All animal handling was in accordance with the instructions and guidelines of the Experimental Animal Ethics Committee, Faculty of Science, Beni-Suef University, Egypt (Approval Number: BSU/FS/2020/18).

A total of 30 apparently healthy adult male albino Wistar rats that initially weighed approximately 85 g were used in the beginning of this study to overcome any sudden death or diseases to rats. Later, we excluded seven rats because they did not achieve serum atherogenic index (AX) > 0.24, required for inclusion. Two rats died after sub-allocation of HFD-fed rats. Table 1 shows the final number of rats used and their allocation. The animals were purchased from Helwan University, Cairo, Egypt and then housed in the Biochemistry Department, Faculty of Science at Beni-Suef University. The rats were acclimatized for one week to exclude any intercurrent infection. Rats were kept in polypropylene cages with well aerated stainless-steel covers in a controlled environment that was maintained under a 12-hour light/dark cycle, a temperature of 24°C (±3°C) and 50–70% humidity. The rats were supplied with a standard diet and had free access to tap water. All efforts were made to reduce the number and suffering of the rats under investigation. These efforts included: 1) continuous checking of animal house air conditioning and continuous and careful supply of drinking water; 2) refreshing the animal house with ordinary air daily for 30 mins; 3) cleaning the cages and the animal house; 4) using the effective anesthesia dose; 5) carrying out euthanasia of animals far away from the other alive rats; 6) carefully disposing of euthanized animals far away from the other alive rats.

Normal standard food was purchased from Mecca Factory, Beni-Suef, Egypt. The high fat diet consists of the normal rat chow mixed with 15% palm oil and 1% cholesterol in accordance with Matos et al., with some modifications ¹³ . Compositions of normal standard and high fat foods are illustrated in Table 2. AS was purchased from Egyptian International Pharmaceutical Industries Co (EIPCO) 10th of Ramadan City, Egypt. All other chemicals were of analytical grade and were obtained from standard commercial suppliers.

Rats were divided into three groups as shown in Table 1. Selection of animals was carried out haphazardly without performing any prior randomization method.

1. Normal control group (CN): Rats were fed a normal diet, ad libitum, for four weeks; the whole period of the experiment. Rats were also given the equivalent volume of corn oil, as a vehicle, by oral gavage daily for the last two weeks.

2. HFD group: Rats were fed an HFD, ad libitum, for four weeks of the experiment, in addition to equivalent volume of corn oil by oral gavage daily for the last two weeks.

3. AS group: Rats in this group were fed a high fat diet for four weeks and treated orally with a dose of 5 mg/kg/day atorvastatin suspended in corn oil for the last two weeks of the experiment. The dose was double of that used by Oktem et al. ¹⁴ .

The 30 rats were allocated to six cages; five rats/each cage. On the first day, we selected a cage for commencing treatment randomly, then, every day we selected the starting cage serially. Selection of an animal inside each cage was arbitrary. The oral gavage started at 10 AM until nearly 12 AM. Rats treatments were carried out at animal house in Faculty of Science. The route of AS administration was by oral gavage, which is easy and suitable for rats.

The first author was aware of most stages of the experiments. Most of the authors and technicians contributed to animal euthanasia and collection of specimens. Therefore, blinding was not achieved during allocation nor treating stages; however, it was achieved during assessment and data analysis stages.

The dose was adjusted every week based on the changes in body weight (b. w.) to ensure a constant dose per kg b. w. of rats over the entire period of study. After two weeks of the onset of the experiment, rats were fasted overnight (10–12 h) and blood samples were collected from the lateral tail vein for screening of the AX according to the formula: log10 (TG/HDL-c). Rats that exhibited AX less than 0.24 were excluded. This criterion was defined before any data was collected. Rats with AX more than 0.24 were allocated to the HFD group and AS-administered group for an extra two weeks. At the end of the experiment, the rats were deprived of food overnight and anaesthetized by inhalation with diethyl ether. Blood samples were collected from the jugular vein and then the rats were killed by sudden cervical decapitation while they were anaesthetized.

