Middle East Fertility Society Journal (2011) 16, 154158

Middle East Fertility Society

Middle East Fertility Society Journal

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ORIGINAL ARTICLE

Sperm quotient in SpragueDawley rats fed graded doses

of seed extract of Momordica charantia
Oshiozokhai Eboetse Yama *, Francis Ikechukwu Duru, Ademola Ayodele Oremosu,
Abraham Adepoju Osinubi, Cressie Carmel Noronha,
Abayomi Olugbenga Okanlawon
Department of Anatomy, Faculty of Basic Medical Sciences, University of Lagos, Idi-Araba, Lagos, Nigeria
Received 18 January 2011; accepted 28 February 2011
Available online 1 April 2011

KEYWORDS
Momordica charantia;
SpragueDawley;
Testes;
Sperm production

Abstract Introduction: Momordica charantia has been investigated for its effect on various organs
and its numerous indications have been cited in literature. There are, however, scanty publications
on its effect on the male reproductive system.
Objective: To evaluate the effects of methanolic seed extract of M. charantia (MC) on the sperm
production (sperm number and motility), testicular volume and testicular testosterone in SpragueDawley (SD) rats.
Materials and methods: Twenty adult male SD rats, weighing 106200 g allotted randomly into
four main groups (A, B, C and D). Groups A, B and C received 15, 25 and 50 mg/100 g b.w/oral
of MC, respectively, daily. Group IV rats (control) were fed equal volume of physiological saline.
The duration of treatment for both extract and physiological saline was 56 days. The animals were
sacriced by cervical dislocation. Testicular volume, sperm count and motility and testicular testosterone estimated.

* Corresponding author. Address: Department of Anatomy, College

of Medicine of the University of Lagos, P.M.B. 12003, Lagos, Nigeria.
Tel.: +234 809321251.
E-mail address: dro_yama@yahoo.com (O.E. Yama).
1110-5690  2011 Middle East Fertility Society. Production and
hosting by Elsevier B.V. All rights reserved.
Peer review under responsibility of Middle East Fertility Society.
doi:10.1016/j.mefs.2011.02.001

Production and hosting by Elsevier

Sperm quotient in SpragueDawley rats fed graded doses of seed extract of Momordica charantia

155

Results: The sperm number and motility were found to be signicantly decreased (p < 0.05) with
increasing dose. Similarly a dose dependent decrease in the testicular testosterone concentrations
and testicular volume (p < 0.05) was also recorded.
Conclusion: M. charantia seed extract suppresses the sperm production in rats. Thus, it could be
developed into a contraceptive agent for men.
 2011 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

2. Materials and methods

Tropical forest plant species have served as a source of medicines for people of the tropics for millennia. Many medical
practitioners with training in pharmacology and/or pharmacognosy are well aware of the number of modern therapeutic
agents that have been derived from tropical forest species. In
fact, over 120 pharmaceutical products currently in use are
plant-derived, and about 75% of these were discovered by
examining the use of these plants in traditional medicine (1).
Yet while many modern medicines are plant-derived, the origins of these pharmaceutical agents and their relationship to
the knowledge of the indigenous people in the tropical forests
are usually omitted. The search for drugs and dietary supplements derived from plants has accelerated in recent years;
2550% of current pharmaceuticals are derived from them,
none are used as anti-fertility agents.
Traditional healers have long used plants to prevent contraception; Western medicine is trying to duplicate their successes (2). Plants are rich in a wide variety of secondary
metabolites, such as tannins, terpenoids, alkaloids, and
avonoids, which have been used in menstrual and pregnancy disorders (3). About four decades ago, there was a
strong interest in looking at plants as sources of new pharmaceutical agents. In fact, many modern pharmaceutical
companies can trace their origins to products originating
from plants. However, advances in molecular biology, genetic engineering, and computational chemistry in the late
1970s and 1980s and, even more recently, advances in combinatorial chemistry (4) created much promise within the
pharmaceutical industry, without the need to explore natures chemical diversity. Some plant-derived compounds
have been found to affect fertility. The use of plant products
to regulate fertility is of an ancient origin. In spite of numerous studies, no plant with conrmed contraceptive efciency
but devoid of toxicity has emerged so far (5). A promising
oral compound would allow metabolism by the liver and allow reduction of the dose below toxic level. Examples of
some plants (herbs) reported to possess anti-fertility properties are date palm, oil palm, gossypol, Carica papaya and
Momordica charantia (2,6,7).
M. charantia is a monoecious climber with oblong, green
coloured fruit that are extensively ribbed (8,9). The fruits are
elongated and resemble a warty gourd or cucumber. They
are emerald-green in colour when unripe and orange-yellow
when ripe. The bitter taste increases as it ripens. It is used traditionally as both food and medicine (9).
Antifertility property has been a subject of signicant evaluation using animal models with interest in developing an
effective oral male contraceptive. The present research was,
therefore, intended at investigating the effect of various concentrations of the crude extract M. charantia seed on sperm
production in SpragueDawley rats.

