JBRA Assist. Reprod. 2020;24(4):454-465
ORIGINAL ARTICLE
doi: 10.5935/1518-0557.20200036
Effects of the methanol root extract of Carpolobia lutea on sperm indices, acrosome reaction, and sperm DNA integrity in cadmium-induced reproductive toxicity in male Wistar rats
1Department of Physiology, University of Medical Sciences, Ondo City, Ondo State, Nigeria
2Department of Physiology, Igbinedion University Okada, Edo State, Nigeria
3Laboratory for Reproductive Physiology and Developmental Programming, Department of Physiology, University
of Ibadan, Ibadan, Oyo State, Nigeria
The authors have no conflict of interest to declare.
ABSTRACT
Objective: Oxidative stress is a mechanism of cadmium-induced reproductive dysfunction. Carpolobia lutea is a free radical scavenger. Our study
investigated the potential protective effects of Carpolobia
lutea root methanol extract against cadmium-induced
reproductive toxicity.
Methods: We obtained the Carpolobia lutea root in Akure, and it was
authenticated at the Forestry Research Institute of Nigeria (FRIN)
herbarium, Ibadan, Nigeria, with FHI number 109784. We used Soxhlet
extraction to obtain its methanol extract. We used thirty male Wistar rats
(150-170g) in this study, (n=5 per group), and treated them as follows:
Control (1 ml/kg normal saline), Cd (2 mg/kg), Cd+MCL (2 mg/kg+100 mg/kg),
Cd+MCL (2 mg/kg+200 mg/kg), MCL (100 mg/kg), MCL (200 mg/kg). We
administered Carpolobia lutea orally for 8 weeks. We
administered a single dose of 2 mg/kg of cadmium intraperitoneally. We
assessed the sperm profile using a computer-aided sperm analyzer. Under
microscopy, we determined the sperm acrosome reaction and the DNA damage. We
measured the seminal fructose level using spectrophotometry, and the data
were analyzed using ANOVA at p<0.05.
Results: Cd+MCL (2mg/kg+200 mg/kg) significantly increased sperm count
(339.0±25.0 vs. 29.0±4.5 million/mL),
motility (80.0±0.2 vs. 55.0±4.9%), viability
(68.7±2.7 vs. 31.3±2.9%) and decreased
abnormal sperm (28.3±1.7 vs. 43.3±2.5%),
relative to the cadmium group. Cd+MCL (2mg/kg+200 mg/kg) significantly
increased acrosome reaction (68.0±7.5 vs. 15.2±2.4%) and seminal fructose level (0.49±0.06 vs. 0.28±0.06 mmol/L) relative to the cadmium
group. Cd+MCL (2mg/kg+200 mg/kg) significantly decreased sperm DNA damage
(14.1±1.6 vs. 35.9±5.3%) in relation to the
cadmium group.
Conclusions: Carpolobia lutea root extract improves the sperm variables
of rats exposed to cadmium.
Keywords: Carpolobia lutea root extract, cadmium, sperm analysis, acrosome reaction, sperm DNA
INTRODUCTION
Infertility affects approximately 15% of all couples trying to conceive. Male
infertility is the sole or a contributing factor in roughly half of these cases, and
no identifiable cause can be found in over 25% of infertile males (Yeşilli et al., 2005). However, the etiology of the male factor
infertility is poorly understood, while some individuals may be genetically
predisposed to be sub-fertile (Reijo et
al., 1996). There are major epigenetic factors implicated as
potential causes of male infertility. The most common idiopathic
oligoasthenoteratozoospermia (OAT) (Hirsch,
2003), which is a condition in which sperm concentration, the proportion
of morphologically normal sperm and the proportion of motile sperm, are all lower
than the World Health Organization reference values (Cooper et al., 2010). Despite extensive research, a
successful treatment for OAT has not yet been developed. Many recent studies have
focused on oxidative stress and its possible role in the pathogenesis of male
infertility. In physiological conditions, spermatozoa produce small amounts of
reactive oxygen species (ROS), and various scavengers act to reduce the
concentration of these ROS in the seminal plasma. However, excessive production
and/or reduced clearance leads to oxidative stress within the sperm, resulting in
reduced motility (Kao et al., 2008) and defective membrane integrity (Agarwal et al., 2003). One of the reactive oxygen
species inducers is environmental exposure to toxicants.