Immediately after weighing the testes, each testis was homogenized in 5 ml cold buffer (100mM potassium phosphate, PH 7.0, containing 2mM EDTA) per gram of tissue. For the biochemical analysis of antioxidants, including reduced glutathione (GSH) levels were determined according to the method of Beutler et al. ¹⁵ . Moreover, glutathione-S-transferase (GST), superoxide dismutase (SOD), and glutathione reductase (GR) activities were also determined ^{16– 18} . Besides that, determination of malondialdehyde (MDA) level, as an indicator for lipid peroxidation, was also estimated according to Ohkawa et al. ¹⁹ .

Determination of testicular GSH content. The protein content of the testicular sample was precipitated by adding an equal volume of 4 % sulphosalicylic acid. After centrifugation, 0.5 ml of the supernatant was added to 4.5 ml of 0.1 mM Bis-(3-carboxy-4-nitrophenyl)-disulfide reagent in 0.1 M phosphate buffer, pH 8. The color intensity was determined spectrophotometrically at 412 nm.

Determination of testicular GST activity. After addition of glutathione (0.5 mM), and sodium phosphate buffer (0.1 M), pH 7.3, to the tissue sample, preincubation was carried out for 5 min at 37°C. Then, 1-chloro-2,4-dinitrobenzene (CDNB, 0.5 mM) was added to the incubation mixture. Thereafter, trichloroacetic acid solution (33%) was added and the mixture was centrifuged. The CDNB conjugate was measured in the supernatant at 340 nm. The quantity of enzyme that enhanced the formation of 1µmol of CDNB conjugate/mg protein/min was considered as one unit of enzyme activity. The molar extinction coefficient was 9.6 mM−1 cm−1.

Determination of testicular SOD activity. The reaction mixture contained the sample, tris/EDTA buffer, 12.1%, pH 8, and pyrogallol 10 mM. SOD prevents pyrogallol auto-oxidation by removing superoxide anions. As described, 0.1 buffer was mixed with 1 ml tissue sample, then 0.05 ml pyrogallol was added. The absorbency was read at 0 and 10 mins after the addition of pyrogallol. One unit of enzyme activity is the amount of enzyme that resist a 50% change in extinction per one min. The modification used to determine SOD activity was the use of pyrogallol instead of cytochrome c. ΔA/min was calculated as follows:

Enzyme activity (U/ml) =

[ΔA/min (sample)]/ [ΔA/min (control) × 50 %]

Then U/ml was converted to U/g tissue.

Determination of testicular MDA content. 1 ml of tissue homogenate was mixed with 2 ml of trichloroacetic acid (TCA, 7.5%) and then centrifuged. 2 ml of the formed supernatant was mixed with thiobarpeturic acid (TBA, 0.7 %) and boiled for 10 mins. The color intensity of the reactants (TBARS) was measured at 532 nm. An extinction coefficient of 156000 M−1 cm−1 was used for calculation.

Serum triglyceride levels were determined according to the method of Tietz ²⁰ . Total serum cholesterol and HDL-cholesterol concentrations were detected according to the procedure of Allain et al. ²¹ . Serum AX was calculated according to the equation [AX= Log(TG/HDL-C)] ²² . AX is a useful measure of response to pharmacological intervention. Total serum testosterone and thyroid stimulating hormone (TSH) were estimated by enzyme-linked fluorescent assay (ELFA).

Determination of serum triglyceride concentration. Serum triglyceride concentration was measured using the Randox triglycerides assay (Cat No. TR 213). The kit included reagent 2 which contained 4-chlorophenol (6 mmol/l), magnesium acetate (5 mmol/l), and tris buffer (50 mmol/l). Reagent 3 contained 4-amino antipyrine (1.0 mmol/l), ATP (1.0 mmol/l), lipases (≥100 U/ml), glyceolkinase (≥120 U/l), glycerol-3-phosphate oxidase (≥2.5 U/ml), and peroxidase (≥4.0 U/ml). Moreover, the working solution was performed by mixing the contents of reagent 3 with 30 ml of reagent 2. On the other hand, reagent 1 contained triglyceride (200 mg/dl) as a standard. As described, 10 μl of the serum or standard was added to 1 ml of the working reagent and then incubated for 5 min at 37°C. Thereafter, the absorbance of sample (Asample) and standard (Astandard) against reagent blank were measured at 456 nm. Triglycerides level (mg/dl) = (Asample/Astandard) × 200.

