G Omotoso, A Oyewopo, R Kadir, S Olawuyi, A Jimoh
garlic, semen parameters, superoxide dismutase
G Omotoso, A Oyewopo, R Kadir, S Olawuyi, A Jimoh. Effects Of Aqueous Extract Of Allium Sativum (Garlic) On Semen Parameters In Wistar Rats.. The Internet Journal of Urology. 2009 Volume 7 Number 2.
Garlic has been in use worldwide since ages, especially as food and for its health benefits. However, concern has been raised on its untoward effects on male reproductive functions. The present study examined the effects of aqueous garlic extract on some semen parameters and erythrocyte superoxide dismutase in Wistar rats. Twenty-four male Wistar rats were grouped into 3, and aqueous extract of garlic was administered orally at different doses (Group B: 500 mg/kg/d; Group C: 1000 mg/kg/d) to the 2 treated groups, and distilled water given to the control group (Group A), for 28 days. Sperm concentration, motility and morphology were studied, and the activity of superoxide dismutase (SOD) was measured. The results of the semen analysis revealed reduction in all the parameters, which was dose-dependent. The percentage of morphologically normal spermatozoa was significantly reduced, as well as sperm concentration, compared with findings in the control animals. Garlic also caused a significant reduction in SOD activity in the blood, and this was dose-dependent, as the least activity was recorded among the high dose group. As people desire to enjoy the maximum beneficial health effects of garlic, its potential to adversely affect the reproductive functions, especially at higher doses, should be borne in mind.
In the present study, the effect of orally administered aqueous extract of
Materials and Methods
Preparation of Garlic Aqueous Extract
The raw garlic plants were collected from Ilorin, Kwara State and authenticated at the Department of Plant Biology, University of Ilorin, Nigeria. The raw garlic cloves were peeled, chopped into small pieces and blended. It was then dissolved in distilled water and kept in the Refrigerator for 12 hours. The solution was thereafter filtered, and the filtrate was concentrated in a water bath at a temperature of 40oC, into the paste form, from which the required dosages (500 mg/kg/d and 1000 mg/kg/d) were prepared.
The experiments were performed in conformity with the Rules and Guidelines of the Animal Ethics Committee of the University of Ilorin.
Experimental Pharmacological Protocol
Twenty-four (24) rats with an average weight of about 116 g were randomly grouped into 3 classes as follows:
Group A:- Control Group given distilled water.
Group B:- Treated Group with aqueous extract of garlic in a dose of 500 mg/kg/d.
Group C:- Treated Group with aqueous extract of garlic in a dose of 1000 mg/kg/d.
Using a feeding tube (size-6), distilled water and aqueous garlic extracts were administered to the control and treated animals respectively for a period of 28 days.
Animal Sacrifice and Sample Collection
Twenty-four hours (24 h) after the last day of administration, the animals were weighed and thereafter sacrificed by cervical dislocation. Blood samples of animals were collected into lithium heparinized bottles. Using a midline abdominal incision, the abdominal cavity was opened to access the reproductive organs. The testes were excised and weighed using an electronic sensitive analytical balance (Gallenkomp FA 2104A).
Assay of Superoxide Dismutase (SOD) Activity
Estimation of superoxide oxidase (SOD) in the blood was based on the method described by Mistra and Fridovish15, using chemical reagents kit produced by Randox Laboratories Limited (UK). Samples were measured spectrophotometrically at 30 sec interval for 3 min, and the data expressed as IU/ml.
Epididymal Sperm Concentration
A modified method of Yokoi and Mayi16 was adopted in counting the spermatozoa from the right epididymis. The epididymis was minced with anatomic scizzors in 5 ml of normal saline, placed in a rocker for 10 min and allowed to incubate at room temperature for 2 min. After incubation, the supernatant was diluted at 1:100 with a solution containing 5 g sodium bicarbonate and 1 ml formalin (35%). The new improved Neuber’s counting chamber (haemocytometer) was used in counting the total number of spermatozoa. About 10 ml of the diluted sperm suspension was transferred to each counting chamber of the haemocytometer and was allowed to stand for 5 min, and thereafter observed under a binocular light microscope.
The fluid from the caudal epididymis was diluted with Tris buffer solution17 to 0.5 ml. An aliquot of this solution was observed under the light microscope at a magnification of x400. The mean motility estimation was reported as the final motility score for each sample.
The morphology of the spermatozoa was determined using the original dilution for motility, diluted 1:20 with 10% neutral buffered formalin (Sigma-Aldrich, Oakville, ON, Canada). The sperm cells were categorised based on the presence of one or more abnormal features such as tail defects (short, irregular, coiled or multiple tails); neck and middle piece defects (distended, irregular, bent middle piece, abnormally thin middle piece); and head defects (round head, small or large size, double or detached head). Findings were expressed as percentage of morphologically normal sperm18.
Data were analysed statistically by application of student’s t-test, using the SPSS version 15 software, presented as mean and standard error mean (SEM), and values of p <0.05 were considered to be statistically significant.
There were no statistically significant changes in the body weight difference of the experimental animals in all the groups, and the ratio of the testis weight to that of the body weight of each animal (Table 1:p>0.05). Semen parameters in all the treatment groups (Groups B and C) decreased when compared with the control group. Only a minimal decrease was observed in the percentage sperm motility of the treated groups, and in the percentage of morphologically normal spermatozoa of the low dose group (Table 2). The percentage of normal spermatozoa was markedly reduced in the animals exposed to 1000 mg/kg/d (high dose) of garlic (69.00 ± 1.22) compared with animals that received a lower dose of 500 mg/kg/d (85.50 ±1.66) and the control animals (86.25 ± 1.75) given distilled water. There was a decrease in sperm motility in the treatment groups (Control: 92.17±0.66; Low dose group: 90.87±0.76; High dose group:90.27±1.13), but this was not statistically significant (p>0.05). Sperm concentration decreased in the treated groups with the animals treated with 1000 mg/kg/d aqueous garlic extract having the lowest value (63.85 ± 3.03) compared with those that received 500 mg/kg/d of the extract (65.35 ± 3.13) and the control group (72.15 ±0.74) that received distilled water. Superoxide dismutase enzyme was markedly reduced in the low dose group (279.75 ± 05.02 IU/ml; p<0.05) compared with the control animals (490.00 ± 20.10 IU/ml), with a much more decreased activity in the group that received a higher dose of 1000 mg/kg/d (188.00 ± 09.82 IU/ml; p<0.05).
The effects of garlic on male gonads, and by extension, the male reproductive function, may appear somewhat different from the usual cytoprotective activities observed by various workers in other tissues and organs of the body19,3; although, the amount of garlic administered, to some extent, could be a factor12. Administration of garlic augments the activities of SOD and also protects the cells against the damaging effects of free radicals, as given by some biochemical and histopathological evidences3. However, on the testes, use of garlic has been noted to compromise some male reproductive functions, as it affects spermatogenesis and testosterone levels, which are vital to reproduction11.
Both at a lower and higher doses of garlic extract, the activity of SOD was noted in this study to decrease with increasing dosage. The endogenous antioxidative functions of the enzyme was compromised, thereby predisposing the cells of the testes to the detrimental effects of various oxygen radicals and other chemically-induced oxidative stress. In this study, both the percentage morphology and sperm count were significantly reduced, although sperm motility was only slightly reduced. Irrespective of the motility, in the event of increasing number of abnormal spermatozoa morphologically, spermatogenesis and other reproductive capability of an intact spermatozoon, could still be compromised. This is because previous studies have shown that use of