Cadmium (Cd) is a heavy metal and a relevant environmental toxicant. The general
population is exposed to cadmium through contaminants found in drinking water and
food. In addition, occupational exposure to cadmium occurs during mining and the
production of batteries and pigments that contain cadmium. Industrial activities,
e.g. smelting and refining of metals, and municipal waste incineration release
cadmium into the atmosphere (Siu et
al., 2009). Tobacco smoke is another source of cadmium
exposure (Blanco et al., 2007). Acute cadmium chloride exposure
causes significant reproductive damage through increased oxidative stress,
histological alteration, (necrosis, edema etc.) and spermatological damage
(decreased sperm motility and sperm concentration, and increased abnormal sperm
cells). Cadmium toxicity is associated with severe damage to various organs,
particularly the testes, in both humans and animals (Fouad et al., 2009). Cadmium impairs the reproductive
capacity by causing severe testicular degeneration, seminiferous tubule damage and
necrosis in rats (Burukoğlu & Bayçu, 2008).
Many natural herbal and nutritional aphrodisiacs enhance sexual drive and pleasure in
both men and women. Studies have validated that some herbs have aphrodisiac activity
(Rajeshwar et al., 2005). Carpolobia lutea G Don (Polygalaceae) is one of
natural herb and nutritional aphrodisiac. It is a shrub or small
tree of up to 5cm-high. It is widely found in tropical Africam, where it is known as
ikpafum in Ibibio; Abekpok ibuhu in Eket; Angalagala in Ibo; Egbo oshunshun in
Yoruba and cattle stick in English. It is used to facilitate delivery and treat male
sexual disorders because of its aphrodisiac effect (Mitaine-Offer et al., 2002). An ethno-botanically
decoction of the root is used by Ibibio’s of Akwa Ibom state of Nigeria as an
aphrodisiac (Ajibesin et al., 2008), and malarial remedy. It has analgesic and androgenic properties,
reputed to cure rheumatism, insanity, fever, skin infection, vermifuge, venereal
diseases, combat sterility and promote childbirth (Muanya & Odukoya, 2008). Carpolobia lutea leaves
are used for the treatment of ulcers and diarrhea (Nwafor & Bassey, 2007), as well as malarial remedy in some part of
Nigeria. The root can also be used as an anti-inflammatory and anti-arthritic agent
(Iwu & Anyanwu, 1982), vermifuge,
facilitate childbirth and to treat sterility and headache (Mitaine-Offer et
al., 2002).
The present study evaluated the Carpolobia lutea root methanol
extract effect on epididymal sperm parameters, sperm capacitation and acrosome
reaction, seminal fructose level and sperm chromatin integrity in cadmium-treated
male Wistar rats.
MATERIALS AND METHODS
Chemical
The cadmium chloride salt came from Loba chemie, PVT, India.
Plant harvest and extraction
The Carpolobia lutea root was obtained from Ijare, a village via Akure, in the Ondo state. The plant was authenticated
at the Forestry Research Institute of Nigeria (FRIN) herbarium, in Ibadan,
Nigeria, with FHI number 109784. The Carpolobia lutea root was
air dried and pulverized. The pulverized Carpolobia lutea root
(5.20kg) was subjected to Soxhlet extraction using pure methanol as the solvent.
Methanol containing the extract was then filtered and the solvent was
vacuum-distilled at 4oC in a rotary evaporator. The remaining extract
was finally dried in a vacuum oven at 30oC for 2 hours to ensure the
removal of any residual solvent. The powdery mass yielded 87.88g (1.69% yields),
which was then stored for the study. The extract’s fresh solution was prepared
in distilled water.
Phytochemical screening
The C. lutea root methanol extract phytochemical screening
determined the presence of chemical constituents such as flavonoids, simple
sugar, alkaloids, tannins, saponins, phlobatannins, cardiac glycosides and
anthraquinones, following the method of Odebiyi
& Sofowora (1978) and Trease
& Evans (1989).
Acute Toxicity Study
The whole animal acute toxicity study was carried out according to the
Organization for Economic Cooperation and Development (OECD) Limit test
guidelines (2001). Nine male rats were used for the study. They were divided
into 3 groups of 3 rats each. The animals were fasted overnight (no food, but
they were given water). Groups 1, 2 and 3 received a single oral dose of 1000
mg/kg, 2000 mg/kg and 5000 mg/kg of C. lutea root
extract, respectively. We observed the animals for 2 hours
for any behavioral and neurological features, then intermittently over the next
72 hours and daily for 14 days, with special attention to any moribound state or
death.
