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ORIGINAL ARTICLE
Sperm quotient in Sprague–Dawley 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;
Sprague–Dawley;
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 Spra-
gue–Dawley (S–D) rats.
Materials and methods: Twenty adult male S–D rats, weighing 106–200 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
sacrificed by cervical dislocation. Testicular volume, sperm count and motility and testicular testos-
terone 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
Middle East Fertility Society Journal (2011) 16, 154–158
Middle East Fertility Society
Middle East Fertility Society Journal
www.mefsjournal.com
www.sciencedirect.com
Results: The sperm number and motility were found to be significantly 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
Tropical forest plant species have served as a source of medi-
cines for people of the tropics for millennia. Many medical
practitioners with training in pharmacology and/or pharma-
cognosy 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 ori-
gins 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 supple-
ments derived from plants has accelerated in recent years;
25–50% of current pharmaceuticals are derived from them,
none are used as anti-fertility agents.
Traditional healers have long used plants to prevent con-
traception; Western medicine is trying to duplicate their suc-
cesses (2). Plants are rich in a wide variety of secondary
metabolites, such as tannins, terpenoids, alkaloids, and
flavonoids, which have been used in menstrual and preg-
nancy disorders (3). About four decades ago, there was a
strong interest in looking at plants as sources of new phar-
maceutical agents. In fact, many modern pharmaceutical
companies can trace their origins to products originating
from plants. However, advances in molecular biology, genet-
ic engineering, and computational chemistry in the late
1970s and 1980s and, even more recently, advances in com-
binatorial chemistry (4) created much promise within the
pharmaceutical industry, without the need to explore nat-
ure’s 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 numer-
ous studies, no plant with confirmed contraceptive efficiency
but devoid of toxicity has emerged so far (5). A promising
oral compound would allow metabolism by the liver and al-
low reduction of the dose below toxic level. Examples of
some plants (herbs) reported to possess anti-fertility proper-
ties 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 tra-
ditionally as both food and medicine (9).
Antifertility property has been a subject of significant eval-
uation using animal models with interest in developing an
effective oral male contraceptive. The present research was,
therefore, intended at investigating the effect of various con-
centrations of the crude extract M. charantia seed on sperm
production in Sprague–Dawley rats.
2. Materials and methods
2.1. Anthology of M. charantia
The fresh fruits of M. charantia were procured locally in a mar-
ket in Lagos State. It was authenticated in the Botany Depart-
ment of University of Lagos (voucher specimen No. FHI
108422). The fruits were dried in an oven at temperatures be-
tween 30 and 40 C for about a week. The dried seeds were ex-
tracted 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 S–D rats were assigned randomly into four
main groups: A, B, C and D. Each comprised five adult male
S–D rats, weighing 106–200 g. They were obtained from the
animal house, CMUL and housed in the Anatomy Depart-
ment CMUL in well-ventilated metal cages under standard
room conditions (temperatures 29–30 C, relative humidity
50–55%). 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 accli-
matize 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 sacri-
ficed 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 connec-
tive 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 modified method of Buege and August
(1978) (10) 0.25 g of testicular tissue sample was homoge-
nized with a mortar and pestle, in 2.5 ml of 0.15 M KCl.
The homogenate was centrifuged at 1000g and the superna-
tant 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
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia 155
for 15 min in a boiling water bath (80–90 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 fluid was deliv-
ered onto a glass slide covered with a 22 · 22 mm cover slip
(11) and examined under the light microscope at a magnifica-
tion of ·400. The microscopic field was scanned systemati-
cally 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 classified as either motile or non-mo-
tile. 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 epi-
didymal 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 dis-
tilled water and making up the final 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 dilu-
tion 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 deter-
mined 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
(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 per-
oxides conjugate was removed and the wells washed. Tetra-
methylbenzidine 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.
