Middle East Fertility Society Journal (2011) 16, 6166

Middle East Fertility Society

Middle East Fertility Society Journal

www.mefsjournal.com
www.sciencedirect.com

ORIGINAL ARTICLE

Eects of Tahitian Noni dietary supplement on

caeine-induced testicular histo-pathological alterations
in adult Sprague-Dawley rats
Rosemary Basiono Bassey a,b,*, Oshiozokhai Eboetse Yama a,
Adewale Abraham Osinubi a, Carmel Cressie Noronha a, Abayomi Okanlawon
a
b

Department of Anatomy, College of Medicine, University of Lagos, Lagos, Nigeria

Department of Anatomy, Faculty of Basic Medical Sciences, University of Uyo, Nigeria

Received 11 October 2010; accepted 9 November 2010

Available online 14 January 2011

KEYWORDS
Noni;
Caffeine;
Coffee;
Testis;
Free radical

Abstract Objective: To investigate the possible ameliorating effect of Tahitian Noni dietary supplement on caffeine-induced testicular histopathological alterations in Sprague-Dawley rats.
Design: This is an experimental animal study.
Methods: Thirty adult male Sprague-Dawley (SD) rats, weighing between 105 and 200 g were acclimatized and grouped into six of ve rats per group. Group 1 was the control. Group 2 received
200 mg/kg of caffeine for 8 weeks, Group 3 received 200 mg/kg of caffeine for 4 weeks and 5 ml/
kg of Noni for another 4 weeks, Group 4 received both 200 mg/kg of caffeine and 5 ml/kg of Noni
for 8 weeks, Group 5 received 5 ml/kg of Noni for 8 weeks, Group 6 received 5 ml/kg of Noni for
4 weeks and 200 mg/kg of caffeine for another 4 weeks.
Results: Tahitian Noni caused a statistically signicant increase in the mean body weight of the SD
rats, opposed to the groups treated with caffeine. There was also a statistically signicant increase in
the testicular weight, sperm count and motility in the SD rats treated with Noni compared to those
treated with caffeine. Caffeine negatively affected the histo-architecture of the seminiferous tubules

* Corresponding author at: Department of Anatomy, College of

Medicine, University of Lagos, Idi-Araba, Surulere, P.M.B. 12003,
Lagos, Nigeria. Tel.: +234 8064853304.
E-mail address: rosemary_bassey@yahoo.com (R.B. Bassey).
1110-5690  2010 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.2010.11.003

Production and hosting by Elsevier

62

R.B. Bassey et al.

with massive loss of spermatogenic cells while the groups exposed to Noni tended towards normal
when compared with the control.
Conclusions: Administration of Tahitian Noni dietary supplement ameliorates the testicular toxicities caused by a high dose of caffeine.
 2010 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction
Caffeine is the worlds most widely consumed psychoactive
substance, but unlike most other psychoactive substances, it
is legal and unregulated in nearly all jurisdictions (1). An estimated 80% of the worlds population consumes a caffeinecontaining substance daily (2).
Caffeine is considered a psychoactive substance since it
stimulates the central nervous system and alters mood and
behavior. Heroin, cocaine, marijuana, nicotine and alcohol
are also examples of psychoactive drugs. Physiological effects
may be seen in adults after as little as one cup of coffee or
two cans of cola (3).
The use and abuse of caffeine are a major public habit
and may be as important a factor as heredity and environment
in the etiology of physiological and psychological disorders
(4).
Infertility affects more than 80 million people around the
globe. It is a ubiquitous phenomenon that transcends race
and nationality (5). Male factor and female factor infertility
each accounts for about 40% of cases of infertility, the remaining 20% is as a combination of male and female (6).
Tahitian Noni or Indian mulberry originated in Tropical
Asia and Polynesia (7,8). It is one of the most common plants
used in herbal remedies. The fruit juice is in high demand in
alternative medicine for different kinds of illnesses, such as
arthritis, diabetes, high blood pressure, muscle aches, menstrual difculties, headaches, heart disease, Human Immuno
Virus/Acquired Immune Deciency Syndrome, cancer, gastric
ulcers, sprains, mental depression, senility, poor digestion, atherosclerosis, blood vessel problems and drug addiction (7).
There have been assumptions on the fertility-enhancing effect of Morinda citrifolia but no work has been done on its effect on the possible testicular toxicities that can be caused by a
lot of substances we ingest, of which caffeine is one of the
foremost.
2. Materials and methods
2.1. Collection of materials
The regular bottled commercial form of Tahitian Noni dietary
supplement produced by Morinda Inc., United States of
America was obtained from a registered Tahitian Noni distributor. The producing companys bottle cap was observed intact
before commencement of use. A 200 g tin of Nescafe, a brand
of coffee made from Robusta beans by Nestle Nigeria Plc was
used for this experiment.