Animals and Experimental Design
We used Wistar adult male rats (150 to 170 gram) housed in well-ventilated rat
cages in the Central Animal House, College of Medicine, University of Ibadan for
this study. We kept them under standard laboratory conditions of 12-hour light
and 12-hour dark cycle and were fed with standard commercial rat pellets
(Ladokun feeds Limited, Ibadan, Nigeria), and allowed access to water ad
libitum. We acclimatized them for two weeks. We weighed the animals
weekly throughout the study. For this study, we followed the guiding principles
for research involving experimental animals, as recommended by the Declaration
of Helsinki, as well as the Guiding principles for the use and care of animals
(World Medical Association & American Physiological Society, 2002). We
randomly divided the animals into six groups, with five animals per group and
treated as follows:
Group 1: Control (1.0 ml/kg normal saline, vehicle);We treated the animals with vehicle and methanol extract of Carpolobia lutea root, orally for 8 weeks and cadmium (single dose intraperitoneally). Twenty-four hours after the last administration, we anesthetized the animals with 50 mg/kg of sodium thiopentone before they were sacrificed. The animals’ testes and epididymis were harvested and used for sperm analysis, sperm capacitation and acrosome reaction, seminal fructose level and sperm chromatin integrity assessments.
Group 2: Cd (2 mg/kg);
Group 3: Cd+MCL (2 mg/kg+100 mg/kg);
Group 4: Cd+MCL (2 mg/kg+200 mg/kg);
Group 5: MCL (100 mg/kg);
Group 6: MCL (200 mg/kg).
Sperm Analysis
We studied sperm concentration, sperm kinetics and motility using the Computer
assisted sperm analyzer (CASA) JH-6004 - Sperm Quality Analyzer. The sperm
viability study (percentage of live spermatozoa) was assessed by microscopy,
using Raji et al.’s method
(2003). We studied sperm morphology by microscopy according to Sarkar et al. (2006).
Sperm Capacitation and Acrosome Reaction
This was assessed by microscopy according to the methods described by Toyoda & Chang (1974) and Feng et al. (2007).
We assessed sperm DNA damage using aniline blue staining techniques
We used microscopy using the methods described by Wong et al. (2008) and Park et al. (2011).
Seminal fructose level
We ran this analysis using spectrophotometry, following the method described by Zahoor et al.
(2010).
Statistical analysis
We expressed our results as mean ± SEM for five animals per group. We used
one-way variance analysis (ANOVA) to assess the statistical significance of the
data. We used Fisher΄s Least Significant Difference (LSD) test for post hoc
analysis (Multiple comparison). P<0.05 was considered
significant.
RESULTS
Acute Toxicity Study
Table 1 shows the acute toxicity of C. lutea root. The male Wistar rats given C.
lutea up to 5,000 mg/kg neither died nor displayed any signs of
toxicity after a 14-day observation.
Table 1. C. lutea root extract acute toxicity using male Wistar
rats
Phytochemical Screening
Table 2 shows the phytochemical screening
of methanol extract of C. lutea root. It shows that flavonoids,
saponins, anthraquinones, alkaloids, tannins, cardiac glycosides, terpenes and
simple sugar were present the in the C. lutea root methanol
extract.
Table 2. Phytochemical screening of C. lutea root methanol
extract
Sperm variables
Sperm Count
Figure 1 shows that sperm count was
significantly decreased in the Cd (2 mg/kg), Cd+MCL (100mg/kg) and Cd+MCL
(200mg/kg) groups when compared with the control group. The sperm count was
significantly increased in MCL (100mg/kg) and MCL (200mg/kg)-treated groups
when compared with the Control Group. In addition, there was significant
increase in the MCL (100mg/kg) and MCL (200mg/kg) Groups when compared with
the Cd (2mg/kg) Group.
Figure 1.
Effect of C. lutea root methanol extract on sperm
count of cadmium-induced reproductive toxicity in male Wistar rats.
Values are expressed as Mean±SEM, n=5. Cd-Cadmium,
MCL-methanol extract of C. lutea. *,+ p<0.05 was considered significant when compared
with control and cadmium groups, respectively.
Sperm Viability
Figure 2 shows that sperm viability was
significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg), Cd+MCL
(200mg/kg) and MCL (100mg/kg)-treated groups, when compared with the Control
Group, while there was a significant increase in the Cd+MCL (100mg/kg) and
Cd+MCL (200mg/kg)-treated groups when compared with the Cd (2mg/kg)-treated
group.
