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Effect of Aqueous Extract of Solanum melongena Fruits (Garden Eggs) on Some Male Reproductive Variables in Adult Wistar Rats

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Abstract

The effects of administration of aqueous extract of Solanum melongena on some reproductive variables of male Wistar rats were exploited in this work. 20 rats were randomized into four groups (Group A, B, C and D) of 5 rats each. Group A serve as the control and was administered 0.9% normal saline, group B, C and D serve as the extract group receiving 200mg/kg, 400mg/kg and 800mg/kg of the extract respectively. The extract was save with LD50 >5000mg/Kg. Sperm counts and motility were quantified; epididymal and general body weights were measured using a weighing balance. The histological studies of the testes and the anterior pituitary were also done. Results of the experiment revealed an increase in the sperm count and sperm percentage motility. There was decrease in body weight gain and epididymal weight across the groups receiving the extract, though not significant. This experiment thus revealed that aqueous extract of Solanum melongena possesses profertility properties which may be beneficial to those who consume it.
Report and Opinion 2015;7(9) http://www.sciencepub.net/report
55
Effect of Aqueous Extract of Solanum melongena Fruits (Garden Eggs) on Some Male Reproductive
Variables in Adult Wistar Rats
1D.U. Jamil, 3M.A. Mabrouk, 2A.W. Alhassan, 2R.A. Magaji
1. Department of Physiology, College of Medicine and Allied Health Professions, Federal University, Dutse, Jigawa
State, Nigeria.
2. Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria.
3. Department of Human Physiology, Faculty of Medicine, Bayero University, Kano, Nigeria.
*Corresponding author: jamilusman@fud.edu.ng or jamso85@yahoo.com, +2348030801260
Abstract: The effects of administration of aqueous extract of Solanum melongena on some reproductive variables of
male Wistar rats were exploited in this work. 20 rats were randomized into four groups (Group A, B, C and D) of 5
rats each. Group A serve as the control and was administered 0.9% normal saline, group B, C and D serve as the
extract group receiving 200mg/kg, 400mg/kg and 800mg/kg of the extract respectively. The extract was save with
LD50 >5000mg/Kg. Sperm counts and motility were quantified; epididymal and general body weights were
measured using a weighing balance. The histological studies of the testes and the anterior pituitary were also done.
Results of the experiment revealed an increase in the sperm count and sperm percentage motility. There was
decrease in body weight gain and epididymal weight across the groups receiving the extract, though not significant.
This experiment thus revealed that aqueous extract of Solanum melongena possesses profertility properties which
may be beneficial to those who consume it.
[D.U. Jamil, M.A. Mabrouk, A.W. Alhassan, R.A. Magaji. Effect of Aqueous Extract of Solanum melongena
Fruits (Garden Eggs) on Some Male Reproductive Variables in Adult Wistar Rats. Rep Opinion 2015;7(9):55-
61]. (ISSN: 1553-9873). http://www.sciencepub.net/report. 8
Keywords: Garden egg fruits (Solanum melongena), Sperm count, Sperm motility, Histology, Testes and pituitary.
1. Introduction
Medicinal plants have long been used by the
peoples of the world for treating different types of
illnesses, but some various substances found in them
may be harmful to the organism. Many plants are
known for their teratogenic and abortive properties
(Norton, 1996; Mengue et al., 2001) while others are
known for their toxic effects on the male reproductive
system (Soufir et al., 1989).
There are several numbers of agents that can
cause adverse effect on the male reproductive system.
These occur by interfering with sexual maturation,
production, and transport of spermatozoa, the
spermatogenic cycle, sexual behavior and fertility
(Kimmel et al., 1995). These agents may also play an
adverse or beneficial roles on the leydig cells, thereby
affecting testosterone production (Mooradian et al.,
1987). Some studies have reported that the toxic effect
of these agents in the epididymis affect fertility
(Klinefelter et al., 1990; Vieira-Filho et al., 2002), by
disturbing the sperm maturation process, and the
functioning of the accessory sex glands (Zenick et al.,
1994).
About 80% of the population in developing
countries use medicinal plants and plant products in
handling some there primary medical problems, due to
their accessibility, availability and affordability (Telefo
et al., 2002; Cherdshewasart et al., 2007). In these
countries, a variety of plants are claimed to have
fertility regulating properties and a few have been
tested for such effects (Cherdshewasart et al., 2007;
Ganguly et al., 2007).
