M.Sc.

RESEARCH UPDATE

ON
AN ASSESSMENT OF THE EFFECT OF AQUEOUS EXTRACT OF
PUMPKIN LEAF (TELFAIRIA OCCIDENTALIS) AND BITTER LEAF
(VERNONIA AMYGDALINA) ON SPERMATOZOA CHARACTERISTICS
IN THE MALE ALBINO WISTAR RATS

BY
NWAOGWUGWU COLLINS CHIKODI
(MAT NO. AAU/SPS/FMBS/PYS/13/M.Sc/04529)

SUPERVISOR: DR H.O. OTAMERE

CO-SUPERVISOR: DR U.C. OSIFO

MARCH, 2017
 INTRODUCTION

Infertility (Berkley, 2004; Swierzewski, 2009).

Pumpkin leaf (Telfairia occidentalis)

Bitter leaf (Vernonia Amygdalina)

(Igile et al., 1994;Horsfall and Spiff, 2005)

 JUSTIFICATION

Telfairia occidentalis has distinctive nutritional and phytochemical

properties which can elicit varied morphological, physiological and
biochemical effects (Longe et al., 1983).

According to Modi et al., (2007), Fluted pumpkin seed oil (FPSO) has been
reported to posses some essential properties (vitamin A, tannins, acid, and
alkaloids) which suppress lipid peroxidation, hence, improving testicular
function (Akintayo, 1997; Bensoussan et al., 1998; Glenville, 2006: Leat,
1983).
 JUSTIFICATION CONTD

The characteristics bitter taste of Vernonia amygdalina has been attributed

to its anti-nutritional contents such as alkaloids, saponins, tannins and
glycosides.

Its antioxidative components have been documented (Jisaka et al., 1992).

Several reports on its anti-malarial (Masaba, 2000; De Madureira et al.,
2002; Tona et al., 2004), antiviral (Vlietinck et al., 1995; Ogbulie et al.,
2007), antitumor (Jisaka et al., 1993) and antidiabetic (Adaramoye et al.,
2009) have been documented
 Objective of Study

This study will examine the effect of aqueous extract of Pumpkin Leaf and
Bitter Leaf on spermatozoa characteristics of male wistar rat.

The specific objectives are;

Sperm motility

Sperm count

Sperm morphology

Testicular Histology
 Research Questions
This proposed experimental work seeks to answer the following question;
Following administration of Pumpkin leaf (Telfairia occidentalis) and Bitter
leaf (Vernonia Amygdalina),

is there ;

an increase in sperm motility and sperm count?

a decrease in sperm motility and sperm count?

changes in sperm morphology?

changes in testicular histology?

do these changes pose any health challenges?

 Significance of the Study
Male reproductive health is of worldwide concern, especially the
inability to produce offspring as it affects the psychology of the
man, his emotions, sexual relationship and his personality (Berkley,
2004).

The most common complications or diagnosis include low sperm

count, decreased motility and abnormal sperm cells (such as dead
cells, big head and double tails) (Swierzewski, 2009).

These challenges if properly understood will go a long way in

addressing some unscientific management pattern which may be
inimical to health and improve male fertility.
 Scope of Study
Animals, in particular wistar rats, will be used in all experiment for the
study.

Humans cannot be used in this study. It involves sacrificing the animals in

the course of the experiment.

The study will be limited to assessment of sperm motility, sperm count,

morphology as well as testicular histology in male wistar rats following
Pumpkin Leaf and Bitter Leaf administration.
 Animals for the study

Twenty four (24) male albino rats (Wistar strain) was used for the study and
were procured from the animal house, College of Medicine, Ambrose Alli
University, Ekpoma, Edo state Nigeria. They were kept in the laboratory of the
Department of Physiology, Ambrose Alli University, Ekpoma, Edo state
Nigeria.
 Acclimatization
The rats were allowed to acclimatize for two weeks before commencement of
the experiment and they were fed with normal rat chow and tap water ad
libitum
 MATERIALS

Male Wistar rats

Pumpkin Leaf

Bitter Leaf

Weighing scale and Hand gloves.

Cotton wool, disinfectants and chloroform.

Syringes-2ml and Canula

250ml beakers and measuring cylinder, spatula for stirring of extract.

Distilled water
 MATERIALS CONTD

Rat feed-(finisher, super deluxe) and Rat cage

Dissecting kit/surgical apparatus, microscope, microscope slides and

sterilized containers.
 ANIMAL GROUPING
The animals were assigned into four (4) groups (A-D) and each group
consist of six (6) male rats.