Figure 2. Effect of C. lutea root methanol extract on
sperm viability of cadmium-induced reproductive toxicity in male
Wistar rats
Values are expressed as Mean±SEM, n=5. Cd-Cadmium,
MCL-methanol extract of C. lutea. *,+ p<0.05 were considered significant when compared
with control and cadmium groups, respectively.
Sperm Abnormality
Figure 3 shows that sperm abnormality
was significantly increased in the Cd (2mg/kg) and Cd+MCL (100mg/kg)-treated
groups, when compared with the Control Group. There was a significant
decrease in sperm abnormality in the Cd+MCL (100mg/kg) group, when compared
with the Cd (2mg/kg) group.
Figure 3. Effect of C. lutea root methanol extract on sperm
abnormality of cadmium-induced reproductive toxicity in male Wistar
rats.
Values are expressed as Mean±SEM, n=5. Cd-Cadmium,
MCL-methanol extract of C. lutea. *,+ p<0.05 were considered significant when compared
with control and cadmium groups, respectively.
Sperm Motility
Figure 4 shows that sperm motility was
significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg) and Cd+MCL
(200mg/kg)-treated groups, when compared with Control animals. In addition,
there was a significant increase in sperm motility in the Cd+MCL (100mg/kg)
and MCL (200mg/kg) groups when compared with the Cd (2mg/kg)-treated
group.
Figure 4. Effect of C. lutea root methanol extract on sperm
motility of cadmium-induced reproductive toxicity in male Wistar
rats
Values are expressed as Mean±SEM, n=5. Cd-Cadmium,
MCL-methanol extract of C. lutea. *,+ p<0.05 were considered significant when compared
with control and cadmium groups, respectively.
Total Sperm Detected
Table 3 shows that the total sperm
detected was significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg)
and Cd+MCL (200mg/kg) -treated groups, when compared with the control
animals. It was significantly increased in the MCL (100mg/kg) and MCL
(200mg/kg) -treated groups, when compared with the Control group.
Furthermore, there was a significant increase in the Cd+MCL (100mg/kg) and
MCL (200mg/kg) -treated groups, when compared with the Cd (2mg/kg)-treated
group.
Table 3. Effects of Carpolobia lutea root Methanol Extract on
total sperm detected, total motile sperm, progressive motility,
non-progressive motility and immotile sperm in Male Wistar Rats
Exposed to Cadmium
Total Motile Sperm
Table 3 shows that the total motile
sperm count was significantly decreased in Cd (2mg/kg), Cd+MCL (100 mg/kg)
and Cd+MCL (200mg/kg) -treated groups when compared with the control
animals, while significant increases were seen in MCL (100mg/kg) and MCL
(200mg/kg) groups when compared with the control group. In addition, total
motile sperm was significantly increased in Cd+MCL (100mg/kg) and MCL
(200mg/kg) groups when compared with the Cd (2 mg/kg) group.
Progressive, Non-progressive and Immotile Sperm
Table 3 shows that sperm progressive
motility was significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg)
and Cd+MCL (200mg/kg) groups when compared with the control group; while
significant increases were seen in the Cd+MCL (100mg/kg) and Cd+MCL
(200mg/kg) when compared with Cd (2 mg/kg) group. Immotile sperm was
significantly increased in the Cd (2 mg/kg), Cd+MCL (100 g/kg) and MCL (200
mg/kg) groups, when compared with the control group, while significant
decreases were seen in the Cd+MCL (100mg/kg) and MCL (200mg/kg) groups when
compared with the Cd (2mg/kg) group.
Sperm Velocity
Table 4 shows that the sperm average
path velocity was significantly decreased in the Cd (2mg/kg), Cd+MCL
(100mg/kg) and Cd+MCL (200mg/kg) groups when compared with the control
group, while it was significantly increased in the MCL (100mg/kg) and MCL
(200mg/kg) treated groups when compared with the control group.
The sperm
average path velocity was significantly increased in the Cd+MCL (100mg/kg)
and Cd+MCL (200mg/kg) groups, when compared with the Cd (2mg/kg) group.