2.8. Statistical analysis
Results were expressed as mean ± standard deviation. Analy-
sis was carried out using analysis of variance (ANOVA) with
Scheffe’s post hoc test. The level of significance was considered
at p < 0.05. All procedures involving animals in this study
conformed to the guiding principles for research involving ani-
mals 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 accept-
able 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 significant
decrease (p < 0.05) in the treated groups compared to control
(Table 1). While within the groups; C treated with 50 mg/100 g
significant difference (p < 0.05) compared to groups A and B
treated with 15 and 25 mg/100 g (Table 1). Similarly an associ-
ated dose dependent significant decrease in testicular testoster-
one concentrations (p < 0.05) compared to control (Table 2)
was observed. This decrease in sperm production or the cessa-
tion 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 produc-
tion cannot proceed to optimal completion without a continu-
ous 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 (gonad-
otropins which are essential for initiation and maintenance of
Table 1 Effects of varying doses of Momordica charantia seed extract on sperm production.
Groups (n = 20) Group details Sperm number (·10
6
) Sperm motility (%)
A 15 mg/100 g b.w 63.5 ± 10.45
a
70.00 ± 7.07
B 25 mg/100 g b.w 40.5 ± 10.45
a
52.00 ± 0.01
a
C 50 mg/100 g b.w 18.01 ± 21.33
a,b
29.20 ± 13.25
a
D Physiological saline 219.2 ± 7.52 93.6 ± 7.89
All values are expressed as mean ± standard deviation; b.w = body weight.
a
Significant difference at p < 0.05 compared to control (group D).
b
Significant difference at p < 0.05 compared to groups A and B.
156 O.E. Yama et al.
spermatogenesis) by inhibiting the latter, the testosterone pro-
duction/supply is compromised, since these hormones (essen-
tially luteinizing hormone) through specific receptors found
on the surface of Leydig cells are known to control testoster-
one production and secretion (16–18). 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 sim-
ilar to the values obtained for the testicular volume in millili-
tres, 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 sig-
nificantly with increasing concentration of the extract. Thus
the extract concentrations of 15, 25 and 50 mg/100 g b.w re-
duced 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 tes-
tosterone concentration and reduced testicular volume and
weight as indicated in our findings (Table 2) signified both
an extensive testicular injury and compromised spermatogene-
sis 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 pro-
duced a better sterility in male rats as compared to the other
doses.
Although the oral ingestion of the fruits is safe as demon-
strated by its’ long-term consumption in Asian cultures (9);a
case of paroxysmal atrial fibrillation 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 hu-
man 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 princi-
ples, toxicological evaluation and its reversibility within a pre-
dictable time frame.
References
(1) Fansworth NR, Bingel AS, Copdell GA, Crane FA, Fong HHS.
Potential value of plants as source of new antifertility agents. Part
II. J Pharmaceut Sci 1975;64:717–53.
(2) Yama OE, Osinubi AA, Noronha CC, Okanlawon AO. Effect of
methanolic seed extract Momordica charantia on body weight and
serum cholesterol level of male Sprague–Dawley rats. Nigerian
Quart J Hosp Med 2010;20(4):209–13.
(3) Technical data on Bitter melon (Momordica charantia), vol. 103;
2002. p. 3–7.
(4) Biere DE, Carlson TJ, King SR. Sharma Pharmaceuticals:
integrating indigenous knowledge, tropical medicinal plants,
medicine, modern science and reciprocity into a novel drug
discovery approach. <http://www.netsci.org/Science/Special/
feature11.html>.
(5) Sharma RS, Rojalakshimi M, Anthony-Jeyaraj D. Current status
of fertility control methods in India. J Biosci 2001;26:391–405.
(6) Fansworth NR, Waller DP. Current status of plant products
reported to inhibit sperm. Res Front Fertil Regul 1982;2:1–16.
(7) Gu ZP, Mao BY, Wang YX, Zhang RA, Tan YZ, Chen ZX,
et al.. Low dose gossypol for male contraception. Asian J Androl
2000;2(4):283–7.
