EFFECT OF SUSTANON@ 250 ON REPRODUCTM EFFICIENCY IN MALE AND FEMALE RATS

Abstract

The present study was conducted to investigate
the effect of ( Sustanon
t
250;
abusing on reproductive efficiency .Thirty two male and thirty two female rats were
used'Theyweredividedintofourgroups(eachgroupconsistedofSmalesand8
females).Thefirstgroupservedascontrolgroupinwhichtheratsdosedby
intramuscular
injection of 10 p I olive oil weekly for !2 weeks '
the rest 3 treated
groups(Gl.G2andG3)inwhichtheratsdosedbyintramuscularinjectionof50.l00
and 150 mg Sustanon
t
250 / kg B.W. respectively
weekly for 12 weeks' then all rats
were terminated to determine testosterone ,FSH
and LH levels in addition to seminal
analysis . The results reveled that testosterone level was significantly
(pS 0'05)
elevated in Gr and G2 . The LH and FSH were significantly
decreased(ps
0.05) in all
animals .The seminal analysis in response to Sustanon
t
250 injection revealed a
significant
.
reduction in the sperm concentration,
viable spenn
percentage and
morphologicallynormalspermpercentage'Anothergroupof16maleand32female
rats were used ,to
determine the reproductive efficiency ,
from which 4 male and 8
femaleweredosedbyintramuscularinjectionwithl0ploliveoilweeklyfor12
rveeks,theyareconsideredascontrolanimalsTherestratsweredosedby
intramuscular
injection of.150 mg Sustanon
@
250 / kg B'W' weekly for 12 weeks '
Thentheyweredividedintofourgroups:groupl(4nontreatedmaleswithSnon
treated females ) ,
group 2 (4 treatedmales with 8 treated females )'
group 3 (4 treated
males with 8 non treated females) and group 4 (4 non treated males with 8 treated
females )
. After they were allowed to mate for 12 weeks ,
the reproductive
parameters
were investigated
which revealed
the fertility percentage as 100% in Gl 'whereasthfemale rats in G2 ,
G3 and
,
G4 showed no pregnancy at all which indicated complete
infertility.

Full text

&
: -* Basrah
journal of veterinary
research ' v{ .]t' 4'1012
proceedings
of the 3d
international
Scientific
conference,
college
of
veterinary
Medicine'
UniversitY
of Basrah-Iraq'
EFFECT OF SUSTANON@
250
ON REPRODUCTM EFFICIENCY
IN MALE AND FEMALE RATS
.Nehaya
M. Al-AubodYt Mohammad
A' Al-Diwan'z
College
of Medicine,
University
of Basrah'
Basrah'Iraq
CollegeofVeterinaryMedicine,UniversityofBasrah,Basrah,Iraq
The present
study
was conducted
to investigate
the effect of ( Sustanon
t 250;
abusing
on reproductive
efficiency .Thirty two male and thirty two female
rats were
used'Theyweredividedintofourgroups(eachgroupconsistedofSmalesand8
females).Thefirstgroupservedascontrolgroupinwhichtheratsdosedby
intramuscular injection of 10 p I olive oil weekly for !2 weeks ' the rest 3 treated
groups(Gl.G2andG3)inwhichtheratsdosedbyintramuscularinjectionof50.l00
and
150
mg Sustanon
t 250 / kg B.W. respectively
weekly for 12
weeks'
then
all rats
were terminated
to determine
testosterone
,FSH and
LH levels in addition
to seminal
analysis . The results reveled that testosterone
level was significantly (pS 0'05)
elevated
in Gr and
G2 . The
LH and
FSH
were
significantly
decreased(ps
0.05)
in all
animals
.The seminal
analysis
in response
to Sustanon
t 250 injection
revealed
a
significant . reduction in the sperm concentration,
viable spenn percentage
and
morphologicallynormalspermpercentage'Anothergroupof16maleand32female
rats were used ,to determine
the reproductive
efficiency , from which 4 male
and 8
femaleweredosedbyintramuscularinjectionwithl0ploliveoilweeklyfor12
rveeks,theyareconsideredascontrolanimalsTherestratsweredosedby
intramuscular
injection
of.150
mg Sustanon
@
250 / kg B'W' weekly for 12
weeks
'
Thentheyweredividedintofourgroups:groupl(4nontreatedmaleswithSnon
treated
females
) , group 2 (4 treatedmales
with 8 treated
females
)' group
3 (4 treated
males
with 8 non treated
females)
and group 4 (4 non treated
males
with 8 treated
females
) . After they
were
allowed
to mate
for 12
weeks
, the
reproductive
parameters
were
investigated
which revealed
the
fertility
percentage
as
100% in Gl 'whereasthe
269

