Animals
Thirty-six (36) adult male Wistar rats (weight range: 160–180 g) were obtained from the Department of Biochemistry, University of Ilorin, Nigeria. They were housed in wooden cages maintained under standardized conditions (12-h light/dark cycle, 27–30 °C, 50–80% relative humidity), and were acclimatized in the laboratory for 2 weeks before the commencement of the study. The rats were fed with standard pelletized rodent diet (Ace Feeds, Ibadan, Nigeria) and water ad libitum. All the animals were well-catered for according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science and approved by the Ethical Research Committee of the University of Ilorin, Nigeria.
Extraction and GC-MS analyses of hemp samples
Mixture of hemp seeds and leaves (75%:15% respectively), both of Benin republic and Nigeria origin, were kindly donated by the National Drug Law Enforcement Agency (NDLEA), Nigeria, for research purpose only. About 200 g of each of these samples was subjected to extraction with 98% ethanol in Soxhlet apparatus for 4–8 h as described earlier [20]. The extract was evaporated to dryness in a rotary evaporator under vacuum to get the oil.
A gas chromatography (GC) from AgilentCo. USA, hyphenated to a mass spectrometer (MS, 5975C) with triple axis detector equipped with an auto-injector (10 μl syringe) was used. Helium gas was used as a carrier gas. All chromatographic separation was performed on capillary column having the following specifications: length- 30 m, internal diameter- 0.25 μm, thickness- 250 μm, treated with phenylmethylsilox. Other gas chromatography mass spectrometry (GC-MS) conditions are: ion source temperature (EI)- 250 °C, interface temperature- 300 °C, pressure- 16.2 psi, cut time- 1.8 min, 1.0 μL injector in splitless mode with split ratio 1:50 and injection temperature of 300 °C. The column temperature started at 35 °C for 5 min and changed to 150 °C at the rate of 4 °C/min for 2 min. The temperature was raised to 250 °C at the rate of 20 °C/min and held for 5 min.
The MS scanning was performed from M/Z85 to M/Z380. The GC-MS solution software provided by supplier was used to control the system and to acquire the data. The separation of the injected samples was carried out on an HP capillary column (HP-5MS). Identification of the compounds was carried out by comparing the mass spectra obtained with those of the standard mass spectra from National Institute for Standards and Technology (NIST) library (NIST II). Identification was based on the molecular structure, molecular mass and calculated fragments. The fatty acids and other organic compounds were identified by comparing their retention times with those of the standards. The quantity of each compound present was expressed as percentage of the total compounds.
Experimental design
Since the hemp sample of Nigerian origin has been repeatedly used in the series of our previous studies [10, 11, 13], we did not repeat its administration in this study, but only compared its composition with that of Benin republic. The 36 rats were blindly randomized into 6 groups (n = 6 per group). Groups I (control) and II received normal saline and bromocriptine (3 mg/kg) respectively. Groups III and IV received 2 mg/kg of BHE [10, 11] alone and in combination with bromocriptine [21] respectively, while groups V and VI received 10 mg/kg BHE [22] alone and in combination with bromocriptine respectively. All treatments were given once daily for a period of 30 days between 8:00 am to 10:00 am via oral gavage.
Animals were anaesthetized a day after the last treatment with sodium pentobarbital (40 mg/kg body weight, im). Thereafter, they were dissected in order to collect blood by cardiac puncture. Whole blood for the determination of the biochemical markers were collected in heparinized sample bottles which were centrifuged at 3500 rpm for about 10 min at -4 °C using a cold centrifuge (Model 8881, manufacturer: Centurion Scientific Ltd., United Kingdom). The separated plasma samples were collected into separate plain bottles and were stored at -20 °C prior to the biochemical analyses.
Determination of semen parameters
The procedure for determination of epididymal sperm parameters, such as; count, motility, morphology, and viability are summarized below as previously described [23, 24],
The testes from each rat were carefully exposed and one of them was removed together with its epididymis. For each separated epididymis, the caudal part was removed and placed in a beaker containing 1 ml of normal saline solution. It was macerated with a pair of sharp scissors and left for few minutes to liberate the sperm cells into the normal saline. Semen drops were placed on a clean grease-free glass slide and two drops of warm 2.9% sodium citrate were added. The improved Neubauer counting chamber was charged with the semen solution and the number of sperm cells, appearing as black dots, was counted in 25 small squares within the central counting area of the counting chamber.
The sperm suspension was diluted in 1 ml of normal saline solution. About 10 L was pipetted onto a clean grease free glass slide. A cover slip was lowered onto the sample on the slide, avoiding air bubbles, and the slide was examined using a microscope with a 40X objective. At least, six widely spaced fields were examined to provide an estimate of the percentage of the progressively motile sperm cells. The sperm cells with progressive motility were estimated and recorded as (N) while the total number of all the sperm cells counted was recorded as (T). Sperm motility (%) was calculated using (N/T × 100%).
The principle for determination of sperm morphology was based on the ability of morphologically normal sperm to appear white in color as the plasma membrane will prevent the dye to enter, while abnormal sperms take up the dye and stain dark color. The microscope slides and the eosin stain were pre-warmed to room temperature. One milliliter of the sperm suspension – normal saline solution was transferred to a test tube and 2 drops of 1% eosin were added and mixed gently for agitation. This was incubated for 45–60 min to allow its proper staining and then re-suspended with a Pasteur pipette. A clean grease-free glass slide was used. Potential damage to the sperm cells should be avoided. One or 2 drops of the stained sperm were placed approximately 1 cm from the end of the slide lying on a flat surface. A second slide was held with the slide’s long edge gently touching across the width of the sperm slide and pulled across to produce a sperm smear. After air-drying the slide, using a microscope at 100X objective, the sperm cells were examined. The sperm along the periphery was normally excluded from the examination because there is a greater tendency for artifacts to occur in these regions. At least, five fields were viewed covering the whole slide. Examples of morphological abnormalities are double-headed, elongated head, pyriform head, bent head, bent tail, bent mid-piece, coiled tail, double tail, headless, tailless, etc. All those with normal morphology were recorded as N while the total number of the counted spermatozoa was recorded as T. The percentage sperm morphology was calculated as (N/T × 100%).
Determination of biochemical parameters
Diagnostic kits for the determination of malondialdehyde (MDA), total anti-oxidant capacity (TAC), superoxide dismutase (SOD), glutathione Peroxidase (GPx), and catalase were obtained from Fortress Diagnostics Limited, United Kingdom. In addition, analytic kits for the determination of GnRH, prolactin, FSH, LH, testosterone, and estradiol were procured from Elabscience Biotechnology Company Ltd. Wuhan, Hubei, China. The analyses were done with spectrophotometer (Spectramax Plus; Molecular Devices, Sunnyvale, CA, USA) according to the manufacturers’ instructions.
Data analyses
Statistical analyses were done using statistical package for social sciences (SPSS) version 16.0 (IBM Corporation, Armonk, NY). Values were expressed as Mean ± S.E.M. of the variables measured. Comparisons among the groups were done by one-way analysis of variance (ANOVA), followed by post-hoc Tukey multiple comparison test. Statistical significance was considered at p < 0.05