Table 4. Effects of Carpolobia lutea root m ethanol extract
on sperm kinetics in Male Wistar Rats Exposed to Cadmium
The sperm curvilinear velocity was significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg) and MCL (200mg/kg) groups, when compared with the Control group, while it was significantly increased in the MCL (200mg/kg) group, when compared with control animals. There was a significant increase in sperm curvilinear velocity of the Cd+MCL (100mg/kg) and MCL (200mg/kg) groups when compared with the Cd (2mg/kg) group.
The straight line velocity was significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg) groups when compared with control animals, while significant increases were seen them in Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg) -treated groups when compared with the Cd (2mg/kg) group.
Lateral Sperm Head Amplitude
Table 4 shows that the lateral sperm head
amplitude was significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg) and
Cd+MCL (200mg/kg) -treated groups, respectively when compared with the control
group. In addition, there was a significant increase in the MCL (200mg/kg)
-treated group when compared with the control group. Alternatively, there was a
significant increase in the MCL (100mg/kg) and MCL (200mg/kg) -treated groups
respectively, when compared with the Cd (2mg/kg)-treated group.
Sperm Beat Cross Frequency
Table 4 shows that the sperm beat
cross frequency was significantly decreased in the Cd (2mg/kg), Cd+MCL
(100mg/kg) and Cd+MCL (200mg/kg) -treated groups, respectively; when
compared with control animals. In addition, there was a significant increase
in the MCL (100mg/kg) and MCL (200mg/kg) treated groups when compared with
the control groups. Alternatively, there was a significant increase in the
Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg) -treated groups, respectively; when
compared with the Cd (2mg/kg) Group.
Sperm Line Moving
Table 4 shows that sperm line moving was
significantly decreased in the Cd (2mg/kg), MCL (100mg/kg) and MCL (200mg/kg) -treated groups, when compared with the control group.
Alternatively, there was a significant increase in the Cd+ MCL (100mg/kg) and
Cd+ MCL (200mg/kg) treated groups, respectively; when compared with the Cd
(2mg/kg) Group.
Sperm Linearity
Table 5 shows that the sperm linearity
was significantly increased in the Cd (2mg/kg), Cd+MCL (100mg/kg) and Cd+MCL
(200mg/kg) -treated groups, when compared with control animals. In addition,
there was a significant decrease in the MCL (100 mg/kg) and MCL (200mg/kg)
groups, when compared with control animals. Alternatively, there was a
significant decrease in the Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg) treated
groups, respectively; when compared with the Cd (2mg/kg) group.
Table 5. Effects of Carpolobia lutea root Methanol Extract on
Sperm kinetics in Male Wistar Rats Exposed to Cadmium
Sperm Straightness
Table 5 shows that sperm straightness
was significantly increased in the Cd (2mg/kg) group, when compared with the
Control group. In addition, there was a significant decrease in the MCL
(100mg/kg) and MCL (200mg/kg) groups, when compared with control animals.
Alternatively, there was a significant decrease in the Cd+MCL (100mg/kg) and
Cd+MCL (200mg/kg) -treated groups, respectively; when compared with the Cd
(2mg/kg) group.
Sperm Wobble
Table 5 shows that sperm wobble was
significantly increased in the Cd (2mg/kg), Cd+100 mg/kg, Cd+MCL (200mg/kg)
and MCL (100mg/kg) -treated groups, respectively; when compared with the
control group. Alternatively, there was a significant decrease in the Cd+MCL
(100mg/kg) and Cd+MCL (200mg/kg) -treated groups, respectively; when
compared with the Cd (2mg/kg) group.
Sperm Mean Move Angle
Table 5 shows that sperm mean move
angle was significantly decreased in the Cd (2mg/kg), Cd+MCL (100mg/kg) and
Cd+MCL (200mg/kg) -treated groups, respectively; when compared with the
control group. In addition, there was a significant increase in the MCL
(100mg/kg) and MCL (200mg/kg) -treated groups, when compared with control
animals. Alternatively, there was a significant increase in the Cd+MCL
(100mg/kg) and Cd+MCL (200mg/kg) -treated groups, respectively; when
compared with the Cd (2mg/kg) group.
Sperm Capacitation and Acrosome Reaction
Table 6 shows that in the acrosome
intact uncapacitated sperm, there was a significant increase in the Cd
(2mg/kg), Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg) -treated groups, when
compared with control animals. On the other hand, there was a significant
decrease in the Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg) -treated groups,
when compared with the Cd (2mg/kg) group. In acrosome-reacted capacitated
sperm, there was a significant decrease in the Cd (2mg/kg), Cd+MCL
(100mg/kg) and Cd+MCL (200mg/kg) -treated groups, when compared with control
animals. On the other hand, the Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg)
-treated groups were significantly increased when compared with the Cd
(2mg/kg) group.