Solanum melongena (garden egg), is a common
and popular vegetable crop grown in the subtropics and
tropics. It is called “brinjal” in India, “yalo” in the
hausa tradition of northern nigeria and aubergine” in
Europe. Eggplant is a perennial plant but grown
commercially as an annual crop (Veeraragavathatham
et al., 2006; Westerfield, 2008).
Solanum melongena is one of the most important
vegetable crops grown on over 1.7 million hectares
world wide. China, India, Bangladesh, Nepal and
Srilanka accounts for about 75% of eggplant
production. It has been established that Solanum
melongena leaves has antipyretic and analgesic effect
(Mutalik et al., 2003), central nervous system
depressant and it is also a multiallergenic vegetable
(Vohora et al., 1984). It fruits is also use for weight
loss, long term control of asthma and as a prokinetic
agent.
Some varieties of the plant are claimed to have
fertility regulating properties and a few have been
tested for such effect. There is dearth of information for
Solanum melongena in that regards. But due to its
chemical constituent that revealed the presence of
dietary fiber, alkaloids, saponins, nasunin, ascorbic
acid, steroids, tannins, flavonoids, proteins and
carbohydrates in both the crown and the fruit (Hanson
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56
et al., 2006; Noda et al., 2000; Tiwari et al., 2009), as
well as it various medicinal claims and it high
consumption in some part of the world especially in the
Egbo tradition of the eastern part of Nigeria. We
decided to search for its fertilty effect. Therefore, the
objective of the current study is to determine the
fertility effect of aqueous extract of Solanum
melongena fruit extract on some reproductive variables
using adult male wister rats. The variables studied in
this research include; Sperm count, sperm motility
body weight change, epididymal weight and histology
of testes and anterior pituitary glands.
2. Materials and Methods
2.1 Plant Material
Fresh fruits of Solanum melongena were bought
from “Kasuwar Sabo” market in Zaria, Kaduna State of
Nigeria, in the month of September. Botanical
identification was performed at the Herbarium section
of Biological Science Department of Ahmadu Bello
University, Zaria, Kaduna State, Nigeria, and given a
voucher number 1939.
2.2 Extraction of plant material
The fruits were shade-dried for 10 days and
ground into powder. An aqueous extract was made
from 1kg of Solanum melongena fruits which was
soaked in distilled water (5L) and the mixture boiled
for 15 minutes. The heated decoction was taken and
allowed to cool at room temperature, filtered and oven-
dried as described by Pierrie et al., ( 2009) to give of
dried aqueous extract 60.2g (yield of extraction,
6.02%). The extraction was carried out in Department
of Pharmacognosy Laboratory, Ahmadu Bello
University, Zaria. The working solution was prepared
at a final concentration of 150mg/ml in distilled water.
2.3 Animals
Male Wistar rats (110–200g) were procured from
the Experimental Animal Unit of the
Vector/Parasitiology Unit of Nigerian Institute for
Trypanosomiasis and Onchocerciasis Research
(NITOR), Federal Ministry of Science and Technical,
Kaduna State Nigeria and used throughout the study.
They were housed in plastic boxes and acclimatized for
two weeks in a controlled environment (temperature
25±2 °C and 12 h dark/light cycle) with standard
laboratory diet and water ad libitum.
2.4 Acute Toxicity Studies
The acute toxicity studies (LD50) of Solanum
melongena fruits was determine using the method of
Lorke, 1983 . the studies was done in two phases. In
the first phase, 9 rats were used. The rats were
randomly divided into three groups having 3 rats in
each group. Group 1 received 10mg/Kg, group 2
received 100mg/Kg and group 3 received 1000mg/Kg
via oral route respectively, and observed for signs of
toxicity and death for 24 hours. In second phase, 4 rats
were used and consist of 4 groups with a rat in each
group. Group 1 received 1000mg/Kg, group 2 received
1600mg/Kg, group 3 received 2900mg/Kg and group 4
received 5000mg/Kg. The median lethal dose (LD50)
was determined at the end of the second phase.
