Twenty healthy adult male Wistar rats weighing between 150 and 200 g and purchased from College of Health Sciences Ogbomosho, Oyo state, Nigeria were used. Animals were acclimatized for 3 weeks under conventional laboratory conditions (temperature 25–27 °C, humidity of 70%, and 12 h light and 12 h dark cycle) and allowed free access to standard diet and water ad libitum. Standard protocol on animal handling and care (according to the declaration of Helshinki) were strictly adhered to. Procedures adopted were also certified by the Lagos State University College of Medicine Animal House Committee.
Chemical and drugs
Vitamin C was purchased from Spring Valley Pharmaceuticals, China. Calcium chloride (CaCl2), glucose, sodium chloride (NaCl), potassium chloride (KCl), sodium hydrogen bicarbonate (NaHCO3), sodium nitroprusside (SNP), phenylephrine (PE), and acetylcholine (ACh) were purchased from Sigma Corporation, St. Louis, USA.
Stress models procedure
The rats were exposed to variable stress models according to the method of Mueller and Bale , with slight modification for predator exposure and noise. Six painless, non-habituating stress models which do not affect feeding were adopted. They include immobility by restraining animal in 50 ml tube for 20 min during light cycle, multiple cage changes at interval of 20 min for 2 h during light/dark cycle, saturated beddings with water overnight causing sleep deprivation, predator exposure by placing a cat in the same cage as the rats with a wire mesh separation, foreign object exposure in cage overnight, and exposure to 100 decibels sound for 4 h during the light cycle.
Experimental design and treatment
Twenty pubertal male Wistar rats (150–200 g) were randomly divided into four groups of five rats each. Groups 1 and 2 were treated with normal saline (vehicle) and vitamin C 7 mg/kg bwt , respectively orally. Groups 3 and 4 were exposed to chronic variable stress with group 3 concurrently treated orally with vitamin C (7 mg/kg bwt). Treatments and stress exposure were for 8 weeks in all groups. All animals were weighed weekly using weighing scale (Highland Adam Equipment, UK).
Serum sample collection
Cardiac puncture technique was used (after injecting 30 mg/kg phento barbital)  to collect a single, good quality volume of blood into a plain bottle from the animals using a 5 ml syringe with needle. The blood was allowed to stand at room temperature for 15–30 min. Thereafter, it was centrifuged using a cold centrifuge (Model SM112, Uniscope Laboratory Centrifuge, England) at 4000 rpm for 15 min. The resulting supernatant designated serum was carefully aspirated using a Pasteur pipette into a plain bottle and stored at − 4 °C
Preparation of the testicular artery strip
The rats were anaesthetized (30 mg/kg phento barbital), before they were sacrificed by cervical dislocation . The testis was surgically removed en bloc with care. The testicular artery was removed from the testis and placed in a petri-dish containing physiological salt solution (PSS). The testicular artery was suspended in a 50 ml chamber of the organ bath. These chambers contained PSS with the following composition (m/mol): NaCl (118.2), CaCl2 (1.6), KCl (4.7), NaHCO3 (15.0), KH2PO4 (1.2), MgSO4 (1.2), and glucose (11.5). The temperature of the organ bath was maintained at 37 °C and the solution was bubbled with a 95% O2 + 5% CO2 gas mixture (pH 7.35–7.40). Each testicular artery strip was anchored with a stainless-steel hook to an electronic transducer (model 7004; Ugo-Basile Varese, Italy) connected to a data capsule model 17400 for recording isometric contractions .
Dose response of testicular artery strip to phenylephrine and potassium chloride
The testicular artery was allowed to stabilize in the physiological solution for 90 min during which it was stimulated three times at 30 min interval with 10-7 M phenylephrine. Cumulative dose response of the testicular artery to phenylephrine (10-9–10−5 M) and 10–60 mM KCl were thereafter determined. Isometric contractions generated were recorded through the data capsule acquisition system.
Dose response of testicular artery strip to acetylcholine and sodium nitroprusside
The testicular artery was allowed to stabilize in the physiological solution for 90 min during which it was stimulated three times at 30 min interval with 10−7 M phenylephrine. Cumulative dose response of testicular artery to acetylcholine (10-9–10-5 M) was determined after pre-contraction with 10-7 M phenylephrine and 60 mmol KCl. The response was ensured to maintain stable level before addition of another dose. Cumulative dose response to sodium nitroprusside (SNP) (10-9–10-5 M) was determined after pre-contracting the testicular artery with 10-7 M of PE and 60 mmol KCl respectively.
Histological preparation of the testis
Sections obtained after preparations were stained with hematoxylin and eosin stains following clearance in xylene and were subsequently dried at 35–40 °C. Photomicrographs were then taken at 200 and 400 magnifications .
Determination of serum superoxide dismutase, catalase, and malondialdehyde activity
Superoxide activity was assayed using a randox kit (Sigma Chemicals Ltd., USA). Catalase activity was determined according to the method of Aebi . Malondialdehyde (MDA) an index of lipid peroxidation was determined using the method as described by Uchiyama and Mihara .
Determination of serum cortisol, testosterone, and sperm parameter analysis
Serum cortisol and testosterone were estimated using enzyme-linked immunosorbent assay (ELISA) method. Cortisol ELISA kit (DRG, USA) and testosterone ELISA kit Monoblind Inc. Lake Forest, CA, USA were used following the manufacturer instruction. Epididymal sperm count, percentage sperm motility, and abnormal sperm morphology were determined as reported by Raji et al. .
Results were expressed as means ± standard error of the mean (SEM). Statistical analysis was done using graph pad prism version 5.0. Data were analyzed using one-way analysis of variance (ANOVA), Newman keuls test was used as post hoc test, and p values less than 0.05 were considered statistically significant.