Document Type : Original Article
The decrease in fertility and male reproduction
is associated with certain special toxic chemicals
in the environment. There is a close relationship
between infertility and vocational toxicity. Lead
is a heavy element in the environment. Its use,
particularly in gasoline, has made it widespread.
In addition, lead is a toxic element whose effects
appear over a long time. The human body
receives lead through food (65%), water (30%)
and weather (15%) (
Lead overexposure may cause anemia, renal disorders,
reproductive abnormalities and neural conditions
such as sudden seizures, behavior abnormalities
or a low intelligence quotient (IQ). Even
exposure to a low level of lead may bring about
changes in the body’s physiological functions (
Studies show that the main target of lead toxicity
is male fertility and the axis of pituitary-testis
reproduction, thus causing infertility, reduction in
sperm levels and morphological changes in people
with excessive lead exposure (
The present study seeks to investigate the oral effects of lead acetate on the parameters related to
sexual behavior in male rats which include: mount
latency (ML), intromission latency (IL), post
ejaculatory interval (PEI), mount frequency (MF),
ejaculatory latency (EL), intromission frequency
(IF), copulatory efficacy (CE), intercopulatory interval
(ICI) and changes in the blood testosterone
hormone level (
The Ethics Committee at Islamic Azad University, Kazerun Branch has reviewed and approved ethical conciderations in the present study.
For the purpose of this study, male and female Wistar rats, supplied by the Animal Breeding Center of Islamic Azad University, Kazerun were used. Rats weighed 200-220 g and were 2.5-3 months old. They were maintained under standard conditions of light, food and temperature.
Since the observation related to parameters of sexual behavior should be performed at night, we darkened a room and placed all rats in that environment while reversing their day and night cycles. The observation and experiment took place in the same location. To observe the parameters of sexual behavior, a glass cage was placed on a tabletop one meter above the floor.
To observe the rats, a 5 watt red lamp was utilized. Light conditions were as follows: 7 am to 7 pm was considered the dark period and 7 pm to 7 am was the light period. Throughout the experiment, male and female rats were maintained separately except for behavioral observation when they were placed together in the glass cage.
Cages were washed and sterilized every other day.
The environmental temperature was controlled at
22 ± 2°C (
To ensure a limited weight range, the rats’ weights
were measured prior to the experiment and male
rats who weighed 200-220 g were selected. In total,
there were 40 male rats divided into the following
five groups: 1. control group (A) received
only condensed food and water during the experiment
with no solvent or medicine, 2. sham group
(B) received 0.2 ml oral solvent (distilled water) in
24 hours, 3. first experimental group (C) received
25 mg/kg oral lead acetate in 24 hours, 4. second
experimental group (D) received 50 mg/kg oral
lead acetate in 24 hours, and 5. third experimental
group (E) received 100 mg/kg oral lead acetate in
24 hours (
Before performing the experiments, active and inactive males were separated. A male rat had 25 minutes to copulate a female rat (intromission or ejaculation). The male rats that were unable to perform the copulation within this period were separated from the other males. Studies have shown that male rats with no sexual experience exhibit increases in the IL, EL and PEI periods.
Thus, in order to obtain sexual experience each male rat copulated several times prior to the main observations with prepared females.
Almost 60 adult female Wistar rats were used on different days. Preparation of female rats was identical in the experimental, control and sham groups.
All female rats were maintained in separation several times before the experience in the glass cage for adaptation. To induce sexual admittance, progesterone (50 μg) and estradiol valerate (100 μg) were used. Forty-eight hours prior to observation, each female rat was injected subcutaneously with 100 μg of estradiol valerate dissolved in olive oil. Female rats that underwent experimentation were left untreated for the next 48 hours. Following the experiment, 1 ml white alcohol was injected into the female rat vagina to prevent pregnancy and kill the sperm.