Table 6. Effects of Carpolobia lutea root Methanol Extract on
Sperm acrosome reaction, sperm DNA integrity and seminal fructose
level in Male Wistar Rats Exposed to Cadmium
Sperm Chromatin Integrity
Table 6 shows that abnormal sperm
chromatin was significantly increased in the Cd (2mg/kg) and Cd+MCL
(100mg/kg) -treated groups, when compared with control animals. On the other
hand, there was a significant decrease in the Cd+MCL (100mg/kg) and Cd+MCL
(200mg/kg) -treated groups when compared with the Cd (2mg/kg) group.
Seminal Vesicle Fructose Level
Table 6 shows that the seminal vesicle
fructose level was significantly decreased in the Cd (2mg/kg) group when
compared with the Control group. On the other hand, there was a significant
increase in the Cd+MCL (100mg/kg) and Cd+MCL (200mg/kg) -treated groups when
compared with the Cd (2mg/kg) group.
DISCUSSION
Cadmium typifies a dangerous environmental, occupational and industrial pollutant.
Several studies with experimental animals have reported that the generation of
reactive oxygen species (ROS) and its interference with the cellular antioxidant
system is one of the major mechanisms by which the toxic effect of cadmium is
mediated (Sen Gupta et al., 2004).
The present study showed the efficacy of Carpolobia lutea root
methanol extract in preventing the toxic effects of cadmium on the rats’
spermatozoa. In this study, we investigated the effects of Carpolobia
lutea root methanol extract on sperm characteristics, sperm
capacitation and acrosome reaction, sperm chromatin integrity and seminal fructose
level in cadmium-induced reproductive toxicity of male rats.
The study showed that C. lutea root is not toxic and it is safe for
oral consumption. The C. lutea root extract contains important
phytochemical compounds such as alkaloids, tannins, saponins, anthraquinones,
flavonoids, cardiac glycosides, terpenes and simple sugar that are similar to the
report by Yakubu & Jimoh (2015).
Flavonoids and tannins are phenolic compounds, and plant phenolics are a major group
of compounds that act as primary antioxidants or free radical scavengers (Li et al., 2009; Sim et al., 2010). Similarly,
terpenoids act as regulators of metabolism and play a protective role as
antioxidants (Soetan, 2008). Saponins are
steroids or triterpenoid glycosides, common in a large number of plants and plant
products that are important in human and animal nutrition. Several biological
effects have been ascribed to saponins, and these include hypolipidemic,
hypoglycemic, anticarcinogenic and antioxidant properties (Elekofehinti et al., 2012). In addition, the
administration of alkaloid compound was reported to decrease lipid peroxidation in
tissues indicating antioxidant-like activity, which alleviates oxidative stress
(Al-Fartosy et al., 2013). Metabolism of simple sugars like glucose will lead to the production
of pyruvate. Pyruvate is a substrate necessary for the activity and survival of
sperm cells (Egbunike et al., 1986). Muanya & Odukoya (2008) also reported that cardiac glycosides and saponins have antioxidant properties.
The present study showed that cadmium significantly reduced sperm motility,
viability, count, while abnormal sperm morphology was increased. The observed
reduction in sperm motility, viability and count might be due to the damaging
effects of cadmium on spermatogenesis. The adverse effects of cadmium on sperm
profile could be ascribed to either the reduction in serum testosterone levels or
generation of reactive oxygen species (Lafuente et al., 2001; Waisberg et al., 2003). Sperm is highly susceptible to
lipid peroxidation (LPO) because of the abundance of unsaturated fatty acids in the
sperm plasma membrane and a very small concentration of cytoplasmic antioxidants
(Aitken et al., 1993). The high level of
LPO can result in oxidative damage to sperm DNA, disrupt membrane functions, impair
motility and possibly have a significant effect on spermatozoa development (Aitken et al., 1989). One of the toxicity indicators due to chemicals
on the reproductive system is a reduction in the level of testosterone (Yoshida et al., 2002).
Testosterone is essential for the maintenance of the structure and function of the
male accessory sex glands. Moreover, reduced or lack of this hormone hinders
spermatogenic function (Boockfor & Blake, 1997). The present result on sperm
profile was in agreement with the findings of El-Missiry & Shalaby (2000) and El-Demerdash et al.