2.5 Experimental protocol
Twenty male adult Wistar rats were randomly
divided into four (4) groups; one (1) control and three
(3) treatment groups. The rats in group A (Control)
were administered with 0.9% physiological saline,
while the treatment groups B, C, and D were
administered with graded doses of the Solanum
melongena fruits extract of 200, 400 and 800 mg/kg
body weight respectively. The animals were dosed
orally once daily for 28 days using rat oral canula. This
method was similar to that described by Oyeyemi, et
al., (2008). At end of the 28th day, the animals were
sacrificed for investigation of some these reproduction
variables.
2.6 Semen Analysis
The rats were anaesthetized using chloroform
soaked in cotton wool placed in a box. Orchidectomy
was performed by open castration method. The testicle
was exposed by incising the tunica vaginalis and the
cauda epididymis were harvested. The cauda
epididymis of rats in each of the experimental group
were removed and minced thoroughly in a specimen
bottle containing normal saline for few minutes to
allow the sperms to become motile and swim out from
the cauda epididymis (Saalu et al., 2008).
2.7 Sperm count and Motility studies
The semen was then taken with 1ml pipette and
dropped on a clean slide, and covered with cover slips.
The slides were examined under light microscope for
sperm motility (Saalu et al., 2008). And with the aid of
the improved Neubauer hemocytometer (Deep1/10mm
LABART, Germany) counting chamber as described
by Pant and Srivastava (2003), the spermatozoa were
counted under the light microscope. Counting was done
in five thoma chambers.
2.8 Histological studies
The testes of all the rats were fixed in 10%
formalin, while the pituitary glands were fixed using
Boiun’s fliud, and processed by the usual method for
paraffin embedding at Anatomy Department in
Ahmadu Bello University, Zaria, Nigeria. Section of 4-
5 µm thickness by microtome was taken, stained with
hematoxylin and eosin stain for histopathological
examination through light microscope by the usual
method described by Akpanatah et al., ( 2003)
2.9 Statistical analysis
Student t-test and one way analysis of variance
(ANOVA) were used to analyze the data. The results
were expressed as mean ± standard error of the mean
(SEM). The difference of the means was considered
significant at p< 0.05.
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3. Results
3.1 Acute toxicity studies
During the experimental procedure, no deaths, no
locomotor activity alteration, no piloerection or any
other clinical signs of toxicity were observed in any of
the groups in both phases even at a dose of 5000mg/kg.
Therefore, LD50>5000mg/Kg, indicate that the extract
is save and non toxic.
3.2 Semen Analysis
3.21 Sperm count
There was significant increase in the sperm count
across the extract groups when compared with the
control group, and the increase was dose dependent
(Table 1).
3.22 Sperm motility
Generally, there was increase in the motility of
sperm cells in all of the extract group when compared
with the control group receiving normal saline in a
dose dependent manner, but the increase was not
significant. There was also an increased number of
progressive motile sperm cells, while it was a decrease
in the non progressive motile and non motile sperm
cells in a dose dependent manner. These observed
changes was only statistically significant for the
progressive and non progressive motile sperm cells in
group receiving 200mg/kg of Solanum melogena
extract (Table 1).
3.3 Change in body and organ weight
There was non significant decrease in the changes
observed in the body and the epididymal weight of
animals across the group receiving the aqueous extract
of the Solanum melogena fruit (Table 1).
3.4 Histological studies
The histology of the testes in the group (A)
receiving normal saline (figure1: A1) appears normal,
with abundant basal cells (primary spermatogonia
cells) (Ps) and presence of sperm cells in the lumen (L)
of the seminiferous tubule. While group (B, C and D)
receiving 200mg/kg, 400mg/kg and 800mg/kg extract
of Solanum melongena respectively, shows increased
spermatogenic activities(arrows) toward the lumen(L)
(figure1: B1,C1 and D1).This is evidenced by the
reduced number of primary spermatogia cells(Ps)
which is an indication that they might have
differentiated to the next level of the spermatogenic
cells. The histology of the anterior pituitary gland in
the control group receiving normal saline appear
normal, with abundant basophilic cells (b) and
acidophilic cells(a)(figure1:A2). There was no
observed change in cytoarchitecture of the anterior
pituitary gland in the group (B, C and D) receiving the
extract (Figure1:B2,C2, and D2). Thus, they all appears
normal.