Department of Biochemistry, College of medicine, University

of Lagos. The rats were housed in well-ventilated metal cages
under standard conditions of temperature (25 5 C) in the
Department of Anatomy, College of Medicine of University
of Lagos. Animals were exposed to a photo period of 12 h
light, alternating with 12 h darkness. They were allowed access
to standard laboratory food and water ad libitum throughout
the experiment. The animals were kept for at least 2 weeks to
acclimatize
to
the
laboratory
conditions
before
experimentation.
2.3. Experimental protocol
The 30 male rats were divided randomly into six groups of ve
rats each. The experimental groups received daily oral doses of
the drugs as follows: Group 1, the control received daily oral
dose of distilled water. Group 2 received 200 mg/kg of caffeine
for 8 weeks, Group 3 received 200 mg/kg of caffeine for
4 weeks and 5 ml/kg of Noni for another 4 weeks, Group 4 received both 200 mg/kg of caffeine and 5 ml/kg of Noni for
8 weeks, Group 5 received 5 ml/kg of Noni for 8 weeks, Group
6 received 5 ml/kg of Noni for 4 weeks and 200 mg/kg of caffeine for another 4 weeks. Body weight was recorded weekly
for every group. All procedures involving animals were performed in accordance with the guidelines guiding the use and
care of laboratory animals and approved by the Departmental
Committee on the use and care of animals.
2.4. Sample collection
Animals were anaesthetized by intra-peritoneal injection of
ketamine (Rotex Medica, Trittau, Germany) (50 mg/kg) (9)
at the end of the experiment.
2.5. Tissue processing for histological work
The organs were processed for histological work as follows:
one testis from each animal was xed in 10% formol saline.
The xed tissues were transferred to a graded series of ethanol
and then cleared in xylene. Once cleared, the tissues were inltrated in molten parafn wax in the oven at 58 C. Serial sections of 5 lm thickness were obtained from a solid block of
tissue, cleared, xed in clean slides, stained with Haematoxylin
and Eosin stains and examined with the light microscope (Figs.
16).
2.6. Testicular weight (morphometry)
The testicular weight was estimated using electronic balance.

2.2. Animals

2.7. Sperm parameters

Thirty adult male Sprague-Dawley rats, 1620 weeks old and

weighing between 105 and 200 g were obtained from the

The cauda epididymis was dissected out; several incisions of

about 1 mm were made and were suspended in 1 ml of Ham-

Effects of Tahitian Noni dietary supplement on caffeine-induced testicular

Figure 1 Section through the testis of Group A (control) at

magnication (i) 100 and (ii) 400 stained with Heamatoxylen
and Eosin.

63

Figure 3 Section through the testis of Group C administered

caffeine for 4 weeks and Tahitian Noni for 4 weeks at magnication (i) 100 and (ii) 400 stained with Heamatoxylen and Eosin.

Figure 2 Section through the testis of Group B administered

caffeine for 8 weeks at magnication (i) 100 and (ii) 400 stained
with Heamatoxylen and Eosin.

Figure 4 Section through the testis of Group D administered

caffeine and Tahitian Noni for 8 weeks at magnication (i) 100
and (ii) 400 stained with Heamatoxylen and Eosin.