(2004), who demonstrated that cadmium can induce lipid peroxidation, testicular
tissue necrosis and apoptosis in rats. Exposure to cadmium can induce germ cell
apoptosis, which may account for the current decline in male fertility (Akinloye et al., 2006). Another
study, carried out by Kasinathan et
al. (1987) showed that cadmium significantly decreased
primary and secondary spermatocytes in the seminiferous tubules. Sen Gupta et al. (2004) reported that reactive oxygen species are involved in cadmium-induced testicular
damage. In addition, cadmium-induced oxidative stress is well established (Stohs et al., 2001; El-Demerdash et al., 2004).
Cadmium administration generates reactive oxygen species at a cellular level (Wang et al., 2004; Kusakabe et al., 2008) and is
associated to increased lipid peroxidation (Croute et al., 2005); hence, cadmium-induced ROS generation
can increase lipid peroxidation, which leads to testicular tissue damage and reduce
spermatogenesis. Indeed, a large proportion of infertile men have increased levels
of seminal ROS (Pasqualotto et al., 2000). The improvement in sperm quality and quantity by the C.
lutea extract and palmitic acid might be attributed to attenuation of
oxidative damage by cadmium and stimulation from testosterone biosynthesis.
During the past decades, the quality and fertility potential of sperm has decreased
dramatically. Sperm motility has a high correlation with fertility and is an early
and sensitive endpoint for evaluating its chemical effects on male fertility (Lifeng et al., 2006). The
efficacy of computer-assisted sperm analyzer (CASA) has been demonstrated for use
with a variety of species in assessing male reproductive quality, as well as the
impact of various treatments on sperm motility. Computer-assisted sperm analyzer
enables an objective assessment of different cell characteristics: motion, velocity,
and morphology (Verstegen et al., 2002). Our results obtained by motion analysis depict a significant
decline in the percentage of spermatozoa with progressive motility, and significant
decrease in average path velocity (VAP), curvilinear velocity (VCL), straightline
velocity (VSL), amplitude of lateral head displacement (ALP), beat cross frequency
(BCF) and significant increase in linearity (LIN), straigthness (STR) and wobble in
cadmium-treated rats. These observations confirm the positive relationship between
cadmium levels and asthenozoospermia, supporting the hypothesis that environmental
cadmium exposure may contribute significantly to reduced sperm motility (Xu et al., 2001; Benoff et al., 2009).
Moreover, reduction in a motility parameter, such as the BCF, has damaging effects
on sperm motility, since they are indicators of sperm vigor (Duty et al., 2004). Important velocity
parameters (VSL, VCL and VAP) directly express sperm motion and decline in sperm
velocity, percentage of motile sperm, BCF and ALH parameters can adversely affect
fertility (Ban et al., 1999; Kato et al., 2001). ALH
is calculated from the amplitudes of the lateral deviations of sperm head about the
axis of progression (Mukhopadhyay et
al., 2010). It is a valuable measurement, as this is one of
the parameters affecting the outcome of in-vitro fertilization and
sperm penetration ability (Verstegen et al., 2002). The C.
lutea root methanol extract ameliorated cadmium-induced toxicity in
sperm kinetics. The action of C. lutea root methanol extract might
be due to its antioxidant ability to mop up the toxic effects of cadmium in the
testes (Salama & El-Bahr, 2007).
To achieve successful fertilization under normal in-vivo conditions, mammalian spermatozoa must consecutively undergo
capacitation and acrosome reaction (Suarez &
Pacey, 2006). The extracellular environment plays a prominent role in
achieving these complex events that enable spermatozoa to achieve fertilizing
ability at the right time and on the right site (Zhou et al., 2008). The coomassie brilliant blue (CBB)
staining technique is most convenient and stable in assessing acrosome reaction than
other methods (Zhang et al., 2005). The result of the study showed that the intact acrosome
uncapacitated sperm was significantly increased in cadmium treated rats. On the
other hand, the co-administration of C. lutea root methanol extract with cadmium
reduced acrosome intact uncapacitated sperm. The acrosome-reacted capacitated sperm
was significantly decreased in the cadmium-treated rats, while significant increase
was seen when co-treated with methanol extract of C. lutea root.