Table 1: Showing change in Body weight (g), epididymal weight (Wepi), Sperm count (x106/ml) and Sperm
motility (%)
Groups initial weight
Wi(g)
final weight
Wf(g)
weight gain
Wgain (g) Wepi(g) Progressive
motility (%)
non
progressive
motility(%)
Non motile
cells (%)
Sperm count(
x106/ml)
A(N/saline) 168.60±9.7 219.20±14.2 50.60±4.6 0.15±0.01 30.00±3.5 41.00±4.3 29.00±5.3 29.00±0.5
B(200mg/kg) 159.60±13.1 190.60±9.6 31.00±6.8 0.14±0.02 50.40±4.8* 26.20±4.3* 23.40±5.6 30.80±0.7
C(400mg/kg) 158.40±14.2 209.00±14.4 50.60±9.0 0.14±0.01 44.00±1.9 32.00±2.5 24.00±1.9 32.20±0.8*
D(800mg/kg) 165 .00±4.3 189.00±14.6 24.00±11.5 0.13±0.01 51.60±1.4 29.80±1.3 18.60±1.0 32.60±0.4*
*P Value<0.05 significant
A1
B1
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C1
D1
A2
B2
C2
D2
Figure 1: Histology of Tetes and Ansterior Pituitary glands (A1, B1, C1 and D1 are testes histology, while A1,
B1, C1 and D1 are the anterior pituitary gland histology) H&E x 250
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4. Discussion
The present study showed a significant increase in
epididymal sperm concentration in the treated group
which was in a dose dependent manner. The observed
increase could be ascribed to the importance of
Solanum melongena as a potent antioxidant and free
radical scavenger (Tiwari et al., 2009). Thus, support
the findings of Saalu et al., (2007a) that potent
antioxidant ameliorate the increased free radicals
generated by the natural and experimental stress,
thereby increasing the spermatogenic activity by
increasing the synthesis of testosterone from the
interstitial cells of Leydig (Saalu et al., 2007a).
Generally, there was increase in motility of sperm
cells in all the extract treated groups as compared to the
control group in a dose dependent manner, but the
increase was not statistically significant. There was
increase in the number of progressive motile sperm
cells in a dose dependent manner, but the increase was
only statistically significant in group receiving
200mg/kg of Solanum melogena extract. The non
progressive motile sperm cells number decrease non
significantly in all the test groups receiving Solanum
melongena extract when compared to the control group
in a dose dependent manner. Except for the group
receiving 200mg/kg extract that the decrease was
significant. The frequency of the non motile sperm
cells decrease in a dose dependent manner when
compared to the control group, though, the decrease
was not statistically significant. This result indicates
that Solanum melongena fruits extract has an effect on
the mitochondria found in the body of the
spermatozoon where energy is been synthesis in the
form of adenosine triphosphate, that increases the
sperm motility (Duke, 1997).
The basophilic cells of the anterior pituitary gland
is responsible for the secretion of follicle-stimulating
hormone (FSH) and luteinizing hormone (LH), which
act on the testes to enhances the process of
spermatogenesis (Moenter et al., 2003). No change was
observed in the cytoarchitecture of the anterior
pituitary glands. The basophilic and the acidophilic
cells in the anterior pituitary gland were intact in all the
extract groups when compared with those in the control
group. Thus, these results revealed to us that Solanum
melongena (garden egg) fruits extract does not affect
the functionality of anterior pituitary gland. Thus,
support the finding of Vohora et al., (1984). There is
no any observable lesion in the histology of the testes
in the entire extract groups when compared with the
control. This is in line with the work of Cody et al.,
Harbone and Williams, Who stated that plants
containing flavonoids are effective in prevention of
lesion, mainly because of their antioxidant properties
(Cody et al., 1986; Harborne and Williams, 2000).
However, in all the test groups, there was observed
increase in spermatogenic activities towards the lumen
of the seminiferous tubule. This increase cellular
activities was from the basement membrane up to the
lumen of the seminiferous tubules of the testes. This
was evidenced by the reduced number of primary
spermatogonia cells. This is an indication that they
might have differentiated to next level of
spermatogenic cells. This was mainly due to the
presence of potent antioxidant like favonoids that
sacavange free radicals and increase testosterone
formation by the interstitial cells of Leydig (Saalu et
al., 2007a).