F-10-solution. Motility and concentration estimation was carried out at room temperature between 24 and 28 C. The

microscopic eld was scanned systematically and each spermatozoon encountered was assessed and for the purpose of this

64

R.B. Bassey et al.

bauer improved haemocytometer. 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. Both chambers of the haemocytometer were
scored and the average count was calculated, provided that
the difference between the two counts did not exceed 1/20 of
their sum (i.e., less than 10% difference). When the two counts
were not within 10%, they were discarded, and the sample
dilution was re-mixed and another haemocytometer was prepared and counted. To minimize the error, the count was conducted three times on each of the samples obtained from each
epididymis. The average of all the six counts, three from each
side, from single rat was constituted as an observation.
2.8. Statistical analysis
Results were expressed as mean standard deviation. Analysis was carried out using one-way analysis of variance (ANOVA) and the Scheffes post hoc test. The level of signicance
was considered at P < 0.05.
3. Results
3.1. Testicular weight

Figure 5 Section through the testis of Group E administered

Tahitian Noni for 8 weeks at magnication (i) 100 and (ii) 400
stained with Heamatoxylen and Eosin.

At the end of the experiment, testicular weight and volume

showed a statistically signicant decrease from 1.7 0.1 (control) to 0.86 0.2 in the groups treated with caffeine only
(Group 2) and 0.7 0.4 in the group treated with both caffeine and Noni (Group 4), and an increase in the groups
administered caffeine then Noni (Group 3), Noni only (Group
5) and Noni then caffeine (Group 6) to 1.2 0.3, 2.1 0.1
and 1.44 0.4, respectively as shown in Table 1.
3.2. Sperm count and motility
The animals administered caffeine only (Group 2) and those
administered both caffeine and Tahitian Noni (Group 4)
showed a statistically signicant decrease from 219.2 3.8
(control) to 70 20.2 and 82 50.3, respectively; and a subsequent increase to 126.8 35.8 in the groups treated with
caffeine then Noni (Group 5) when compared to control as
shown in Table 1(P < 0.05).
There was also a slight decrease in the sperm percentage
motility from 95.6 4.0 (control) to 40.0 10.3 in the group
administered caffeine only (Group 2) and 33.0 19.2 in those
administered both caffeine and Tahitian Noni (Group 4), and
a subsequent increase in groups administered caffeine then
Noni (Group 3), Noni only (Group 5) and Noni then caffeine
(Group 6) to 68.6 16.9, 73.6 17.6 and 78.6 4.1, respectively, as shown in Table 1 (P < 0.05).
3.3. Mean body weight

Figure 6 Section through the testis of Group F administered

Tahitian Noni for 4 weeks and caffeine for 4 weeks at magnication (i) 100 and (ii) 400 stained with Heamatoxylen and Eosin.

study, motility was classied as either motile or non-motile.

The procedure was repeated once and the average reading
was taken. The sperm count was determined using the Neu-

The mean body weights decreased signicantly (P < 0.05)

from 224.08 1.2 (control) to 149.42 11.1 in the group
treated with caffeine only (Group 2) and 94.02 13.6 in the
group treated with caffeine and Noni (Group 4). There was
a consistently signicant (P < 0.05) increase in the mean body
weights of the groups treated with Noni (Table 1) as compared
to control.

Effects of Tahitian Noni dietary supplement on caffeine-induced testicular

65

Table 1 Effect of caffeine and Tahitian Noni dietary supplement on testicular weight, sperm count, sperm motility and mean body
weight of Sprague-Dawley rats.
Treatment groups
1
2
3
4
5
6

Control
Caeine (8 weeks)
Caeine (week 14), Noni (week 58)
Caeine + Noni (8 weeks)
Noni (8 weeks)
Noni (week 14), Caeine (week 58)
a
b

Testicular weight (g)

a

1.7 0.1
0.86 0.2a,b
1.2 0.3a
0.7 0.4a
2.1 0.1a,b
1.44 0.4a

Sperm motility (%)

a

95.6 4.0
40.0 10.3a,b
68.6 16.9a
33.0 19.2a,b
73.6 17.6a
78.6 4.1a,b

Sperm count (106/ml)

Mean Body weight (g)

219.2 3.8a
70 20.2a,b
126.8 35.8a,b
82 50.3a,b
213.8 6.7a
155.2 38.9a

224.08 1.2a
149.42 11.1a,b
151.62 9.0a,b
94.02 13.6a,b
110.1 7.5a,b
128.28 4.1a,b

Mean Standard deviation.