Development of culture systems that allow capacitation and fertilization in-vitro has made it possible to determine ions required
precisely for capacitation and acrosome reaction. Calcium ion is a prime regulator
of sperm motility, capacitation and initiator of acrosome reaction processes (Schuh et al., 2004; Publicover et al., 2007). Also
low concentrations of Na+ are necessary for sperm capacitation. C. lutea root methanol extract may prevent cadmium to replace
the metals cofactors from their active site or to bind to a deactivating site of the
enzyme itself and disrupt or interrupt activity, which can lead to oxidative stress
(Casalino et al., 1997).
The extract might also cause the plasma membrane to act as a barrier and also,
activate catalytic enzymes (Alvarez & Storey,
1984).
The standard sperm analysis is the preferred and the most crowd-pleasing laboratory
test in the diagnosis of male fertility. It evaluates sperm concentration, motility,
morphology and viability. However, it is well known that normal results of sperm
analysis cannot eliminate men from causes of couples’ infertility (Lewis et al., 2008). Today, it
is well known that the quality and integrity of sperm chromatin is very important in
the reproductive capability of men because sperm DNA is known to contribute to half
of the embryo’s genomic material. Our study showed that there was a significant
increase in abnormal sperm chromatin in the cadmium-treated groups. In in-vitro systems, cadmium binds weakly to DNA (Valverde et al., 2001), and
there are many other cellular bio-ligands to which cadmium has high affinity to bind
with, in particular the SH groups of thiols such as metallothioneins (Klaassen et al., 1999).
Therefore, the direct attack of DNA by cadmium may cause mutation. The most common
cause of sperm DNA damage is oxidative stress (Barroso et al., 2000; Kemal Duru et al., 2000) and cadmium has the capacity
to induce oxidative stress (Bal & Kasprzak,
2002; Nemoto et al., 2009). In exposed cells and tissues, cadmium affects cellular thiol redox
balance that leads to decreased intracellular glutathione content and reduced
activities of cellular antioxidant enzymes (i.e. superoxide dismutase, peroxidase
and catalase), which in turn results in the buildup of reactive oxygen species (ROS)
and an increase in intracellular oxidative stress (Casalino et al., 1997; Nemmiche et al., 2011). The ROS might damage DNA
through modification or deletions of bases, frame shifts, DNA cross-linkages,
chromosomal rearrangement, single and double strand DNA breaks, and gene mutations
(Aitken & Krausz, 2001; Spiropoulos et al., 2002; Sharma et al., 2004).
Reactive oxygen species are produced in sperm through leakage of electrons from the
mitochondrial electron transport chain (Vernet et al., 2001), NADPH oxidase (Baker & Aitken, 2005) and generation of nitric oxide (Balercia et al., 2004). The
ameliorated DNA-damaged sperm by C. lutea extract might be due to
its ability to maintain the level of zinc, an important regulator of DNA
replication, transcription, and protein synthesis, influencing cell division and
differentiation (Chia et al., 2000).
Another factor, which is essential for spermatozoa metabolism and motility, is
fructose, which serves as an energy source for spermatozoa. It is produced mainly by
the seminal vesicles, with some contribution from the ampulla of the ductus deferens
(Schoenfeld et al., 1979). Determination of seminal fructose concentration has been used to
examine obstructive azoospermia and inflammation of male accessory glands (Manivannan et al., 2005). The
result of this study showed that the seminal fructose level was significantly
decreased in cadmium-treated rats. The diminution in seminal fructose level is in
line with the Coppens (1997) report, which
affirm that inflammation may lead to atrophy of the seminal vesicles and low seminal
fructose concentration, and when ejaculatory ducts are blocked; fructose
concentration in seminal plasma usually decreases and may become undetectable.
Manivannan et al. (2005) also reported that fructose concentration
in seminal plasma of patients with obstructive azoospermia is usually absent or
significantly lower than that in men of normal fertility. Also, (Kise et al., 2000; Kumar et al., 2005) reported
that the absence or reduced seminal fructose has been found in patients with
congenital vas deferens-seminal vesicle developmental defect. On the other hand,
there was significant increase in the seminal fructose level when cadmium was
co-treated with C. lutea root methanol extract. The action of C. lutea methanol extract root may be attributed to its
antioxidant properties.
In conclusion, the results of this study show that Cadmium has deleterious effects on
male reproductive profile in experimental rat models, enough to cause infertility. Carpolobia lutea counteracts Cadmium-induced damage by
ameliorating the toxic effects of Cadmium, thus enhancing reproductive
activities.
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