Evaluation of The Deleterious Effects of Aqueous Fruit Extract of Abelmoschus Esculentus (Okro Fruit) on Some Male Reproductive Parameters in Sprague Dawley Rats

Abstract

Objective: The reversible reproductive impact of oral administration of methanol fruit extract of Abelmoschus esculentus was assessed in pubertal male Sprague Dawley rats.
Design: The animals were divided into the following groups; Control group (six rats), Extract-treated (seven rats), One week of recovery group (five rats), two weeks recovery group (five rats). A dose of 70mg/kg body weight/day of methanol fruit extract of A. esculentus was given orally to the extract treated, one-week recovery and two-weeks recovery groups respectively for a period of four weeks (28days). The recovery groups were left untreated after the four weeks for one and two weeks respectively.
Results: Methanol fruit extract of A. esculentus significantly reduced (P0.05). Methanol fruit extract of A. esculentus reduced the mean weight of testes; this is supported by the histological studies which showed testicular atrophy. There was significant reduction in the weight of the prostate gland. On the other hand, methanol fruit extract of A. esculentus significantly increased body weight (P

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Journal of Phytology 2009, 1(6): 461–468 ISSN: 2075-6240
© Journal of Phytology, 2009 Available Online: www.journal-phytology.com
REGULAR ARTICLE
EVALUATION OF THE DELETERIOUS EFFECTS OF AQUEOUS
FRUIT EXTRACT OF ABELMOSCHUS ESCULENTUS (OKRO
FRUIT) ON SOME MALE REPRODUCTIVE PARAMETERS IN
SPRAGUE DAWLEY RATS
I.I. Olatunji-Bello1, T. Ijiwole2, F.O. Awobajo2*
1Department of Physiology, College of Medicine, Lagos State University, Ikeja
2Department of Physiology, College of Medicine, University of Lagos, Nigeria
SUMMARY
Objective: The reversible reproductive impact of oral administration of methanol fruit
extract of Abelmoschus esculentus was assessed in pubertal male Sprague Dawley rats.
Design: The animals were divided into the following groups; Control group (six rats),
Extract-treated (seven rats), One week of recovery group (five rats), two weeks recovery
group (five rats). A dose of 70mg/kg body weight/day of methanol fruit extract of A.
esculentus was given orally to the extract treated, one-week recovery and two-weeks
recovery groups respectively for a period of four weeks (28days). The recovery groups
were left untreated after the four weeks for one and two weeks respectively.
Results: Methanol fruit extract of A. esculentus significantly reduced (P<0.05) gross sperm
motility, sperm count, life/dead ratio and it increased the occurrence of abnormal sperm
cell in the semen sample. Methanol fruit extract of A. esculentus had no effect on
epidydimal volume neither did it have any significant effect on both testosterone and
luteinizing hormone serum levels (P>0.05). Methanol fruit extract of A. esculentus reduced
the mean weight of testes; this is supported by the histological studies which showed
testicular atrophy. There was significant reduction in the weight of the prostate gland. On
the other hand, methanol fruit extract of A. esculentus significantly increased body weight
(P<0.05). These effects were reversed after two weeks recovery period.
Conclusion: These results showed that oral administration of methanol fruit extract of A.
esculentus produced a reversible reduction in male fertility assessment parameters in male rats.
Keywords: Abelmoschus esculentus, Male hormonal profile, Sperm motility, Sperm count, Male Fertility.
I.I. Olatunji-Bello et al. Evaluation of The Deleterious Effects of Aqueous Fruit Extract of Abelmoschus Esculentus (Okro Fruit) on Some Male Reproductive Parameters in
Sprague Dawley Rats. J Phytol 1 (2009) 461-468
*Corresponding Author, Email: funmi_bajo@yahoo.com

I.I. Olatunji-Bello et al./J Phytol 1 (2009) 461-468
1. Introduction
There has been a renewed attention in the
use of traditional medicine globally in the past
decade. This is with varying degree of
dependence in various country of the world,
with developing countries not left out [1]. The
easy accessibility and affordability is part of
the reasons for such broad use.
Several vegetables that are eaten or used for
medicinal purposes have been implicated with
adverse effects on both male [2,3] and female
reproductive functions [4,5]. Usually, in the
diagnosis and treatment of infertility among
couples the male factor is now been taken
serious and important. This is because several
reports have put the male factor in infertility
among married couples at different
percentages which are significant. Onwudiegu
and Bako [6] reported that it accounts for up
to 70%, while Idrisa et al reported [7] 46%.
Razzak and Wais [8] put their own value at
36.8%.
Abelmoschus esculentus is a vegetable and
also a medicinal plant with wide application in
folk medicine. It is a shrub that grows to about
two meters with yellow flower and succulent
seed pod emanating from the flower. Both the
leaves and the seed pod are major ingredients
in several local dishes across West Africa
countries. The seed pod is used as soup
thickening. The roasted seed can serve as
coffee substitute [9]. Extracts from the seed
was reported to be used as antispasmodic [10].