P < 0.05 compared to control.

3.4. Testicular histopathological ndings/summary

The microscopic examination of the prepared histological sections of the testes showed moderate disorganization in the cells
of the spermatogenic series and areas of extensive necrosis in
the group treated with caffeine; and the other groups tending
towards normal.
4. Discussion
Many agents have been shown to have deleterious effects on
the spermatozoa or the cyto-architectural pattern of the testes.
Some of these agents include caffeine, nicotine, steroids, alcohol, anesthetic agents, and insecticides. Researchers have expressed their concern about the rising cases of male
spermatozoa abnormalities (10).
In a study by Weinberger et al. severe bilateral testicular
atrophy with aspermatogenesis or oligospermatogenesis was
found in 85100% of the rats fed on caffeine or theobromine
(11). Increases in the incidence of abnormal sperm have been
reported after the treatment of male albino rats with caffeine-containing analgesics (12). Ezzat and el-Gohary in their
study concluded that long term intake of caffeine induces suppression of spermatogenesis (13). The combination of coffee
drinking with smoking was linked with diminished sperm
motility and increase in the percentage of dead sperm (14).
Parazzini et al. found an increasing risk of poor semen quality
with an increasing coffee consumption (15).
In this study, a high dose of caffeine manifested a principal
impact on the male reproductive system of Sprague-Dawley
rats antispermatogenic effects. This is reected in the cessation of spermatogenesis as seen in the results obtained.
This result is similar to that observed by Ax et al. who observed that after the treatment of roosters with caffeine for
14 days, fertility was signicantly lower than before. Semen
output and sperm concentration were markedly reduced to
1721 days after treatment, and no semen could be collected
from the roosters after they had received caffeine for 30 days.
Testicular histology showed that spermatocyte division ceased
and spermatogenesis was abnormal (16).
Of the many causes of male infertility, oxidative stress has
been identied as one factor that affects fertility status and
thus has been extensively studied in recent years (17). As the
administration of caffeine has caused reduction in the spermatogenesis and steroidogenesis, it may alter the sexual behavior and cause infertility.
Increased Reactive oxygen species (ROS) level has been
correlated with decreased sperm count and motility (18,19).

However, the exact mechanism through which ROS causes

the decrease is not understood (17).
Tahitian Noni dietary supplement was found to increase
the cauda epididymal sperm count, testicular weight, mean
body weight of the rats since it acts as a powerful immune
modulator; hence it helps in keeping the endocrine system in
a well-balanced condition and so may help in cases of sterility.
In a case of low semen production, Noni may help to improve
this condition. If the infertility is due to a high increase in free
radicals damage, Noni may also help to improve this condition as it has high power anti-oxidant properties.
Epidemiological studies have demonstrated that consuming
fruits and vegetables reduced free radical-induced oxidative
damage (20). Under experimental conditions, the free radical
scavenging activity of Tahitian Noni was shown to be 2.8 times
that of vitamin C, 1.4 times that of Pycnogenol, and 1.1 times
that of grape seed powder. Therefore, Tahitian Noni has a
great potential to scavenge reactive oxygen free radicals (21).
Noni is also able to affect so many systems through its ability to promote new cellular growth and to repair damaged cells
(22). There is, however, a dearth of literature on the exact
mechanisms by which Tahitian Noni dietary supplement produces these effects.
5. Conclusion
The present investigation shows the possible therapeutic efcacy of Tahitian Noni in modulating testicular toxicities that
can be induced by a high dose of caffeine. This is an experimental animal work and as such is not justied for use as a
therapy for male infertility.

Acknowledgements
The technical assistance of Mrs. Patricia Onuoha in the preparation of the histological slides is acknowledged. Special
thanks also to Dr. N.A. Awolola, FMCPath for his kind
and independent review of the slides.
References
(1) Fredholm B, Battig K, Holmen J, Nehlig A, Zvartau E. Actions
of caffeine in the brain with special reference to factors that
contribute to its widespread use. Pharmacol Rev 1999;51:83133.
(2) Best B. Is caffeine a health hazard? Am J Psychiatry
1999;156:2238.