Group A served as the control group

Group B, C and D served as the experimental groups.

 EXPERIMENTAL PROCEDURE

Oral-gastric canula was used to administer the filtrates of Pumpkin leaves

and Bitter leaf to the rats that were used in this study.

Group A (control) received distilled water while group

B received 0.5ml (500mg/kg per body weight) of aqueous extract of

Pumpkin leaves and

group C received 0.5ml (500mg/kg per body weight) of aqueous extract of

Bitter leaf respectively for 14days.

Group D also received 0.5ml (500mg/kg per body weight) of aqueous

extract of Bitter leaf and aqueous extract of Pumpkin leaves for 14 days
 SAMPLE COLLECTION
After 14 days, the rats was anaesthetized and place on dorsal recumbent.

The testicles were surgically removed through a lower abdominal incision.

The epididymis were trimmed off the testes and sample was collected from
the caudal through an incision made with a scalpel using a Pasteur pipette.

Semen examination was done to evaluate spermatozoa characteristics such as

motility, live-dead ratio, concentration and morphology.

The spermatozoa concentration was also evaluated using the improved

Neubar chamber method of counting.

Testes were collected and preserved with 10% formalin for histological
examination
 DATA ANALYSIS

The data obtained from this study were analyzed by statistical package
(SPSS), version 17. The statistics include mean Standard error of mean
and analysis of variance (Anova), p< 0.05 was considered significant.
 RESULTS
Table 4.1: Percentage Spermatozoa Motility of Male Wistar Rat fed with Pumpkin Leaf and
Bitter Leaf Extract

Groups % Sperm Motility

A - Control 87.502.74

B Rats fed Pumpkin Leaf 84.178.01

C - Rats fed Bitter Leaf 84.173.76

D- Rats fed combined leave 91.333.83

n=6 (p>0.05)
 Results contd
Table 4.2: Sperm Count of Male Wistar Rat fed with Pumpkin Leaf and Bitter Leaf
Extract

Groups Sperm count (Million/ML)

A - Control 57.504.18a

B Rats fed Pumpkin Leaf 66.335.47b

C - Rats fed Bitter Leaf 68.335.16b

D- Rats fed combined leave 69.507.71b

n=6 *p<0.05
 Results contd
Table 4.3: Percentage Dead Sperm Cells, Immature Sperm Cells and Normal Spermatozoa
Morphology of Male Wistar Rat fed with Pumpkin Leaf and Bitter Leaf Extract

Groups Total Dead Cells Total Immature Total Normal Cells

(%) Cells (%) (%)

A - Control
12.502.74ab 32.504.18a 56.505.82a

B - Pumpkin Leaf
15.838.01a 19.179.17b 73.338.16b

C - Bitter Leaf
18.833.76a 18.335.16b 75.005.48b

D - both combined
8.673.83b 16.337.12b 77.834.71b

n=6 *p<0.05
 Results contd
Table 4.4: Mean Values of Abnormal Sperm Cells of Male Wistar Rat fed with Pumpkin Leaf and
Bitter Leaf Extract

Groups Tailless Headless Bent Curved Curve/Mid

Head (%) Tail (%) Tail (%) Tail (%) Piece (%)

A 6.675.16a 2.672.25 1.172.04 0.832.04 1.332.16

B 3.332.58ab 0.832.04 0.832.04 0.000.00 1.672.58

C 1.332.16b 0.832.04 1.172.04 0.832.04 0.832.04

D 2.832.48ab 0.000.00 0.000.00 0.170.41 1.332.16

n=6
 HISTOLOGICAL FINDINGS

PLATE 1: Photomicrograph of Group A testes tissue section

(H&E; X100) showing normal histological features with
intact seminiferous tubules (ST) and interstitial spaces (IS)
PLATE 2: Photomicrograph of Group B testes tissue section
(H&E; X400) showing intact testes cytoarchitecture with mild
vacuolations in the seminiferous tubules (arrows).
PLATE 3: Photomicrograph of Group C testes tissue section
(H&E; X400) showing testes cytoarchitecture with locations
of scanty cell population in some seminiferous tubules.
PLATE 4: Photomicrograph of Group D testes tissue section (H&E;
X100) showing intact testes cytoarchitecture with distinct
seminiferous tubules (ST) and interstitial spaces (IS).
 Discussion
In the present study however, aqueous extract of Telfairia Occidentalis or
Vernonia Amygdalina leaves or their combinations was observed to produce
variety of effects on sperm physiological parameters and on testicular
histology.

