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OJHAS Vol. 8, Issue 4: (2009
Oct-Dec) |
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| Effect of Neem Oil on
Sperm Mitochondrial Activity |
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Prashant Patil,
Department of Physiology,
Laxminarayan Medical college and Research centre, Bhopal,
Gaikwad RD,
Department of Physiology,
Shantiram Medical College, Nandiyal
Sawane MV,
Department of
Physiology,
Jawaharlal Nehru Medical College, Wardha,
Waghmare VS,
Department of
Physiology,
People’s College of Medical Sciences, Bhopal |
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Address For Correspondence |
Prof &
Head,
Department of Physiology,
Laxminarayan Medical college and
Research
centre,
Kolar Road, Bhopal - 462042
E-mail:
prashantpatil1411@gmail.com |
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Patil P, Gaikwad RD, Sawane MV, Waghmare VS. Effect of Neem Oil on
Sperm Mitochondrial Activity. Online J Health Allied Scs.
2009;8(4):12 |
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Submitted: Oct 13, 2009;
Accepted:
Nov 19, 2009; Published: Apr 30, 2010 |
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| Abstract: |
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It is a known
fact that neem oil has some effect on motility of sperm. Motility
of sperm depends on mitochondrial activity present in mid-piece of
sperm. In the present study, the mitochondrial activity of sperm was evaluated
after treating semen with the different quantities of neem oil. The
mitochondrial activity was also evaluated after subjecting the semen
samples for different incubation periods keeping the quantity of semen
as well as that of neem oil same. Tests were done on thirty
normozoospermic semen samples with motile score more than
75%. It was found that as the quantity of neem oil increases, the mitochondrial activity
decreases significantly (P < 0.001). Similar results were
found, when same quantity of neem oil was treated with same quantity of semen,
but incubating for different time durations. The mitochondrial
activity decreases significantly (P < 0.001) from one minute to twenty
minutes. So, it indicates that as the contact period between neem oil and semen
increases the mitochondrial activity decreases significantly.
Key Words:
Normozoospermia, Neem Oil, Sperm Mitochondrial Activity |
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Indigenously
available neem oil in its natural form has spermicidal and anti-implantation
effect.1 Two derivatives of neem oil – sodium nimbinate
and sodium nimbidinate have been found to possess weak spermicidal
action in vitro.2 It was found that neem oil decreases sperm
motility instantly.3 In view of this, the present study was
undertaken to find the effect of neem oil on sperm mitochondrial activity.
The study was
carried out in semen laboratory of Department of Physiology, Govt.
Medical
College, Nagpur. Semen sample of normozoospermic men were obtained
after
five days of abstinence. After proper liquefaction, at room
temperature,
sperm motility was checked. Thirty samples with more than 75%
motile
spermatozoa were taken for test, while the remaining samples were
discarded. Neem oil was procured from Khadi Gramudhyog shop. Two types of
tests
were carried out. In first type of test, the quantity of neem oil
was
changed and incubation period was kept constant. [Table 1]
In second type
of test, neem oil and semen quantities were kept constant and they were
incubated for different time durations. [From one min. to twenty mins.] [Table
2]
After
incubation
(in first case for 5 min. and in second case from one min. to twenty
mins), one drop of solution from respective test tube was drawn and
SMAI (Sperm Mitochondrial Activity Index) was performed.
Table 1: Incubation period
kept constant (n=30)
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Test tube no |
Quantity of semen (ml) |
Quantity of neem oil (ml) |
Incubation period (min) |
SMAI score |
P value |
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1 |
0.1 |
nil |
5 |
73.32 ± 3.31 |
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2 |
0.1 |
0.1 |
5 |
68.52 ± 1.61 |
<0.001 |
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3 |
0.1 |
0.2 |
5 |
44.18 ± 4.69 |
<0.001 |
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4 |
0.1 |
0.3 |
5 |
32.07 ± 2.15 |
<0.001 |
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5 |
0.1 |
0.4 |
5 |
20.17 ± 1.97 |
<0.001 |
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6 |
0.1 |
0.5 |
5 |
10.98 ± 2.09 |
<0.001 |
Table 2: Quantity of neem
oil kept constant (n=30)
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Test
tube no |
Quantity of semen (ml) |
Quantity of neem oil (ml) |
Incubation period (min) |
SMAI score |
P value |
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1 |
0.1 |
0.1 |
1 |
62.23 ± 2.12 |
<0.001 |
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2 |
0.1 |
0.1 |
5 |
51.13 ± 2.18 |
<0.001 |
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3 |
0.1 |
0.1 |
10 |
41.88 ± 2.84 |
<0.001 |
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4 |
0.1 |
0.1 |
15 |
21.92 ± 2.65 |
<0.001 |
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5 |
0.1 |
0.1 |
20 |
13.32 ± 3.52 |
<0.001 |
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6 |
0.1 |
Nil |
20 |
74.15 ± 3.06 |
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SMAI Test4
Method
- One drop of semen
and one drop of Nitrobule Tetrazolium (NBT) solution was put in a
cavity
slide.
- The cavity slide
was covered with cover slip.
- It was kept in a
Petri dish with moist cotton on the sides.
- It was incubated
at 37º C for 30 minutes.
- It was mix well
and a smear was prepared on a pre-cleaned labeled slide.
- It was allowed it
to air dry.
- Then the slides
were fixed in neutral formalin for 2 hours.
- After 2 hrs slides
were wash with distilled water and allowed to dry.