An insulin syringe with a feeder was used for drug injections that were administered every morning at a given time. Having injected the 25, 50 and 100 mg/kg lead acetate, parameters related to sexual behaviors were recorded through per second observations for 120 minutes. The following parameters were recorded: mount latency (ML) or the interval between placing a female animal near a male animal and the first copulation attempt. Copulation is the behavior in which the male animal is mounted on the back of the female. Intromission latency (IL): the interval between placing a female animal near a male animal and the first male intromission. Intromission is a copulatory behavior in which the male animal genital organ enters the female vagina. Ejaculatory latency (EL): the interval between placing a female animal near a male with ejaculation. In ejaculation, which is often accompanied by intromission, the male animal mounts on the back of female animal. The male genital organ enters the female vagina rapidly and stays there for a moment. Rapid and rhythmic movements can be observed on the back of the male animal.
It is worth noting that the intromission accompanied by ejaculation is longer than the usual and common intromissions. Additionally, the pressure that the male animal exerts into the female animal is greater in this condition. Post ejaculatory interval (PEI): the interval between the ejaculation and the first next copulation intromission. In this period, the male animal is separated from the female counterpart. Mount frequency (MF): the number of times that the male animal copulates with the female animal without intromission. MF is calculated in the copulation series before ejaculation. Intromission frequency (IF) refers to the number of times that the male copulates with intromission and performs this action in a copulation series before ejaculation.
Copulatory efficacy (CE) is not obtained by observation, but is measured by the formula CE= IF MF+IF
It shows that copulation is completely successful.
Intercopulatory interval (ICI) is the interval between
two copulations accompanied by intromission
and is calculated by the following formula:
We measured each of the above mentioned factors in different copulatory series.
A copulatory series is the interval between the first male animal activities for copulation until ejaculation, followed by their separation.
During the observation time (about 120 minutes for every male animal), several copulation series were observed and factors were determined by indices. After the 28 day treatment with lead acetate, blood samples obtained from the rats’ hearts were collected in test tubes under mild ether anesthesia at the end of day 28.
After about 15 minutes, test tubes that contained blood samples were gathered and placed in an incubator at 37°C for 30 minutes.
After coagulation the tubes were placed into the centrifuge at 5000 rpm. Sera was pipetted by Pasteur pipettes and transferred into new, labeled tubes sealed with parafilm and stored frozen until the measurement of testosterone by RIA.
SPSS was used from data analysis. ANOVA analyzed means. In case the means were different, TUKEY test was used for comparison of pairs. Each mean value was compared with the control group. P≤0.05 was considered significant.
The investigation into the effects of different doses
of lead acetate on the concentration of testosterone
indicated that the groups who received 50 and
100 mg/kg lead acetate significantly decreased
(p≤0.05) when compared with the control group
The effects of different doses of lead acetate on testosterone hormone levels in the experimental and control groups (*p≤0.05).
The effects of different doses of lead acetate on
ML in different series (ML1, ML2, ML3), indicated
a significant increase (p≤0.05) in the groups that
received doses of 50 and 100 mg/kg lead acetate
compared to the controls (
Comparison of ML factor between the experimental groups receiving different doses of lead acetate and control group in the first (ML1), second (ML2) and third (ML3) copulatory series (*p≤0.05).
The investigation of the effects of different doses
of lead acetate on IL in different copulation series
showed that IL1 in the first copulation series in the
groups receiving 25, 50 and 100 mg/kg lead acetate
significantly increased when compared with the
control group. In the second and third copulation series,
factors IL2 and IL3 showed significant increases
in the experimental group (50 and 100 mg/kg) compared
to the control group (p≤0.05) but not in the
group that received 25 mg/kg lead acetate (
Comparison of IL factor between the experimental groups receiving different doses of lead acetate and control group in first (IL1), second (IL2) and third (IL3) copulatory series (*p≤0.05).
Statistical test results related to factor EL in the
EL1, EL2 and EL3 copulatory series show that the
groups which received 50 and 100 mg/kg of lead
acetate had significant increases (p≤0.05) when
compared with the control group (
Comparison of EL factor between the experimental groups receiving different doses of lead acetate and control group in first (EL1), second (EL2) and third (EL3) copulatory series (*p≤0.05).
The results related to PEI in the first and second
copulation series showed significant increases
(p≤0.05) in the experimental groups that received
50 and 100 mg/kg lead acetate than the control
Comparison of PEI factor between the experimental groups receiving different doses of lead acetate and control group in first (PEI1), second (PEI2) and third (PEI3) copulatory series (*p≤0.05).