2.1. Anthology of M. charantia

The fresh fruits of M. charantia were procured locally in a market in Lagos State. It was authenticated in the Botany Department of University of Lagos (voucher specimen No. FHI
108422). The fruits were dried in an oven at temperatures between 30 and 40 C for about a week. The dried seeds were extracted and taken to the Pharmacognosy Department of
College of Medicine University of Lagos (CMUL), where they
were weighed, and the percentage yield/concentration of 230 g
of M. charantia in 1000 ml of methanol prepared.
2.2. Animals
Twenty adult male SD rats were assigned randomly into four
main groups: A, B, C and D. Each comprised ve adult male
SD rats, weighing 106200 g. They were obtained from the
animal house, CMUL and housed in the Anatomy Department CMUL in well-ventilated metal cages under standard
room conditions (temperatures 2930 C, relative humidity
5055%). They were exposed to a photoperiod of 12 h light,
alternating with 12 h darkness; fed rat chow (Livestock feeds
Plc. Ikeja, Lagos, Nigeria), and clean tap water were provided
ad libitum. The animals were kept for at least 2 weeks to acclimatize to the laboratory condition before experimentation.
2.3. Experimental protocol and autopsy schedule
The groups A, B and C were treated daily with 15, 25 and
50 mg/100 g body weight of MC orally, respectively. Group
IV rats (control) were fed equal volume of physiological saline.
The duration of treatment for both extract and physiological
saline was 56 days. A metal canula was used for the gavaging
and done between 13 and 16 h daily. The animals were sacriced 24 h after the last dose. Cervical dislocation was used
to induce brief anaesthesia, this followed a ventral laparotomy
to deliver testes per abdomen. The harvested testes and Cauda
epididymis were neatly dissected out, cleared of fat and connective tissue and weighed. Testicular weight and volume, sperm
number and motility including testicular testosterone (from
testicular homogenate) were all estimated.
2.4. Testicular homogenate: the supernatant processing
This is done by the modied method of Buege and August
(1978) (10) 0.25 g of testicular tissue sample was homogenized with a mortar and pestle, in 2.5 ml of 0.15 M KCl.
The homogenate was centrifuged at 1000g and the supernatant collected. An aliquot of 2 ml of thiobabituric acid
(0.375%, 1 mol/l), 15% trichloroacetic acid was added to
1 ml of the tissue supernatant and mixed vigorously, heated

156

O.E. Yama et al.

for 15 min in a boiling water bath (8090 C). The samples

were cooled in ice cold water and centrifuged at 1500g for
15 min.
2.5. Testicular gravimetry
The testicular volume was estimated by water displacement
method (Archimedes principle). The testicular weight was by
electronic balance (2).
2.6. Sperm number and motility analysis
Several small cuts were made in the C. epididymis which was
then placed in a sterile universal specimen bottle, containing
1 ml of normal saline to allow motile sperm to swim up from
the epididymis. Five microlitres of epididymal uid was delivered onto a glass slide covered with a 22 22 mm cover slip
(11) and examined under the light microscope at a magnication of 400. The microscopic eld was scanned systematically and each spermatozoon encountered was assessed.
Motility was determined by counting the number of immotile
spermatozoa and subtracting from the total count 100%.
The motility was simply classied as either motile or non-motile. The procedure was repeated and the average of the two
readings taken.
The sperm number was determined using the Neubauer
improved haemocytometre. A dilution ratio of 1:20 from
each well-mixed sample was prepared by diluting 50 ll of epididymal spermatozoa suspended in physiological saline with
950 ll diluent. The diluent was prepared by adding 50 g of
sodium carbonate and 10 ml of 35% (v/v) formalin to distilled water and making up the nal solution to a volume
of 1000 ml (11). Both chambers of the haemocytometer were
scored and the average count calculated, provided that the
difference between the two counts did not exceed 1/20 of
their sum (i.e., less than 10% difference). When two counts
were not within 10%, they were discarded, the sample dilution re-mixed and another haemocytometer was prepared
and counted. To minimize error, the count was conducted
three times on each epididymis. The average of all the six
counts (three from each side) from a single rat was taken
and this constituted one observation for the sperm number.
2.7. Testicular testosterone assay
Testosterone (T) in the homogenate supernatant was determined by the enzyme immunoassay technique based on the
principle of competitive binding between T and T-horseradish
peroxidase conjugate for a constant amount of rabbit anti-T