There was no any disturbance in the
cytoarchitecture of pituitary gonadotrophic cells which
secrete stimulating hormones responsible for
reproductive endocrine functions. These also oppose
the finding of Elbetieha et al., (2001), who stated that
an increase or decrease in the weight of reproductive
organs is under hormonal control and could suggest a
disturbance of the reproductive endocrine functions.
The effect of aqueous extract of Solanum
melongena in this study shown that Solanum
melongena fruits has a potential to decrease mean body
weights of rats, although the changes in the mean body
weight was not statistically significant, when compared
to the control group. This is agreement with the work
of Edijala et al., (2005), who also recorded that garden
egg plant reduce weight gain by reducing the serum
total cholesterol, triglyceride and increase serum HDL-
cholesterol in his comparative study of the effect of
Solanum melongena (garden egg) fruit, oat and apple
on serum lipid profile. There was slight decrease in
weight of epididymis in the test groups in a dose
dependent manner when compared with the control
group, though it was not statistically significant despite
the increase in cellular activity in the testes. This
oppose the findings of Shittu et al., (2007) that says
increased cellular activities are key factor to be
considered in the evaluation of organ weights.
5. Conclusion
This work reveals that oral administration of
aqueous extract of Solanum melongena fruits possesses
profertility properties which may be beneficial to those
who consume it. These profertility properties can be
exploited in male fertility therapy.
Acknowledgement
The Author wishes to acknowledge the
contribution of academic and the non academic staff of
department of Human physiology, Ahmadu Bello
University, Zaria, Nigeria throughout the course of the
study.
Corresponding author
Dauda Usman Jamil
Report and Opinion 2015;7(9) http://www.sciencepub.net/report
60
Department of Physiology
College of Medicine and Allied Health Professions
Federal University, Dutse
Jigawa State, Nigeria.
Email: jamilusman@fud.edu.ng
or jamso85@yahoo.com,
Phone: +2348030801260
Co- Authored by
Professor Muhammad A. Mabrouk
Department of Human Physiology
Faculty of Medicine
Bayero University, Kano
Nigeria.
Dr Alhassan Abdulwahab
Department of Human Physiology
Faculty of Medicine
Ahmadu Bello University, Zaria
Nigeria.
Rabiu AbduSSALAM Magaji PhD
Department of Human Physiology
Faculty of Medicine
Ahmadu Bello University, Zaria
Nigeria.
Email: rabiumagaji@yhoo.co.uk
Phone:+2348023558721
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9/17/2015
... The extraction of the plant material was done by modification of the method reported by Jamil et al. [31]. The fruits were thoroughly washed in sterile water and were shade-dried to a constant weight in the laboratory. ...
... Also, the gonadosomatic index (GSI) value of HgCl 2 -treated and SM þ HgCl 2 group was significantly lower than that of control and SM treated groups. This is in disagreement with the report that the aqueous extract of SM fruits has the potential to decrease mean body weights of rats [31]. This also disagrees with the report that garden eggplant reduces weight gain by reducing the serum total cholesterol, triglyceride and increase serum HDL cholesterol in the comparative study of the effect of SM (garden egg) fruit, oat, and apple on serum lipid profile [58]. ...
... 3]. This is in accordance with the report of Jamil et al. [31], that aqueous extract of SM fruit did not induce any lesion in the histology of the testes after twenty-eight days of administration. These also concur with the work of Cody et al., Harbone and Williams, who stated that plants containing flavonoids are effective in the prevention of lesion, mainly because of their antioxidant properties [60e62]. ...
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Background Solanum melongena (SM) is commonly known as the garden egg fruit or eggplant. It can be eaten fresh or cooked and has a large history of consumption in West Africa. This study focused on interventions of aqueous extract of SM (garden eggs) fruits on Mercury chloride (HgCl2) induced testicular toxicity in adult male Wistar rats. Methods Thirty-two adult male Wistar rats were randomly divided into four groups (A-D) of eight (n = 8) rats each. Group A Served as control and was given 10 ml/kg/day of distilled water, Group B- 500 mg/kg B.W of SM, Group C received 40 mg/kg B.W HgCl2 and Group D- 500 mg/kg B.W of SM and 40 mg/kg B.W HgCl2). The administration was done by gastric gavage once a day, for twenty-eight consecutive days. Testicular weight, semen analysis revealing the sperm count and sperm motility were assessed, gross parameters of the testis and testicular histology were assessed. Testicular oxidative stress markers viz a viz malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) were also assessed. Results assessment of the histological profiles of the testes showed a derangement of the cytoarchitecture and deterioration of sperm quality after HgCl2 administration and a marked improvement was observed after SM administration. Similarly, SM was associated with increased antioxidant parameters (SOD, CAT, GPx, and GSH) and decreased MDA in SM + HgCl2 rats. Conclusion It was concluded that S. melongena offers protection against free radical mediated oxidative stress of rats with mercury chloride induced testicular toxicity.