(8) Bates DM, Merrick LC, Robinson RW. Minor cucurbits. Evolu-
tion of crop plants. New York: John Wiley & Sons; 1995, p. 110.
(9) Alternative Medicine Review. Description of Momordica charan-
tia, a member medicine. Monograph, Clinical report, vol. 12(4);
2007. p. 360–3.
(10) Buege JA, Aust SD. Microsomal lipid peroxidation. Methods
Enzymol 1978;52:302–10.
(11) Barratt CLR. On the accuracy and clinical value of semen
laboratory tests. Hum Reprod 1995;10:250–2.
(12) Tietz NW. Clinical guide to laboratory tests. 3rd ed. Philadelphia:
W.B. Saunders Company; 1995.
(13) American Physiological Society. Guiding principles for research
involving animals and human beings. Am J Physiol Regul Integr
Comp Physiol 2002;283:R281–3.
(14) Mohri H, Suter DA, Brown-Woodman PD, White IG, Ridley
DD. Identification of the biochemical lesion produced by alpha-
chlorohydrin in spermatozoa. Nature 1975;255(5503):75–7.
(15) Osinubi AA, Adeyemi A, Banmeke A, Ajayi G. The relationship
between testosterone concentration sperm count and motility in
fertile Nigerian males. Afr J Endocrinol Metab 2003;1:43–5.
(16) de Krester DM. Local regulation of testicular function. Int Rev
Cytol 1987;10:89–112.
(17) de Krester DM, Kerr JB. The cytology of the testis: the
physiology of reproduction. New York: Raven Press; 1988.
(18) Jarrow JP, Chen H, Rosner W, Trentacoste S, Zirkin BR.
Assessment of the androgen environment within the human testis:
minimally invasive method to obtain intratesticular fluid. J
Androl 2001;4:640–5.
(19) Abney T. The potential roles of estrogens in regulating Leydig
cell development and function: a review. Steroids 1999;
64:610–7.
(20) Sharma A, Verma PK, Dixit VP. Effect of semecarpus anacar-
dium fruits, on the reproductive function of male albino rats.
Asian J Androl 2003;5:121–4.
(21) Lohiya NK, Manivannan B, Mishra PK, Pathak N, Sriram S,
Bhande SS, Panneerdoss S. Chloroform extract of Carica papaya
seeds induces long-term reversible azoospermia in langur monkey.
Asian J Androl 2002;4(1):17–26.
Table 2 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 15 mg/100 g b.w 16.14 ± 0.78 0.90 ± 0.08 0.91 ± 0.08
B 25 mg/100 g b.w 13.50 ± 1.43
a
0.56 ± 0.30
a
0.57 ± 0.31
a
C 50 mg/100 g b.w 10.30 ± 0.95
a
0.32 ± 0.27
a
0.33 ± 0.29
a
D Physiological saline 18.01 ± 0.83 1.14 ± 0.22 1.14 ± 0.23
All values are expressed as mean ± standard deviation; b.w = body weight.
a
Significant difference at p < 0.05 compared to control (group D).
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia 157
(22) Mahmoud AM, Goemaere S, El-Grem Y, Van-Pottelbergh I,
Comhaire FH, Kaufman JM. Testicular volume, in relation to
hormonal indices of gonadal function in community-dwelling
elderly men. J Clin Endocr Metab 2003;88:179–84.
(23) Takihara HL, Cosentino MJ, Sakatoku J, Cockett ATS. Signif-
icance of testicular size measurement in anthropology: II.
Correlation of testicular size, with testicular function. J Urol
1987;137:416–9.
(24) Ismail E, Serkan O, Emine CE, Sabri OC. A case of atrial
fibrillation due to Momordica charantia (bitter melon). Ann Saudi
Med 2010;30(1):86–7.
(25) Basch E, Gabardi S, Ulbricht C. Bitter melon (Momordica
charantia): a review of efficacy and safety. Am J Health Syst
Pharm 2003;60(4):356–9.
158 O.E. Yama et al.