Specifically, the present study showed that Telfairia Occidentalis at a dose of

500mg/kg has an increasing effect on sperm count, but a reducing effect on
sperm motility and a disruptive effect on testicular histology.

In accordance with this findings, Oyeyemi et al. (2000) has previously reported
Telfairia Occidentalis to reduce motility of spermatozoa at 500mg/kg dosage
and Saalu et al. (2010) reported higher doses (400 and 500mg/kg/day/oral) to
provoke varying degrees of testicular degeneration, deranged sperm
parameters and worsened testicular oxidative status.
 Discussion contd
Although the present study observed improved sperm morphology (such as
increased normal sperm cells and reduced immature and disfigured sperm cells)
in Telfairia Occidentalis treated group compared to the control, and this is
contradicting to the report of by Saalu et al. (2010).

Similarly, 500mg/kg aqueous leaf extract of Vernonia Amygdalina was

observed to increase sperm count increase sperm morphology but reduced
sperm motility and altered testicular histological-architecture.

In contrast with these findings, Longe et al. (1983) and Oyeyemi et al. (2008)
have reported significant improvement in sperm parameters in Vernonia
Amygdalina ingestion.

Specifically, Saalu et al. (2013) showed that low doses (50 and 100mg/kg) of
Vernonia Amygdalina increased sperm qualities.
 Discussion contd
Thus the findings of the present study may be due to the higher dose ingested
(500mg/kg) which is asserted to have provoke sperm quality and testicular
histology.

In view of the assertion, tannins (which is a constituent of Vernonia

Amygdalina), though classified as antioxidant; at high dose it has been
reported to act as pro-oxidant and increase lipid peroxidation (Taitzoglou et al.,
2001; Nworgu et al., 2007). Thus the present study observations with Vernonia
Amygdalina are in line.

On the other hand, the administration of the combined aqueous extracts of

Telfairia Occidentalis and Vernonia Amygdalina was observed to produce
significant improvement in sperm motility, sperm count, sperm morphology
and an intact testicular histological architecture compare to group A (control),
B (Pumpkin) and C (Bitter leaf).
 Conclusion
The overall results obtained in this study suggest that Telfairia
Occidentalis and Vernonia Amygdalina Leaf extract improved
some sperm parameters. However, it was observed to be best
when the leaves were combined in which there was an
improvement in all the sperm parameters as well as testicular
histology.
 Recommendation
Consequent upon the result of this study, the findings of aqueous extract of
Telfairia Occidentalis leaf and Vernonia Amygdalina leaf on sperm motility
disagrees with the fact that plant in adequate amounts is associated with
numerous health benefits considering the impact of the separate leaf extract
on sperm motility. The following are recommended

Therefore, there is need for men to limit the consumption of Telfairia

Occidentalis leaf and Vernonia Amygdalina leaf. A moderate consumption of
should be considered.

The combined Telfairia Occidentalis leaf and Vernonia Amygdalina leaf

should be applied in treatment of infertility.

Further studies should concentrate on the mechanism by which the combined

extract improved sperm parameters and testicular histology
 Contribution to knowledge
The findings of this study explicitly elucidate the following:

Herbal drugs in this instance Pumpkin leaf and Bitter leaf brings about alterations
in sperm quality which can be harnessed for human benefits and research purposes.

This research has unfolded that the consumption of Pumpkin leaf and Bitter leaf
improves male fertility in wistar rats which may likely translate to same outcome in
human

The semen parameters investigated in this study will occupy a position of relevance
in male fertility studies.

The effect of combined aqueous extract of Pumpkin Leaf and Bitter Leaf on sperm
motility, morphology, sperm count and testicular histology will be elucidated and
contribute to therapeutic considerations in male infertility.
 References
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Akubue, P.I., Kar, A. and Nnachetta, F.N. (1980).Toxicity of extracts of roots

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Domar, A.D., Zuttermeister, P.C. and Friedman, R. (1993). The psychological

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(2005). Hypoglycaemic activity of Telfairia occidentalis in rats.
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Oyeyemi, M.O., Leigh, O.O., Ajala, O.O., Badejo, A.O. and Emikpe, B.O.
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