Determination of serum cholesterol concentration. Serum cholesterol concentration was measured using the Randox Cholesterol assay (Cat No. CH 201). The cholesterol kit included reagent 1 which contained cholesterol (200 mg/dl) as a standard, while reagent 2 contained 3,5dichloro-2-hydroxybenzene sulfonic acid (4mmol/l), and tris buffer (50mmol/l) detergent (0.2 %). Reagent 3 contained cholesterol esterase (≥160 U/l), cholesterol oxidase (≥ 120 U/l), peroxidase (≥2000 U/l), and 4-aminoantipyrine (0.45 mmol/l). The working solution was prepared by mixing reagent 3 with 30 ml of reagent 2. The procedure for determining of serum total cholesterol level was similar to that of triglyceride determination, except the absorbance measurement was at 500 nm.

Determination of serum HDL-cholesterol concentration. Serum HDL-cholesterol concentration was measured using the Randox HDL-cholesterol assay (Cat No. CH 203). Briefly, 50 μl of phosphtungestic acid (13.9 mol/l) was added to 500 μl of sample to precipitate LDL, VLDL and chylomicron fractions in the presence of magnesium ions, allowed to stand for 10 min, centrifuged for 10 min at 4000 rpm. Then, the supernatant was collected to determine the cholesterol content as described before. HDL-cholesterol concentration was calculated as follows:

HDL-cholesterol concentration (mg/dl) = A _sample × 180.

Determination of serum total testosterone level. Serum total testosterone level was measured using the VIDAS® Testosterone II kit (Cat No. 414320, bioMérieux, France). The antibodies used were monoclonal anti-testosterone antibodies produced in mice. All assay steps were performed automatically by the instrument. The strip consisted of 10 wells covered with a labeled foil seal. The last well of each strip was a cuvette in which the fluorometric reading was performed. The wells in the center section of the strip contained the various reagents required for the assay.

The wells reagents included: well-1 (the sample well), well-2 (Conjugate): phosphate buffer, bovine albumin, alkaline phosphatase-labeled anti-testosterone antibody, and preservative (300 μL), and well-3 (Pre-treatment solution): phosphate buffer, bovine albumin, dissociation agent, preservative (600 μL). Additionally, wells-4, -5, and -6 were empty, while wells-7, -8, and -9 were (wash buffer) contained tris, surfactant, and preservative (600 μL). Well-10 (reading cuvette with substrate) contained 4-Methyl-umbelliferyl-phosphate (0.6 mmol/L), diethanolamine (DEA, 0.62 mol/L or 6.6%, pH 9.2, and 1 g/L sodium azide 300 μL).

Briefly, the procedure aimed, firstly, to isolate testosterone from the carrier proteins in the sample by using the pre-treatment solution. The pre-treated sample was transferred to a well containing a testosterone antibody (conjugate) labelled with alkaline phosphatase. For the anti-testosterone-specific antibody sites, the antigen in the sample and the testosterone antigen attached to the SPR ® inner wall. During the washing steps, unbound pieces were removed. The substrate (4-Methylumbelliferyl phosphate) was cycled in and out of the SPR ® during the final detection stage. The hydrolysis of this substrate was catalyzed by the conjugate enzyme into a fluorescent product (4-Methylumbelliferone), whose fluorescence was measured at 450 nm. The fluorescence intensity was inversely proportional to the testosterone concentration present in the sample. The results were automatically determined by the instrument at the end of the assay with regard to the calibration curve stored in memory.

Determination of serum TSH level. Serum TSH level was measured using the VIDAS® TSH kit (Cat No. 30400, bioMérieux, France). The protocol was like that of total testosterone determination except we used mouse monoclonal anti-TSH antibodies instead of anti-testosterone antibodies.

Data are expressed as mean ± SE. The statistical differences between continuous variables was analyzed using analysis of variance (ANOVA). Statistical analysis was performed using SPSS v.24 (SPSS Inc., released 2007, SPSS for Windows, version 16.0, Chicago, IL). Values with p < 0.05 were considered significant.

Harvard Dataverse: Replication Data "The Ameliorative Effect of Atorvastatin on Fertility and Testicular Oxidant/Antioxidant System of HFD-Fed Male Albino Rats". https://doi.org/10.7910/DVN/9LEX1O ²³ .

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).