ASCORBIC ACID AND ALPHA-TOCOPHEROL
AMELIORATE CHLOROQUINE-INDUCED IMPAIRMENT
OF SPERM MOTILITY AND VIABILITY IN MALE RATS
T.M. SALMAN AND O.P.AJAYI
DEPARTMENT OF PHYSIOLOGY AND BIOCHEMISTRY,
COLLEGE OF MEDICINE, UNIVERSITY OF ILORIN, ILORIN,
NIGERIA.
ADDRESS FOR CORRESPONDENCE: T.M. SALMAN,
DEPARTMENT OF PHYSIOLOGY AND
BIOCHEMISTRY, COLLEGE OF MEDICINE,
UNIVERSITY OF ILORIN, ILORIN, NIGERIA
E-MAIL:drsalman111@yahoo.com.
1
ABSTRACT
The effects of two antioxidants, ascorbic acid (vitamin C) and
alpha-tocopherol (vitamin E) were studied on sperm motility, viability
and counts in chloroquine-treated rats. Adult male rats were divided
into five groups which were administered normal saline, chloroquine
only, chloroquine and vitamin C, chloroquine and vitamin E, and
chloroquine and vitamins C and E respectively for 35 days. The drugs
were administered orally at the doses of 20mg/kg, 14.3mg/kg and
9.3mg/kg body weight respectively for chloroquine, vitamin C and
vitamin E. The results showed that chloroquine significantly reduced
sperm motility (P<0.05) and viability (P<0.05). Chloroquine also
caused an apparent but insignificant reduction in sperm counts. When
ascorbic acid was co-administered with chloroquine, the reduction in
sperm viability became insignificant while the reduction in sperm
motility remained significant. However, the reduction in both sperm
motility and viability caused by chloroquine was insignificant in the
groups of rats in which alpha-tocopherol alone and the combination of
2
both ascorbic acid and alpha-tocopherol were co-administered with
chloroquine. The results suggest that these antioxidants could
ameliorate the reduction in sperm quality induced by chloroquine.
Key words: Ascorbic acid, Alpha-tocopherol, Chloroquine, Sperm
motility, Viability, male rats.
3
INTRODUCTION
Chloroquine is regarded as one of the most common
antimalarial drugs worldwide1.
The induction of reversible male
infertility in experimental animals and humans resulting from
treatment with chloroquine and other antimalarial agents had since
attracted the attention of researchers. For instance, chloroquine had
been reported to cause reductions in sperm motility, fertilizing
capacity of epididymal sperm2 and fertility in male rats3; complete
obliteration of leydig’s cell response to leutropine and leutropine-like
activity4 and inhibition of basal and stimulated testosterone secretion
in decapsulated testes in-vitro5. Similar reductions in sperm motility,
viability, counts, serum testosterone levels and fertility had been
observed in rats treated with the extracts of quassia amara6,
Azadirachta indica7 and Alstonia boonei8; all of which have welldocumented antimalarial properties.
Furthermore, the positive influence of some antioxidant
vitamins such as ascorbic acid and alpha-tocopherol on sperm quality
and fertility in animals and humans had also caught the attention of
4
researchers. For example, ascorbic acid had been observed to protect
human epididymis and spermatozoa against deoxyribonucleic acid
damage9,. It also improves semen quality and sperm motility in
smokers10. The water-soluble reactive oxygen specie (ROS) scavenger
had also been reported to improve fertility in boar11. Moreover, alphatocopherol, a lipid-soluble vitamin also enhances spermatogenesis and
prevents loss of spermatogenesis
12, 13, 14
, improves semen quality and
cell viability in chicken 15. This vitamin also increases the percentage
of normal sperm in men16 and protects sperm cells from
morphological damage 17. Also, by combining their hydrophilicity and
lipophilicity, ascorbic acid and alpha-tocopherol may significantly act
in-vivo to reduce damage to spermatozoa by peroxidation18.
However, in spite of expanding literature on the adverse effects
of antimalarial agents on reproductive functions and the positive
influence of these vitamins, attempts have not been made to
investigate the effects of these vitamins on the antifertility activities of
these antimalarial agents. The present paper therefore reports the
5
effects of ascorbic acid and alpha-tocopherol on sperm quality in
chloroquine–treated rats.