The investigation of lead acetate effect on the
MF,series was not significantly different (p≤0.05)
among all the experimental groups and the control
Comparison of MF factor between the experimental groups receiving different doses of lead acetate and the control group in first (MF1), second (MF2) and third (MF3) copulatory series (*p≤0.05).
A comparison of statistical test results related to IF
factors (IF1, IF2 and IF3) between the experimental
groups that received 25 and 50 mg/kg lead acetate
and the control group did not show a significant
decrease (p≤0.05), however the experimental
group that received the 100 mg/kg dose significantly
decreased (p≤0.05) compared to the control
Comparison of IF factor between the experimental groups receiving different doses of lead acetate and control group in first (IF1), second (IF2) and third (IF3) copulatory series (*p≤0.05).
Also, statistical comparison of the results related
to CE1 showed that the group which received 100
mg/kg lead acetate significantly decreased (p≤0.05)
compared to the controls. No significant difference
was observed between the experimental groups administered
25 and 50 mg/kg lead acetate, and the
control group. In the second and third copulation
series, no significant difference in CE parameters
was observed between the groups receiving doses
of 25, 50 and 100 mg/kg lead acetate and controls
Comparison of CE factor between the experimental groups receiving different doses of lead acetate and control group in first (CE1), second (CE2) and third (CE3) copulatory series (*p≤0.05).
Statistical comparison of the results related to ICI
parameter (ICI1, ICI2, ICI3) indicated a significant
decrease (p≤0.05) in the groups administered 50
and 100 mg/kg lead acetate compared to the control
group, however in the group that received 25
mg/kg lead acetate, no significant difference was
observed compared to the control group (
Comparison of ICI factor between the experimental groups receiving different lead (*p≤0.05).
acetate levels and control group in first (ICI1), second (ICI2) and third (ICI3) copulatory series.
Decreases in fertility and reproduction are associated
with toxic chemicals in the environment;
there is a close correlation between infertility and
vocational toxicity (
Considering the present findings, testosterone
hormone levels in the experimental groups that received
50 and 100 mg/kg lead acetate significantly
decreased compared to the control group (
The IL factor indicates intromission latency.
Groups that received doses of 25, 50 and 100 mg/
kg lead acetate showed significantly increased IL
in the first copulatory series. However in the second
and third copulatory series, this increase was
observed in IL only at doses of 50 and 100 mg/
As previously mentioned, lead decrease the level
of dopamine level in the preoptic area. Dopamine
mediates the performance of and in fact, erection is
the precense of nitric oxide (NO) (
The neurosteroid production from adrenal and gonadal
steroid endocrine sources may affect male
sexual behavior through GABAA receptors. This
receptor is located in the preoptic area. It is essential
and necessary for sexual attraction, intercourse,
erection and ejaculation. These steroid gonadal
sources occur in the brain through testosterone aromatization,
which is essential for sexual behavior
and sensitivity. Neurosteroids also may modulate
activities related to smell in order to sexually attract
Decreased enzymatic activities in the olfactory
bulb cause disturbances in smell-related behaviors
and decreases in sexual function (
The groups that received 25 and 50 mg/kg lead acetate
had no significant change in CE when compared
with the control group, whereas the group
that received 100 mg/kg lead acetate showed a significant
decrease only in the first copulatory series
As mentioned before, lead acetate leaves an unsuitable
effect on copulatory efficacy and causes a
disorder in sexual function by decreasing the mentioned
parameters, in particular IF. Long term consumption
of industrial metal salts such as magnesium
sulfate, aluminum chloride, lead acetate and lead chloride create adverse effects on sexual behavior,
fertility and the adult male rat reproductive
system. It also decreases copulatory efficacy (
We may conclude that 50 and 100 mg/kg lead acetate increases ML, IL, PEI and EL parameters and causes a disorder in sexual motivation and performance. In addition, 100 mg/kg lead acetate causes a significant decrease in IF and CE parameters which is indicative of a disorder in copulation. Lead acetate causes decreased sexual motivation and reproductive activities due to the decreased testosterone hormone secretion, which is probably due to its effect on the central nervous system and disordered regulation and secretion of neurotransmitters.