66
(3) Craig A. Caffeine may trigger most migraines. Back Experts
2008;1:23.
(4) Bolton S, Null G. Caffeine: psychological effects, use and abuse.
Orthomol Psychiatry 1981;10:20211.
(5) Anate M, Akeredolu O. Attitude of male partners to infertility
management in Ilorin. Niger Med Pract 1991;21:2632.
(6) Randolph LM. The principles of clinical cytogenetics. 2nd ed.
Totowa NJ: Humanan Press; 2004, p. 247.
(7) Wang M, West B, Jensen C, Nowicki D, Palu A, Anderson G.
Morinda citrifolia (Noni): a literature review and recent advances
in Noni research. Acta Pharmacol Sin 2002;23:112741.
(8) Abbott I, Shimanzu C. The geographic origin of the plants most
commonly used for medicine by Hawaiians. J Ethnopharmacol
1985;1:21322.
(9) Marcon L, Hale BF and Robaire B. Reversibility of subchronic
exposure to cancer chemotherapeutics, bleomycin, etoposide and
cisplatin (BEP) on spermatogenesis, fertility and progeny outcome in the male rat. J Androl 2008 [Epub ahead of print].
(10) Kuku SF, Osegbe ND. Oligo azoospermia in Nigeria. Arch
Androl 1989;22:2323.
(11) Weinberger MA, Friedman L, Farber TM, Moreland FM, Peters
EL, Gilmore CE, et al. Testicular atrophy and impaired spermatogenesis in rats fed high levels of the methylxanthines caffeine,
theobromine, or theophylline. J Environ Pathol Toxicol
1978;2:687706.
(12) Ekaluo UB, Udokpoh AE, Udoa UU, Ajang RO. Comparative
toxicity of ve commonly used analgesics on sperm count and
sperm head abnormalities. Global J Pure Appl Sci 2005;11:814.
(13) Ezzat AR, el-Gohary ZM. Hormonal and histological effects of
chronic caffeine administration on the pituitary-gonadal and
pituitary-adrenocortical axes in male rabbits. Funct Dev Morphol
1994;4:4550.

R.B. Bassey et al.

(14) Marshburn PB, Sloan CS, Hammond MG. Semen quality and
association with coffee drinking, cigarette smoking and ethanol
consumption. Fertil Steril 1989;52:1625.
(15) Parazzini F, Chatenoud L, Benzi G, Di Cintio E, Dal Pino D,
Tozzi L, et al. Coffee and alcohol intake, smoking and risk of
multiple pregnancy. Hum Reprod 1996;11:23069.
(16) Ax RL, Collier RJ, Lodge JR. Effects of dietary caffeine on the
testis of the domestic fowl, Gallus domesticus. J Reprod Fertil
1976;2:2358.
(17) Makker K, Agarwal A, Sharma R. Oxidative stress and male
infertility. Indian J Med Res 2009:35767.
(18) Agarwal A, Ikemoto I, Loughlin KR. Relationship of sperm
parameters with levels of reactive oxygen species in semen
specimens. J Urol 1994;152:10710.
(19) Armstrong JS, Rajasekaran M, Chamulitrat W, Gatti P, Hellstrom WJ, Sikka SC. Characterization of reactive oxygen species
induced effects on human spermatozoa movement and energy
metabolism. Free Radic Biol Med 1999;26:86980.
(20) Wang MY and Su C. Cancer preventive effect of Morinda
citrifolia. The proceedings of the Strang International Cancer
Prevention Conference, 2000 Nov 1011, New York.
(21) Wang MY, Su C, Nowicki D, Jensen J and Anderson G.
Protective effect of Morinda citrifolia in carbon tetrachlorideinduced liver injury model: a light and electron microscopic study.
The Proceedings of the Eicosanoids and other Bioactive Lipids in
Cancer, Inammatory and Related Diseases, the 7th Annual
Conference, 2001 Oct 16. Loews Vanderbilt Plaza, Nashville,
Tennessee, USA.
(22) Morton J. The Ocean-going Noni, or Indian Mulberry (Morinda
citrifolia, Rubiaceae) and some of its colorful relatives. Econ
Bot 1992;46:2416.