6
MATERIALS AND METHODS
Animal model: Wister strain albino rats (160-200g) obtained
from the Central Animal House, College of Medicine, University of
Ilorin, were used for the study. The rats were housed in wire mesh
cages under standard conditions (Temperature, 25-290C, 12hrs light
and 12hrs darkness cycles) and fed with standard rat pelleted diet and
water. The study was generally conducted in accordance with
recommendations from the declaration of Helsinki on guiding
principles in the care and use of animals.
Drugs:
Tablets
of
chloroquine
and
ascorbic
acid
(Tuyil
Pharmaceutical Industry, Nigeria) and alpha-tocopherol (G.A.
Pharmaceuticals, Athens, Greece) were dissolved in normal saline and
used for the study.
Experimental design: 25 male rats were divided into five groups of
five (5) animals per group. Group 1 consists of rats which received
normal saline and served as the control. Groups II, III, IV and V were
treated with chloroquine only, chloroquine and ascorbic acid,
chloroquine and alpha-tocopherol; and chloroquine, ascorbic acid and
7
alpha-tocopherol respectively. Drug administration was by oral route
and the doses were 20mg/kg, 14.3mg/kg and 9.3mg/kg body weight
for chloroquine, ascorbic acid and alpha-tocopherol respectively.
Sperm motility, viability and counts: The rats were anaesthetized
using ether 24hrs after the last administration of the treatment and the
caudal epididymis was immediately dissected out. An incision (about
1mm) was then made in the caudal epididymis. Epididymal volume
was 0.2ml per rat. A little drop of sperm fluid was squeezed onto the
microscopic slide and 2 drops of normal saline were added to
mobilize the sperm cells. Epididymal sperm motility was then
assessed by calculating motile spermatozoa per unit area and was
expressed in percentage. Epididymal sperm counts were done by
homogenising the epididymis in 5ml of normal saline. A further
dilution of 1/200 was made and the sperm counts were done using the
counting chamber in the haemocytometer. 25 squares of the
haemocytometer give a total area of 1/square mm and the layer of
liquid is 0.1mm thick. Thus, the sperm in 0.1mm3 of diluted sperm
covers 25 squares. Five (5) of the 25 squares were counted. So, for
8
every ml dilution, 1/200 × 1/1000 × 1/5 was counted. This value was
then multiplied by 200 × 1000 × 5 or 1,000,000 to give the estimated
counts per ml. The results were then expressed as million/ml of
suspension. The sperm viability was also determined using Eosin /
Nigrosin stain as earlier described 7 .
Statistical Analysis: Data were expressed as mean + SEM and
analyzed using Duncan’s test and student’s t-test where necessary.
P<0.05 was considered significant.
9
RESULTS
Sperm motility significantly reduced (P<0.05) from 73.00 +
2.10 percent in the control rats to 57.50 + 3.23 percent and 60.60 +
1.63 percent in rats that were treated with chloroquine only and those
treated with chloroquine and ascorbic acid respectively. This
represents a reduction of 21.2 percent and 17.0 percent in sperm
motility respectively in these groups of rats when compared with the
control. However, the decrease in sperm motility in the rats that were
treated with chloroquine and alpha-tocopherol and those treated with
chloroquine, ascorbic acid and alpha-tocopherol was not significant
(P>0.05) as the percentage motility of 66.50 + 4.31 and 68.25 + 4.59
observed in these rats were similar to that of the control (Table 1).
The percentage sperm viability also reduced significantly
(P<0.05) from 34.60 + 2.10 percent in the control rats to 26.50 + 2.53
percent in the rats that were treated with chloroquine only. This
represents a reduction of about 24 percent in sperm viability in these
rats when compared with the control. There was no significant
(P>0.05) reduction in sperm viability in the rats which received
10
ascorbic acid with chloroquine and those that received alphatocopherol with chloroquine. The observed sperm viability of 30.40 +
1.57 percent and 32.80 + 2.33 percent in these groups of rats were
similar to that of their control counterpart (Table 1).
