Collection of fungi
Fresh fruiting bodies of P.tuber-regium were collected from a forest close to University of Nigeria, Nsukka. They were identified by a taxonomist at the International Center for Ethnomedicine and Drug Development (INTERCEDD) Nsukka, Nigeria. The mushrooms were air dried, pulverized and stored in airtight containers until use. The voucher specimen for the same is conserved under the reference number INTERCEDD/050.
Male Sprague Dawley rats with average weight of 170–180 g were obtained from the Animal House of University of Port Harcourt, Rivers State Nigeria. The animals were maintained under standard laboratory conditions at ambient temperature of 25 °C ± 15%, with darkness cycle of 12 h. They were allowed access to standard commercial pellet diet and water ad libitum. All the experimental studies conformed to the guidance for care and use of animals in experimental studies of the University of Port Harcourt Ethical protocols.
Acute toxicity test
Various doses of P. tuber-regium were administered to male Sprague Dawley rats per oral (50–5000 mg kg−1 b.w). The animals were observed for gross behavior, neural and autonomic toxicity as described on OECD guidelines . There was no mortality or toxic signs recorded in this period even up to 5000 mg/kg dose.
The animals were divided into six (6) groups (n = 10). They were treated as follows;
Group I Control (received olive oil 3 mL/kg i.p. twice weekly for 13 weeks in addition to feed and water).
Group II Carbon tetrachloride treated (received 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly for 13 weeks)  modified.
Group III Rats were treated with 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly in addition to 100 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks .
Group IV Rats were treated with 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly in addition to 200 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks.
Group V Rats were treated with 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly in addition to 500 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks.
Group VI Rats received olive oil 3 mL/kg i.p. twice weekly in addition to 500 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks.
Measurement of organ weights
At the end of the 13th week post treatment, the animals were sacrificed under ether anesthesia and the right testes removed, carefully trimmed of fats and rinsed with ice cold saline before being weighed using a digital weighing balance. The relative organ weights were determined as follows;
Relative organ weight (ROW) = Organ weight/Animal weight x 100.
Part of the testes were homogenized in phosphate buffered saline for lipid peroxidation and antioxidant studies while part were preserved in 10% formalin for histological evaluations.
Collection of Blood samples
At the end of the 13th week post treatment, blood samples were withdrawn from the medial canthus of the eye using micro hematocrit tubes into a clean test tube . The blood was allowed to coagulate for 30–60 min, then centrifuged at 1800 x g relative centrifugation force (RCF) for 30 min to effect separation of serum from blood cells. The serum was stored below 0 °C in 1.5 mL tubes until used for hormonal analyses.
Sperm motility was determined by macerating the epididymis in mortar and pestle and mixing it with 1 ml of phosphate-buffered saline (37 °C) in order to release the sperm cells, and a drop of the suspension was quickly placed on a glass microscope slide, covered with a glass slip and examined using a light microscope at × 40 magnification. At least 200 sperm cells were counted and the number of motile sperm cells reported as a percentage of the total cells . Sperm viability was assessed microscopically from smears that were prepared by mixing equal volumes of eosin-nigrosin stain with epididymal suspensions, incubation for 30 s, distributing the stained suspensions on glass slides, and drying in air. After 30 s, a drop of the mixture was placed on a glass slide and spread out to make a smear, and this was allowed to air-dry. The smear was examined under x100 oil immersion using a light microscope. Live sperm were left unstained whereas dead sperm stained pink or red. At least 100 spermatozoa were evaluated and the sperm vitality recorded as Live to Dead sperm ratio . The caudal epididymal sperm reserves and testicular counts were determined using the standard hemocytometric method .
Lipid peroxidation and antioxidant assays
At the end of the 13th week post treatment, one gram of testis tissue was homogenized in 9 ml ice cold Phosphate buffered saline (pH 7.4) to make 10% homogenates. The homogenates were centrifuged at 1800 x g RCF for 15 min at 4 °C and the supernatants were used to determine the levels of malondialdehyde (MDA), ascorbic acid, alpha tocopherol, and total glutathione as well as superoxide dismutase, catalase, and peroxidase enzyme activities. Lipid peroxidation was quantified as malondialdehyde . Ascorbic acid was assayed colorimetrically using the 2,4-dinitrophenylhydrazine method . Alpha tocopherol was analysed colorimetrically with 2, 4, 6 – tripridyl - s – triazin and FeCl3 after extracting with absolute ethanol and xylene as described by Martinek . Superoxide dismutase (SOD), catalase, total glutathione and peroxidase were assessed using a commercial kit (Biovision, Mountain View, CA, USA) obtained from a local representative and assayed according to the manufacturer’s protocol.
Serum analysis of hormones
Serum analysis of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, estrogen and prolactin were estimated using commercial kits (Biocheck Inc, 323 Vintage Park Dr, Foster City, CA 94404) obtained through a local representative and assayed according to the manufacturer’s protocol.
Portions of the testis from all animals were sliced and dehydrated with a range of concentrations (70%, 80%, 90% and absolute) of alcohol and then cleared with xylene (twice) before embedding in paraffin. The embedded tissue blocks were section with Shandon AS 325 rotary microtomes into approximately 5 μm thick section and slides were prepared with the sections. The tissues were stained with Ehrlich’s haematoxylin and eosin blue.
All statistical analyses were done on GraphPad Prism version 5.02 (www.graphpad.com/scientific-software/prism). Data were expressed as mean ± standard deviation (SD). ANOVA test was used to analyze the difference among various treatments with least significance difference (LSD) at 0.05 followed by Bonferroni’s posttest.