- These slides were
viewed using oil immersion lens.
- The extent of NBT
precipitate of 100 spermatozoa as standard, substandard, low,
residual
and nil were counted.
- The percentage under
each grade were multiplied with arbitrary value of 1, 0.7, 0.3, 0.1
and 0 for standard, substandard, low, residual and nil respectively.
- The total of all
was the sperm mitochondrial activity index.
Such tests were carried out
on 30 normozoospermic subjects in Dept. of Physiology, Govt Medical
College, Nagpur. Stastical analysis was done by Student’s t test.
In the first
test, where incubation time was kept constant (5 min), it was seen that
test tube with nil concentration of neem oil had 73.32 ± 3.31
sperm mitochondrial activity index. As the concentration of neem
oil
was increased from 0.1 ml to 0.5 ml, the significant results were
obtained
for SMAI.[Table 1] In first test tube with 0.1 ml concentration
of neem oil, SMAI score was 68.52 ± 1.61. It was highly significant
when
compared with test tube having no neem oil (test tube no. 1) (P <
0.001). Similarly, as the concentration of neem oil was increased
in
other test tubes, it showed a significantly decreased SMAI score as compared to
test tube no. 1 (P < 0.001).
When
concentration
of neem oil was kept constant and incubation time was varied from 1
to 20 mins, it showed the following result.[Table 2] In test tube
no.
6 (with no neem oil) the SMAI score was 74.15 ± 3.06. This shows
that
there was no obvious change in SMAI score after 20 mins of
incubation. In test tube no. 1 to 5, SMAI score started decreasing significantly
when incubation period was increased. When incubation period was
20
mins SMAI score was 13.32 ± 3.52 (neem oil conc 0.1 ml). When
these
results were compared with test tube no. 6 they were found to be
statistically
highly significant (P < 0.001). This shows that neem oil
decreases
the SMAI scores significantly with increasing incubation periods.
It is reported
that neem oil has spermicidal effect and it reduces the motility of
sperms.5-8 Also, it increases the uterine
contractions
as reported by Mitra9, after application to Guinea pig
uterus.
Again, the suppression of ovulation by Azadiractin (a component of neem
oil) is reported by Renbold & Sieber in locusts.10
The present
study is mainly concerned with motility of sperm. Motility of
sperm
depends on flagella. It is reported that neem oil has anti
flagellal
action.11 Flagellal motility is due to mitochondrial
activity
in mid-piece of sperm. In the present study mitochondrial activity
was
measured after treating the semen with neem oil. It shows that
with
increase in quantity of neem oil, the mitochondria of sperm are affected
more, thus affecting the sperm motility adversely. (Table
1).
When same
quantities
of semen and neem oil were incubated for different time durations it was found
that the sperm mitochondrial activity decreased significantly as the incubation
period increased. SMAI score was 74.15 ± 3.06 in
sample without neem oil, while it was 13.32 ± 3.52 in sample with same
quantity of neem oil when incubated at 37º C for 20 min (Table
4). This indicates that there is reduced motility of sperm after
reacting
with neem oil for 20 min. The neem oil is possibly acting upon the
enzymes
in mitochondria that are responsible for production of ATP. Thus
the
depletion of ATP is resulting in reduction of sperm motility.
The above study is helpful
in showing that neem oil is a potent agent having adverse action on
mitochondria and thus reducing sperm motility. Spermicidal action
of
neem oil was also reported by K C Sinha et al.1,6,7,13 Abundant
availability of neem oil can make it a favourable agent of contraception
in India. It can be an agent in non hormonal pre and post coital
contraceptive. Further studies are needed.
- Sinha KC et al.
Anti-implantation effect of neem oil. Ind. J M. Res. 1984;80;708-710.
- Sharma VN, Saxena KP. Spermicidal action of Sodium Nimbinate.
Ind Jour.
M. Res
1959;47;322-324.
- Sinha KC, Tiwari
RS. Neem oil as Vaginal Contraceptive. Ind Jour. M. Res
Jan1984;79;131-136.
- Gopalkrishna et
al. Mol. Andrology 1990;3;243-253.
- Sharma VN et al. Ind Jour. M. Res 1959;47; 3.
- Kasturi M, Ahmad
RN et al. Mode of long term anti-fertility effect of
intra-uterine neem
treatment. J Basic Clin Physiol Pharmacol. 2002,13(4):311:28.
- Sharma SK, Sairam
M et al. Neem oil inhibits two cell embryo development and
trophectoderm
attachment and proliferation in vitro. Contraception. Dec 1996;54(6):373–8.
- Juneja SC, Williams
RS et al. Immunocontraception undergoing promising trials. J
Assist Reprod Genet. Aug 1996;13(7):576–85.
- Mitra CR. In-Natural
Pesticides from neem tree, proceedings of 1st International
conference, Rottach Egren, Federal Republic of Germany, 16-18 June
1980.
pg no. 97.
- Rembold H, Seiber
KP. In-Natural Pesticides from neem tree, proceedings of 1st
International conference, Rottach Egren, Federal Republic of Germany,
16-18 June 1980. pg no. 75.
- Chopra et al. Indigenous
Drugs of India. 1982. Pp. 962.
- Newton P et al. A new family planning tool to slow population growth. IDRC
Rep. Jan
1993;20(4):16–18.
- Bardhan J, Riar
SS et al. Neem oil – a fertility controlling agent in rhesus
monkey. IJPP. Oct 1991;35(4):278–80.
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