... The extraction of the plant material was done by modification of the method reported by Jamil et al. (2015). The fruits were thoroughly washed in sterile water and were shade-dried to a constant weight in the laboratory. ...
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Background Estradiol valerate (EV) is known to cause hypothalamic-pituitary dysregulation of the gonadotrophin-releasing hormone. This study focused on the impact of aqueous extract of Solanum melongena (SM) on EV-induced ovarian-pituitary dysfunction in female Sprague-Dawley rats. Methods Thirty-two healthy female Sprague Dawley rats weighing (8 weeks old; weight 150–200g) were randomly divided into four groups of eight (n=8) rats each. Group A received 2 ml of distilled water, Group B received a single dose of 4 mg/kg body weight (bwt) of EV (i.p), Group C received 500 mg/kg bwt SM extract orally and Group D received a single dose of 4 mg/kg bwt of EV (i.p) and 500 mg/kg bwt of SM orally, the experiment lasted for 21days. The following parameters body weight, pituitary gland weight, ovary weight, ovary, and pituitary gland histomorphology, total antioxidant status (TOS), total oxidant status (TOS), oxidative stress index (OSI), total cholesterol (TC), triglyceride, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), progesterone, FSH, LH, Testosterone, and estradiol were assessed. Results Estradiol valerate decreases the number of healthy follicles, progesterone, estradiol, HDL, and TAS. And increase degenerated cystic follicles, TT, FSH, LH, triglyceride, TC, LAL, TOS, and OSI. Pituitary gland showed degenerative changes, characterized by neutrophil infiltrations and loss of cellular projections marked improvement was observed in the ovary, pituitary gland, hormone, and biochemical parameters after SM administration. Conclusion Solanum melongena offers protection against EV-induced ovarian-pituitary dysfunctions evidence by restoration of oxidative stress markers, steroidogenesis, and histopathological alterations back to control level.
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Effect of Garcinia Kola (G. kola) on various organs has been widely reported in literature, however, there is a dearth of literature on its effect in the reproductive system. 45 adult Sprague-Dawley (S-D) rats were used. The animals were divided into two experimental groups, ‘A' and ‘B' and a control group ‘C'. The experimental groups ‘A' and ‘B' received oral dose 100mg/kg b.w. of crude extract G. kola seeds six days/week for 2 and 8 weeks respectively. Histological sections prepared at the end of the study period revealed no significant changes in the seminiferous tubular cytoarchitecture. Increased secretions were observed in the lumen of the prostate gland and seminal vesicle when compared with the control. There was a slight reduction in the luminal spermatozoa volume within the epididymis in the test groups. Semen analysis revealed a significant reduction of sperm concentration in the eight weeks group. Sperm concentration values of 60.28 ± 1.08 x 106/ml were obtained for the control, 58.23 ± 0.89 x 106/ml and 46.14 ± 1.80 x 106/ml for the two and eight weeks experimental groups respectively (p<0.05). The hormonal assay showed a significant increase in serum peripheral levels of testosterone when compared with control. Mean values of 2.93 ± 0.53ng/ml, 2.96 ± 0.05ng/ml and 5.13 ± 1.70ng/ml were obtained for control, 2 and 8 weeks experimental groups respectively. There was a reduction in testicular weight and attendant increases in weights of the prostate gland and seminal vesicle in the experimental animals. These results suggest that G. Kola seed produces a reduction in sperm concentration which is duration dependent. KEY WORDS: Garcinia Kola; hormonal milieu; male reproductive organs. [Nig. Jnl Health & Biomedical Sciences Vol.2(1) 2003: 40-46]