Although, there was an apparent increase in sperm viability in
the group of rats in which both ascorbic acid and alpha-tocopherol
were co-administered with chloroquine, the increase was not
significant (P>0.05). However, the sperm viability was 15%, 24% and
44% respectively higher(P<0.05) in the rats that were given ascorbic
acid, alpha-tocopherol and the combination of both ascorbic acid and
alpha-tocopherol when compared with those treated with only
chloroquine.
There was no significant decrease (P>0.05) in sperm counts in
all the treated groups when compared with their control counterpart
(Table 1).
11
DISCUSSION
The present study observed a significant reduction in sperm
motility and viability in the chloroquine-treated rats while the
reduction in sperm counts was not significant. This is consistent with
the known antifertility effects of chloroquine. Chloroquine reduced
sperm motility and fertilizing capacity of epididymal sperm 2,3 without
a significant reduction in epididymal sperm counts. The reduction in
sperm motility and viability observed with chloroquine treatment in
this study suggests that chloroquine was able to penetrate into the
testes. Although, testicular chloroquine concentration was not
estimated in this study, earlier studies had shown that chloroquine was
concentrated in the testes of guinea pigs19 and was secreted in human
semen
20
. The decrease in sperm motility caused by chemical agents
had earlier been attributed to their ability to permeate the blood-testes
barrier 21.
The observation that the significant reduction in sperm viability
with chloroquine treatment became insignificant while sperm motility
12
remained significantly reduced when ascorbic acid was administered
with chloroquine suggests that the vitamin could not effectively
prevent the antifertility effects of chloroquine at the dose used. On the
other hand, the preservation of normal sperm motility and viability in
chloroquine-treated rats by alpha-tocopherol alone indicates that the
antioxidant might effectively prevent the antifertility effects of
chloroquine. This is consistent with the findings that alpha-tocopherol
(Vitamin E) is effective in the treatment of infertility associated with
asthenozoospermia and alteration in sperm motility 22. The prevention
of a reduction in sperm motility when both ascorbic acid and alphatocopherol were administered with chloroquine was actually the effect
of alpha-tocopherol since in the first instance, ascorbic acid alone
failed to do so while alpha-tocopherol did.
The observation that the increase in sperm viability was highest
in the rats in which both ascorbic acid and alpha-tocopherol were
administered with chloroquine when compared with those treated with
chloroquine only, suggests that both vitamins are synergistic in their
actions. This is also consistent with the reported ability of ascorbic
13
acid to resuscitate alpha-tocopherol, preventing its damage during
vigorous antioxidant activities23 and reports that ascorbic acid protects
cell membranes and lipoprotein particles from oxidative damage by
regenerating the antioxidant form of alpha-tocopherol
24
.
The
observations in this study might be due to the antioxidant properties of
these vitamins.
The results therefore indicate that these vitamins could prevent
the impairment of sperm motility and viability induced by chloroquine
in male rats. Although, the mechanism of chloroquine antifertility
effects remains unknown, these vitamins; as antioxidants, are known
to act by mopping up reactive oxygen species and preventing lipid
peroxidation
25, 26
. However, whether or not chloroquine induces the
formation of free radicals also remains unraveled. In view of these
findings, further investigations into the mechanism of antifertility
effects of chloroquine and possible mechanism of prevention by these
vitamins will be required.
14
ACKNOWLEDGEMENT
The authors are grateful to Mr. A. U. Akapa and Mr.O. A
Olayiwola for their technical and secretarial assistance.
15
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20
TABLE1: EFFECTS OF ASCORBIC ACID AND ALPHATOCOPHEROL ON SPERM MOTILITY, VIABILITY, AND
COUNTS IN CHLOROQUINE-TREATED RATS
Treatment
groups
Motility (%)
Viability (%)
Counts
(Million/ml)
Control (normal 73.00 + 2.10
saline)
Chloroquine
57.50 + 3.23*
34.60 + 2.10
40.20 + 2.54
26.50 + 2.53*
36.50+ 2.80
Chloroquine and 60.60 + 1.63*
ascorbic acid
Chloroquine and 66.50 + 4.31
alpha-tocopherol
Chloroquine,
68.25 + 4.59
ascorbic acid and
30.40 +1.57*
37.60 + 3.33
32.80 +2.33*
37.80 +1.97
38.25 + 2.93*
38.50 +3.62
alpha-tocopherol
* P<0.05
21