Because of the high fibre content, the stem is
used in making paper [11]. As a green
vegetable, it is a good source of Iron, Vitamin
C, Calcium and dietary fibre [12].
In folk medicine in south west Nigeria, men
are advised against the consumption of A.
esculentus seed pod. Reasons given include its
anti-male fertility potentials. It was to this
effect that we therefore decided to investigate
the likely effect of methanol-seed-pod extract
on A. esculentus on some male reproductive
functions using laboratory animal model.
2. Materials and methods
Animal and Treatment
Adult male Sprague Dawley rats weighing
between 120-270g were used for this study.
They were housed in standard plastic cages in
a clean animal room. Clean water and feed was
provided ad libitum throughout the
experimental period. The animals were
grouped into three groups. Extract treated
groups were administered 70mg/kg body
weight of methanol fruit extract of A. esculentus
orally per day for 28days. The one and two
weeks recovery groups were also treated with
the same dose of the extract for twenty eight
days before been allowed to recover for the
specified number of days (7days and 14days
respectively). The control group received equal
volume of distilled water. All animals were
allowed free access to water and feed
throughout the experimental period. All
animals were weighed weekly throughout the
experimental period.
Fresh okra fruits were purchased from
Ojuwoye market in Mushin, Lagos state,
Nigeria. The fruits were authenticated at the
Department of Pharmacognosy, Faculty of
Pharmacy, University of Lagos. The fruits were
oven dried at a temperature of 300C pounded
in a mortal and extraction carried out using
soxhlet extractor. The resultant yield used in
this experiment was stored under 40 C, and
prepared in distil water daily for use. At the
end of the experimental period allowed each
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group of animals were anesthetized with 25%
Urethane chloralose (0.5ml/100g i.p.) and the
following test carried out;
Semen analysis
Semen analysis was carried out under room
temperature to avoid heat or cold shock effect on
the sperm cells. The testes and the epididymids
were carefully and quickly removed. The caput of
the right epididymis was quickly transferred to
pre-warmed slide (270C), lacerated with a razor
and semen milk released for motility study.
Subjective Gross motility score was done
according to the method of Raji and co workers
[2]. The left epididymis was immersed in 5mls of
saline in order to measure the epididymis volume.
The epididymis organ then mashed in a mortar
and sperm count carried out using improved
Neubbauer haemocytometer while adjustment
was made for the volume of saline added. Few
drops of eosin-nigrosin stain was added to the
slide containing semen milk after motility score, in
order to prepared for sperm morphology study.
Morphology study was done according to the
method of Oyeyemi and co workers [13].
Histological studies
The testis, prostate gland, heart, liver, and
kidney were also removed, weighed and
preserved in formal saline for histological
studies. The tissues of the testes and
epididymis embedded in paraffin wax. Section
of 5 µm thick were cut and stained in
haematoxylin and eosin. The testis
histopathology was performed according to
Russell [14,15].
Hormonal assay
Blood samples were collected from the
animals into sterile bottles via cardiac
puncture. Blood samples were centrifuged at
300rpm for 20 minutes to obtain the serum
used for hormonal assay. Assay was carried
out using enzyme immunoassay kit supplied
by Immunometrics U.K.
Statistical analysis
All data were presented as Mean ± SEM
and analyzed using one way ANOVA. Level of
significance was placed at P< 0.05. Bar chart
was also used for graphical representation.
3. Results
1. Results of Phytochemical analysis of the fruit
extract of Abelmoschus esculentus
4. Discussion
By WHO standards, male fertility
assessment is routinely carried out by semen
analysis which yields information about the
integrity of the semen sample needed to
provide spermatozoa for fertilization of the
ovum [15]. Oral administration of methanol
fruit extract of Abelmoschus esculentus caused a
significant weight gain in rats at the second
week of treatment only compared with the
Control group of rats that received equal
volume of water. Beyond this point both
Control and extract treated rats recorded
normal growth signified by increased weight.
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I.I. Olatunji-Bello et al./J Phytol 1 (2009) 461-468
Fig 1
Fig. 2.
Fig 3.
Fig 4.
Fig 5.
Fig 6.
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I.I. Olatunji-Bello et al./J Phytol 1 (2009) 461-468
Fig. 7.