Basrahjournal ofveterinary
research,
vol. 11, 4,20L2
Proceedings
of the 3'd international Scientific Conference,
College of Veterinary Medicine,
Universiry of Basrah-Iraq.
female
rats in G2 , G3 and
, G4 showed no pregnancy at all which indicated
complete
infertility.
INTRODUCTION
Sustanon@
250 is one of the androgenic
- anabolic
steroids
that usually
abused
by athletes and young females
and males
to have
a muscular
body and increase
their
sportive
performance
(1). The present
study aimed
to investigate
the adverse
effects
induced by Sustanon abuse on reproductive efficiency , that determined
through
measure
some
reproductive..hormonss( including testosterone
, LH and
FSH ) , semen
analysis
,fertility percentage
and
other
reproductive
parameters.
Testosterone
is derived
from cholesterol
( 2) produced
and secreted
by the interstitial leydig cells ,which
constitute
about
20 o/o
of the mass of adult testis
(3). It has a range of biological
actions.
the classical effects
of testosterone are induction
and
maintenance of secondary sexual
characteristic
and preservation
of libido ,sense
of well being ,lean mass ,bone
density(4)
and stimulation of protein
synthesis
(5). Testosterone
is found in the body in
three forms as free , Sex Hormone
- Binding Globulin (SIIBG) and these that are
impotently bounded to albumin or cortisol binding globulin (6).Circulating
bound
and
free testosterone
are collectively referred to as total testosterone
(7).LH and FSH are
glycoprotein
secreted
from the anterior lobe of pituitary gland as a response
to the
release
of gonadotropic
releasing
hormone
(GnRtI) from the hypothalamus(8).
(FSH)
has been implicated
in Leydig cell function through its action on Sertoli cells (9) to
produce
the sperm
cells
(10).It
is essential
for initiation
of spermatogenesis
(11).
LH
had been
called interstitial cell - stimulating hormone
(2) , it stimulates
Leydig cell
production
of testosterone
, It acts synergistically
with FSH
(12).
MATERIALS AND METHODS
Testosterone ,foilicle stimulating hormone (FSH) and lutenizing
hormones(LH)
were assayed by using enzyme
- linked immunosorbent assay
(ELISA)
kits specific
for rat hormones, manufactured
by USCN Life Scince
Inc. in China.
The
sperms
concentration and spenn viability were counted
according to method of Evan
and
Maxwell (13) by using
Neubauer hemocytometer
chamber which use for RBC and
WBC count . The abnormal spermatozoa were counted
in the same slide
that was used
270

Basrahjournal
ofveterinary
research
' vol'
11'
+'iOtZ
',
Proceedings
of
the 3'd
international
Scientific
Conference,
College
of
Veterinary
Medicine'
Universiry
of
Basrah-Iraq'
to measure
the dead
sperrnatozoa
using
account
200 sperms
under
the light microscope
using 100
X power according
to Sakamoto
and
Hashimoto ( 14)'
The reproductive
effects
caused
by weekly intramuscular
injection of high
dose
of sustanon
@
250 (150 mg / kg body
weight
) for male
and female
rats ( 16
male
rats
and 32 female
rats) divided
into 2 groups
1- Control group ( 8 males
and 16 females
rats
) injected
weekly with only 10 ptl
olive
oil
2- Treated
group (8 males
and 16 females
rats ) injected
with 150 mg / kg body
weight sustanon
t 250
At the end of treatment
period (12 weeks
) the animals
fuither divided into 4
groups as
follows : -
r Group 1 consists
of 4 non
treated
males with 8 non
treated
females
'
e Group
2 consists
of 4 treated
males
with 8 treated
females
.
r Group
3 consists
of 4 treated
males
with 8 non
treated
females.
r Group
4 consists
of 4 non
treated
males
with 8 treated
females
.
The animals
left for 12 weeks
for mating
before
measuring
the parameters
to
determine
the reproductive
effrciency
(number of pregnant
females
' fertility percent
,
number of life birth , weight of life birth , abnormalities
of life birth , number
of
corpora
lutea
and
number
of implanting
sites
The results
were expressed
as mean
* SD. The comparisons
between
groups
were performed with analysis
of variance
(ANOVA) by using computerized
sPSS
program
(Statistical
Program
for Social
Sciences).
P<0.05
was considered
to be the lest
limit of significance.
Least significant difference
test (LSD) was calculated
to test
difference
between
means
(groups) for (ANOVA) SPSS
(1998)'
RESULTS
Effect
of Sustanon@250
Injection on Some
Hormones
In Male Rats.
The testosterone
level in male
rats treated
with (sustanon@250;
50 and 100mg
/kg B.W were significantly increased
(p S O.OS)
compared
with testosterone
level of
control
males
(table 1) whereas
the group
treated
with 150
mg /kg B'W does
not seem
27L

