Drugs and chemicals
Ganoderma lucidum extract composed of cracked spores and fruiting bodies, branded ReishiMaxGLpTM, was procured from Pharmanex Inc. (United States of America). Sildenafil was purchased from Pfizer, USA, while progesterone and estradiol benzoate were procured from Sigma Aldrich, Pakistan.
Animals
Healthy adult rats (Wistar strain) bearing weight of 150–200 g were purchased from the animal house of Dow University of Health Sciences, Pakistan. Polypropylene cages have been used for keeping the animals under controlled conditions at room temperature (25–30 °C) with light-dark cycle of 12\12 h. Standard diet and water ad libitum were given to the rats. Handling of the animals was done according to the requirements mentioned in “Guidelines for the care and use of laboratory animals 8th edition” [17]. Prior approval of the ethical review committee of Ziauddin University was taken before starting this research study.
Animal groups
Male rats were divided into four groups (n = 6):
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Group I: Control group, given distilled water (10 ml/kg) orally for 40 days.
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Group II: Treatment group, given extract (150 mg/kg) for 40 days.
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Group III: Treatment group, given extract (300 mg/kg) for 40 days.
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Group IV: Standard drug group, given sildenafil citrate (5 mg/kg) for 40 days.
Preparation of male rats
Sexual behaviour training was given to male rats for 10 days. The animal that did not show any sexual interest was replaced by another sexually active rat [18, 19].
Preparation of female rats
Oestrus (heat) was developed artificially in female rats by giving progesterone (0.5 mg / 100 g body weight) and estradiol benzoate (10 microgram/100 g body weight) over the subcutaneous route sequentially before mating. Before conducting the experiment the receptivity of female rats was ensured [18, 19].
A number of parameters such as mounting behaviour, mating performance, orientation activities of male rats towards the surrounding, towards female and towards themselves were observed for the assessment of aphrodisiac activity of the extract.
Mounting behaviour test
The climbing property of male rats onto the female rats is known as “Mounting”. To assess the number of mounts, the mounting behaviour test was performed. Males, treated with the extract, were paired with non-oestrus female rats. The behaviour of the animals was noted for 3 h on the 10th, 20th and 40th day of the study. Male rats were placed individually in each cage. After 15 min of acclimation, non-oestrus female was introduced into each cage and number of mounts were noted at the beginning of 1st hour for next 15 min. Every female was then given a resting period of 105 min by pulling it from each cage. The females were again introduced into the cages and the number of mounts were again noted for 15 min before the end of 3rd hour of the test. The test was performed at room temperature of 25–27 °C between 9 a.m. to 12 p.m. [18, 19].
Determination of mating performance
In this test every male rat was settled separately in a glass cage. After the acclimation period of 15 min, five oestrus females were admitted into each cage and they cohabited overnight. For the detection of any sperms in females, microscopic investigation of the vaginal smear of every female rat was performed. Sperm positive females were noted in every group [18, 19].
Test for libido
In this test sexually experienced male rats demonstrating vigorous sexual performance were selected. Mounting Frequency (MF) was observed in these rats in the evening of 10th, 20th and 40th day. Before initiating the observation, the sheath of the penis was retracted to expose the penis of rats and xylocaine 5% ointment (AstraZeneca Pharma) was applied at the interval of 5, 15 and 30 min. Each male rat was allocated separately in a cage and female rats were admitted in the similar cage. The total number of intromissions and ejaculations were observed [20].
Determination of the orientation activities of male rats
In this test the behavioural activities of the male rats, towards the surrounding, towards themselves and towards females were examined. The rats were observed and the number of climbings, genital groomings, lickings and anogenital sniffings were counted for one hour [21].
Preparation of serum for testosterone analysis
The blood was collected via cardiac puncture using chloroform anaesthesia. Using sterilized syringe, 5 ml of blood was collected in appropriately labelled blank tubes containing no anti-coagulant. The tubes were given 5 to 10 min for coagulation before they were subjected to centrifugation. The needles were changed for every single animal. After coagulation these tubes were centrifuged at 3000 rpm for 10 min. The supernatant formed after centrifugation was sucked and collected in a clean, empty tube for testosterone assay [22].
Testosterone analysis
Quantitative determination of total testosterone was done by the competitive imunoenzymatic colorimetric method mentioned in the manufacturer’s test procedure (Diametra testosterone ELISA kit). The absorbance was noted at 420 nm against a reference wavelength of 620–630 nm.
Statistical analysis
One-way ANOVA followed by Tukey’s test was used for analysing the data. Data were expressed as mean ± standard error of mean. A statistical level of 0.05 or less was accepted as significant.