Fig. 8
Plate 1. Histological picture of a section of
testicular tissue from the control group
administered water. The histology is normal, with
the presence of mature sperm cells in the intact
seminiferous tubules (ST) and Leydig cell (L) in the
interstitial space (IS). (Eosin-Nigrosin stain)
Plate 2.Section of testicular tissue from rats treated
with methanol fruit extract of Abelmoschus
esculentus showing varying degrees of degeneration
of seminiferous tubule (A & B) and empty
interstitial spaces characterised with disappearance
of the leydig cells (C). (Eosin-Nigrosin stain)
Fig 3.Section of testicular tissue from rats allowed
recovering for one week from 28days oral
administration of methanol fruit extract
Abelmoschus esculentus. There was thickening of
the basement membrane (A), Degeneration of setoli
cell (B), Persistence of empty interstitial space (C)
and low level of spermatogenesis (D) .
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I.I. Olatunji-Bello et al./J Phytol 1 (2009) 461-468
Plate 4.Section of testicular tissue from rats
allowed recovering for two weeks from 28days oral
administration of methanol fruit extract
Abelmoschus esculentus. There was evidence of
mild vascular congestion with interstitial oedema
and the presence of lymphocytes indicating repair.
Spermatogenesis
This gave an indication of non interference
of the methanol fruit extract of Abelmoschus
esculentus with acceptance of rat feed and
possible no adverse effects on digestion which
could have resulted in reduction in weight.
However, there was a reduction in the mean
weight of the testes of treated rats compared
with Control, although, this reduction was not
significant (Fig. 2)(Control 14.73±0.97, Treated
13.04±1.29, 1wk recovery15.3±0.52, 2wks
recovery 17.66±0.94). Histological studies of
the testes further revealed a progressive
degeneration of the basement membrane of the
germ cell characterised by loss of leydig cells
responsible for secretion of testosterone and
also a reduction in spermatogenesis (Plate 2).
Previous Authors who have worked on
medicinal plants with adverse effects on male
reproduction have reported similar disruption
of testicular histomorphology with consequent
arrest of spermatogenesis [16]. The weight of
the prostate gland which is an accessory organ
of male reproduction, responsible for secreting
20% by volume of semen sample and some
other nutrients required for spermatozoa
viability into the semen was also significantly
reduced (p<0.05) in treated rats. The viability
of the male reproductive organs have been
linked to the continuous presence of the male
hormone; testosterone [17, 18]. The significant
reduction in the serum level of testosterone in
treated rats may therefore, be responsible for
the atrophy of the cells of these organs and
consequently the recorded reduction in the
weight (Fig 2).
The results of assessment of the semen
quality showed that methanol fruit extract of
Abelmoschus esculentus significantly (p<0.05)
reduced the gross sperm motility in all the
treated rats. The alteration in the activities of
the prostate gland as well as tissue toxicity of
the extract may contribute to the reduction in
the motility. This effect was however reversed
after the 2 week recovery period, thou the
recovery was not total. (Fig 2 and 4)(Control
90.83± 0.83, Treated 52.86±3.6, 1wk recovery
75±2.24, 2weks recovery 80±1.58). In addition,
sperm concentration in the semen samples was
significantly reduced (p<0.05) in the treated
and 1wk recovery group (Fig 3). The reduction
in the weight of the testes of the treated rats
coupled with the testicular atrophy as revealed
by histological slides (Plate 2) proffer the
reasons for the reduction in sperm count.
Further work on the quality of the sperm
cell revealed that there was a significant
reduction ( p<0.05) in the % life/Dead ratio of
sperm cells in the semen sample from extract
treated, 1wk recovery as well as 2wk recovery
groups of rats compared to the control (Fig 5)
(Control 96.5±0.67, Treated 90.71±1.3, 1wk
recovery 84±1.87,2wks recovery 90±1.58). This
further explains the reason for the reduction in
sperm motility recorded (Fig.3). A closer
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I.I. Olatunji-Bello et al./J Phytol 1 (2009) 461-468
microscopic assessment of the semen smear
samples revealed a significant increase in the
presence of abnormal sperm cells in the extract
treated rats (Control 9.54±0.15, Treated
12.99±0.43). Prominent among the
abnormalities observed are those that has to do
with the tail which further explain the
reduction in the gross motility of the semen
sample in extract treated rats.
Administration of methanol fruit extract of
Abelmoschus esculentus also significantly
reduced (p<0.05) the circulating serum
testosterone level in all treated rats compared
to the control (Control 0.69±0.05, Extract
Treated 0.29±0.02, 1wk recovery 0.17±0.01,
2wks recovery 0.16±0.01). This corroborate the
reports of other Authors who have worked on
drugs and medicinal plants that adversely
affect male reproduction, in which a reduction
in histomorphology of the testis precipitates a
reduction in circulating serum testosterone
level [19,3]. The treated animals were not able
to recover from this deleterious effect within
the 2 weeks recovery period allowed.
The results of these study showed that four
weeks oral administration of methanol fruit
extract of Abelmoschus esculentus will adversely
affect male reproductive functions in rats. The
antifertility effect was characterised by a
reduction in semen quality and quantity,
reduction in serum male hormonal level as
well as atrophy of the testes.
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