Basrahjournal ofveterinary research,
vol. 11,
4,20L2
Proceedings
of the 3'd international Scientific Conference,
College of Veterinary Medicine,
University of Basrah-Iraq.
to be affected
by sustanon@250 injection compared
with control group and other
treated
groups
.
The
LH and FSH levels decreased significantly
(p S 0.05)in
all injected male
rats with (sustanon@250
) 50,100
and 150 mg /kg B.W compared with control male
rats. No significant difference (p > 0.05) in LH and FSH level among
treated
male
groups
were
recorded.
Table ( I ) Effect of Sustanon@2s0 Injection
on Some
Ilormones in Male Rats
means
* SD
N=8
Different letters in the same column
represent
significant
difference
at (pS0.05).
In Female Rats
NTestosterone
nglml
LH
mIU/ml
FSH
mIU/ml
Control
(0.lmlolive
oil)
4.453
b
it.049
2.9t7
a
10.333
2.626
a
J.0.492
Groupl
(50mg/kg
B.w.)
6.375
a
*0.751
1.803
b
*0.753
t.737
b
+0.t23
Group2
(100mglkgB.W.)
6.t39
a
*0.t23
1 aaa
I.JJJ
b
'+0.421
t.484
b
+0.557
Group3
(150mg/kgB.W.)
I
4.471
b
*1.454
0.72r
b
+0.087
1.143
b
+0.237
LSD t.604 1.1 13 0.603
272

' Barr"h
journal
ofveterinary
research
' vol'
11'
4'2072
', Proceedings
of
the
3'd international
Scientific
Conference'
College
of
Veterinary
Medicine'
UniversitY
of Basrah-Iraq.
The injection of sustanon@250
in female rats is shown to cause
significant
increase ( p s 0.05
) in testostefone
level of all treated
groups
compared
with control
group
(table
2) . There were no significant
differences observed
among
three
treated
groups
The LH level in female rats injected with (sustanon@250
) showed a
significant
decrease
(p 5 0.05) in treated
groups
(50,100 and 150 mg/kg B'W)
compared
with control females
. It seems
that (sustanon@250
) could decreased
the
LH
level significantly (p S 0.05) according to increase
the dose 'The FSH level
significantly
decreased
(p <'0.05) by iirjection
of (sustanon@z5o;
50 , 100 and 150
mg/kg
B.w compared
with control females
.while no significant
differences
(p z 0'05)
were
observed
among
all treated
groups.
Table ( 2 )Effect of Sustanon*250
Injection on Some
Hormones in Female
Rats
meanst SI)
N=8
Different
letters
in the
same
column
represent
significant
difference
at
(pS0.05)
6949
a
+t.044
2.694
b
+0.699
Control
group
(0.lmlolive
oil)
5.469
b
r0.460
6.270
a
*0.782
Groupl
(50mg/kg B.W.)
4.s93
b
+0.470
6.701
a
+0.245
Group2
(100mg/kg
B.W.)
4.232
b
+0.806
6.230
a
+0.510
Group3
(150mgikg
B.W.)
273

Basrah
journal of veterinary research
' vol' 1
1
' 4'2012
, proceedings
of the 3d international Scientific Conference,
College
of Veterinary Medicine,
UniversicY of Basrah-Iraq.
Seminal
AnalYses
The effect
of sustanon@25g
injection for 12 weeks
with 3 different
doses
( 50
, 100
and 150
mg / kg B.w.) on seminal
analyses
as shown
in table 3 revealed
that
the
spenn
concentration,viability
pefcentage
and
normal
spenn
morphology
percentage
in
male rats injected
with (sustanont2sg
) 50 ,100 and 150 mg /kg B.W were
significantly
decreased
( p s 0.0s ) compared
with that of control male
rats . while the
percentage
of abnormal
sperm
morphology
increased
significantly (p S 0'05 ) in all
treated
groups
compared
with control
group
( table
3)'
Table ( 3 )The Bff""t of Sustanon@250
Injection on Seminal
Analyses
means
* SD
Sperm
concentration
Xl06/mm3
viable
%
Normal
%
Abnormal
Yo
Control
group
(0.lmlolive
oil)
2902
a
+
0.653
7r,40
a
L0.027
82.0
a
+0.034
18.0
b
+0.034
Groupl
(50mg/kg
B.W
)
1.402
b
*0.341
6t.24
b
+0.092
47.29
b
+0.027
52.71
a
+0.027
Group2
(100mg/kg
B.W)
1.055
b
+0.064
55.25
b
+0.102
45.41
b
+0.031
s4.59
a
+0.031
Group3
(150mg/kg
B.W)
1.055
b
+0.221
52.52
b
+0.077
42.68
b
+0.040
47.22
a
+0.040
LSD 1.490 t9.21 09.56 7.r7
N:8
Different letters
in the same
column
represent
significant
difference
at (p50.05)
274

