some adaptations made by Erthal et al. ^{Reference Erthal, de Siervo G.E.M., Frigoli, Zaninelli, Verri and Fernandes31} The enzyme present in the normalized homogenates (1 mg/mL) was added to a microplate in which a reaction mixture containing sodium carbonate buffer (50 mM, pH 10.2), nitroblue tetrazolium (NBT) (96 mM), and Triton X-100 (0.6%), previously incubated for 2 min with sodium hydrochloride hydroxylamine (NH₂OH.HCl) (20 mM, pH 6.0), was added. The final volume in the microplate well was adjusted to 200 μL. The method is based on the quantification of complexes formed by superoxide anions in addition to NBT and NH₂OH.HCl of yellowish color, with the reduction of NBT, forming a bluish solution, which is then read at 560 nm for 2 min at intervals of 15 s. Results were expressed as U/protein mg.

Glutathione S-transferase activity

GST activity was determined as described by Keen et al. ^{Reference Keen, Habig and Jakoby38} The method is based in the formation of a thioether from the interaction of GSH with 1,chloro-2,4-dinitrobenzene, with its absorbance read at 340 nm during 5 min at 40 s intervals. Values were expressed in mM/protein mg.

Statistical analysis

All parameters were submitted to the Shapiro–Wilk test for normality and thus classified into parametric or nonparametric data. Data were compared using an unpaired t-test or the nonparametric Mann–Whitney test. Differences were considered significant when p < 0.05.