Table 1

2.8. Statistical analysis

Results were expressed as mean standard deviation. Analysis was carried out using analysis of variance (ANOVA) with
Scheffes post hoc test. The level of signicance was considered
at p < 0.05. All procedures involving animals in this study
conformed to the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the
Guiding Principles in the Care and Use of Animals (13) and
were approved by the Departmental Committee on the Use
and Care of Animals in conformity with international acceptable standards.
3. Results and discussion
The result of the effects of graded doses of M. charantia seed
extract on sperm production ( C. epididymal sperm number
and motility) reveals a dose dependent statistically signicant
decrease (p < 0.05) in the treated groups compared to control
(Table 1). While within the groups; C treated with 50 mg/100 g
signicant difference (p < 0.05) compared to groups A and B
treated with 15 and 25 mg/100 g (Table 1). Similarly an associated dose dependent signicant decrease in testicular testosterone concentrations (p < 0.05) compared to control (Table 2)
was observed. This decrease in sperm production or the cessation of spermatogenesis may be linked to the extract directly
suppressing the gonadal androgens resulting in a sub-optimal
testosterone levels. This supports the fact that sperm production cannot proceed to optimal completion without a continuous androgen supply (14). This is also in consonance with
recent studies which showed that no human subject having a
lower than normal testicular testosterone levels had a sperm
count greater than 20 million/ml or motility greater than
50% (15). The extract may also have had a circumlocutory
association with hormones of the extra-testicular axis (gonadotropins which are essential for initiation and maintenance of

Effects of varying doses of Momordica charantia seed extract on sperm production.

Groups (n = 20)
A
B
C
D

(12). Goat antirabbit IgG-coated wells were incubated with

T standards, controls, samples (supernatants of testicular
homogenates), T-horseradish peroxides conjugate reagent
and rabbit anti-T reagent at 37 C for 90 min. Unbound T peroxides conjugate was removed and the wells washed. Tetramethylbenzidine was added and incubated, resulting in the
development of blue colour. The colour development was
stopped with the addition of 1 MS HCl, and the absorbance
measured spectrophotometrically at 450 nm. A standard curve
was obtained by plotting the concentration of the standard
versus the absorbance and the T concentrations calculated
from the standard curve.

Group details
15 mg/100 g b.w
25 mg/100 g b.w
50 mg/100 g b.w
Physiological saline

All values are expressed as mean standard deviation; b.w = body weight.
a
Signicant difference at p < 0.05 compared to control (group D).
b
Signicant difference at p < 0.05 compared to groups A and B.

Sperm number (106)

a

63.5 10.45
40.5 10.45a
18.01 21.33a,b
219.2 7.52

Sperm motility (%)

70.00 7.07
52.00 0.01a
29.20 13.25a
93.6 7.89

Sperm quotient in SpragueDawley rats fed graded doses of seed extract of Momordica charantia
Table 2

157

Effects of graded doses of Momordica charantia seed extract on testicular testosterone (TT), weight (TW) and volume (TV).

Groups (n = 20)

Treatment group

TT (mmol/l)

TW (g)

TV (ml)

A
B
C
D

15 mg/100 g b.w
25 mg/100 g b.w
50 mg/100 g b.w
Physiological saline

16.14 0.78
13.50 1.43a
10.30 0.95a
18.01 0.83

0.90 0.08
0.56 0.30a
0.32 0.27a
1.14 0.22

0.91 0.08
0.57 0.31a
0.33 0.29a
1.14 0.23

All values are expressed as mean standard deviation; b.w = body weight.
a
Signicant difference at p < 0.05 compared to control (group D).

spermatogenesis) by inhibiting the latter, the testosterone production/supply is compromised, since these hormones (essentially luteinizing hormone) through specic receptors found
on the surface of Leydig cells are known to control testosterone production and secretion (1618). Although the levels of
gonadotropins were not estimated in this study the observed
reduction in the number of sperm number and motility may
indicate lowered availability of the gonadotropins.
The mean testicular weights in grams of the testes were similar to the values obtained for the testicular volume in millilitres, giving a mean testicular density of one; this follows the
same pattern in all. The mean testicular weight and volume
of rats fed with physiological saline were 1.14 0.22 g and
1.14 0.23 ml (Table 2). These values became decreased signicantly with increasing concentration of the extract. Thus
the extract concentrations of 15, 25 and 50 mg/100 g b.w reduced the weight of the testes to 0.90 0.08, 0.56 0.30
and 0.32 0.27 g and the volume to 0.91 0.08,
0.57 0.31 and 0.33 0.29 ml, respectively (Table 2). This
reduced testicular weight and volume indicate a wide spread
destruction (19) which could be the depleted protein elements
in these testes (20,21). Similarly the testicular volume has been
shown to associate positively with testosterone level, as well as
testicular function (22,23). This means that the decreased testosterone concentration and reduced testicular volume and
weight as indicated in our ndings (Table 2) signied both
an extensive testicular injury and compromised spermatogenesis and male infertility.
It is concluded from these obtained data that methanolic
seed extract of MC at an oral dose of 50 mg/100 g/day produced a better sterility in male rats as compared to the other
doses.
Although the oral ingestion of the fruits is safe as demonstrated by its long-term consumption in Asian cultures (9); a
case of paroxysmal atrial brillation was reported recently
with the use of the extract (24). However, the toxicity, and
safety margin of the seed must be assessed in well-designed human trials. Other reported toxicities include hypoglycaemic
coma and convulsions in children, a favism-like syndrome,
and increases in gamma-glutamyltransferase and alkaline
phosphatase levels in animals (25).
Thus the future use of MC extract as a contraceptive agent
would dependent on successful isolation of the active principles, toxicological evaluation and its reversibility within a predictable time frame.
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