Basrah
journal
of veterinary
research
, vol' 1 1
' 4'2012
Proceedings
of the 3'd
international
Scientific
Conference,
College
of Veterinary
Medicine'
University
of Basrah-Iraq.
The sperm was considered
morphologically
abnormal if it had one of the
following features
: rudimentary
tail , absence
of tail, round head and / or detached
head.
Reproductive
fficiencY
After l2weeks of mating
period of female
and
male
rats
treated
previously
for
12 weeks
with 150
mg / kg B.W.Sustanon@250
injected
weekly as shown
in table (4)
eight pregnancy
cases
were recorded
. This indicated T00%
fertility percent to give
5.25
+.0.75 litters
weighted5.l * 0.35
and after
killing the mothers
the number
of
implanting
sites
were
5.1
+ 0.20
and
the number
of corpora
lutea
were
6 *0'20'
Table (a)The Reproductive Efficiency Results
of Mating Rats After L2
weeks
of
intramuscular
injection
of 150
mg / kg B.W. of Sustanon@250
means
* SD
0
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tm :treated
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ntm: non
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male
ntf : non
treated
female
27s

Basrahjournal
ofveterinary
research, vol. 11,
4,2012
Proceedings
of the 3'd
international Scientific Conference,
College
of Veterinary Medicine,
University of Basrah-Iraq.
The other
3 groups
, which include
either treated male
with non treated
female
or treated female
with non treated
males
or treated both
males
and females
" showed no
pregnancy
in all females indicating
complete
infertility.
DISCUSSION
The sperm
concentration
in male
rats of all treated
groups
were significantly
decreased
compared
with the sperm
concentration
of control male rats(table
3). This
finding agrees
with previous
studies
which found that the abuse
of supraphysiologieal
doses
of anabolic
androgenicfteroids
(AAS ) resulted in transient
sever
oligospermia
in
males
because
the AAS induced
reversible
hypogonadotrophic
hypogonadisim
(15,
16;
and 17).The low sperm
count
of treated
rats
could be
due to reduction
in FSH
level
induced
by testosterone
injection , because
the FSH is essential
to .initiate the
spermatogenesis
in Sertoli cells (2) and for nourishment of sperms( 12 ). The
percentages
of viable spenn and that of morphologically normal spenns were
significantly
decreased
compared
with control
group,
while the percentage
of dead and
morphologically
abnormal
sperms
were significantly increased
compared with control
group(table
3) .The findings
of Torres
{alleja et al. ,2001
(18) that abuse
of AAS
alone not only reduced the spenn count but also impairs the percentage
of
morphologically
normal semen
.The reason for the decreasing percentage
of viable
and morphologically normal spenns and inueasing the percentage
of dead and
morphologically
abnormal
sperms
,was
due
to the testosterone
injection which led to
hormonal
disturbance
and increased
ROS formation as supported
by increased
MDA,
and
because
the mammalian
spermatozoa
are
more vulnerable
to lipid peroxidation
due
to their cell membrane
rich with lipids especially
poly unsaturated
fatty acids( PUFA)
are the main substrates
for lipid peroxidation
, that may provoke severe
functional
disorder
of sperm
(19) .MDA levels
were
negatively
correlated
to sperm
concentration
and
motility which
was
consistent
with previous
reports
(20and2l) .lnother studies,
it
was reported
that peroxidation
reaction
affects membrane
structure
and fluidity and
causes
damage to axonemal proteins leading to a permanent
impairment in sperm
motility ( 22; 23 ; and 24).A according
to (25; 26; and 27) the RoS, in addition
to
reacting
with the polyunsaturated
fatfy acids,
might also react
with DNA nucleotides
(particularly 8-hydroxy-2' deoxyguanosine
) of speffns leading to base
276

Basrah
journal
of veterinary
research
, vol. 1 1
' 4,2012
Proceedings
of the 3'd international
Scientific Conference,
College
of Veterinary
Medicine,
University
of Basrah-Iraq.
modifications(oxidative
DNA damage)
and DNA fragmentation
resulting
in altering
the
sperm
quality and its impact on reproductive
outcomes
.After 12 weeks of mating
period of female and male rats treated
previously for 12 weeks with 150 mg / kg
B.W.sustanon@250
injected
weekly to study
the reproductive
efficiency above
dose
of
sustanon@25g
on rats . It seems
that the sustanon@250
defiantly had
negative
effects
on
fertility percentage
of treated
males and / or females
.It is known ( 2) that the FSH
receptor -hormone formation at the Sertoli cell results in the initiation of
spermatogenesis
process
, thgrefore
, the reduction
in FSH level in treated
male serum
may
reduce
the level of fertility , thus the reason
for treated
rats infertility could
be due
to impaired spermatogenesis
caused by damage
of leydig cells as a result of
testosterone
injection and therefore very low sperm count in treated rats which
supports
by seminal
analysis.
The production of abnormal
quantities of ROS
is thought
to be involved in male infertility ( 28). Sperm
exposed
to superoxide
anions are
apparently
dysfunctional
by lipid peroxidation and altered
membrane
function , along
with impaired
metabolism
, morphology and
motility (29) . A according
to (25;26; and
27) the ROS, in addition to reacting
with the polyunsaturated
fatty acids,
might also
react with DNA nucleotides
(particularly 8-hydroxy-2' deoxyguanosine)
of sperms
leading to base modifications(oxidative
DNA damage) and DNA fragmentation
resulting
in alter the spenn quality and its impact
on reproductive
outcomes
.Sperm
DNA damage is thought to be detrimental
to all reproductive tests outcomes,
particularly to pregnancy
rates(26
;27; 30 and 31). Many hypotheses
explained
the
origin of sperm DNA damage such as defective chromatin packaging during
spermiogenesis
(32; 25; and
33) .The
infertility of treated
male in the present
study
was
in match
with some studies
(16 ; l7and, 34).The
treated
female
rats
infertility may be
attributed
to the effect
ofsustanon@250
on ovary
secretion
(estradiol
and
progesterone
)
and
gonadohopein hormones(
LH and FSH ) due
to damage
of ovarian
tissue in which
seems
as a sort atrophy
, therefoi.e
granulosa
cells which responsible
for expression
of
the androgen
receptors to promote follicle development
and oocyte
maturation
would
be affected
. Thus androgens
could involve in the regulation of granulosa cell death
and hence
follicular atresia
(35). The oxidative stress
( induced
by sustarion@250
)
affects
natural fertility (36). Other studies.have
suggested
that ROS are involved in
various causafive factors of female infertility as peritoneal factor otubal factor,
277

Basrahjournal
ofveterinary
research,
vol.
11,
4,20L2
Proceedings
of
the 3'd
international
Scientific
Conference,
College
of Veterinary
Medicine,
University
of Basrah-Iraq.
endometeriosis
and unexplained
infertility (37 and
38 ). According to Polak
et al. (39)
the antioxidants
concentrations
in women with unexplained
infertility is less than
those
in fertile women , the above authors found that increased
ROS -induced lipid
peroxidation
damage
resulting
in infertility. The present study
revealed
that the abusing
of Sustanon@
250 cause
serious
physiological changes
in the most body structures
,particularly
the reproductive
organs
;thus
affected
the fertility .
.013..1+ll
l$ls &Ul
(r! 4J*,ulll SsLiSll
eJe (sustanon@250)
flrAi*l Jiiil
UlgJloJr u--.. t'.J+'ll*-U. L"H
6l!16 i-r-Jl, "
J,-+ll
ar-ots.'
;'Lll aJS
,jlJ'll, 6r-Jlr i)-illi*1. , qt-tl+,ll
c+ll4$
i,a)till
(sustanon@250)
pl'rr..,t
!-i J$L 4-lJJ iJ"-.Jl A*b / g-l'slr ,.+tll
iJS ; qttslt :*t-p!l c,p6l
,ili_pJl JJSrJ
dtul ,.+
+J-tstt ieli$l ,rJo
, 6.\. a+-,'l
J! ,",^.",
,Jl liJidr.ll 61i.,;;Jl
dr .Fl fY J JSi r.f tl)ij-l i cJ:!t ti+ll d
!r*-i 6.:r,l-9
6-.;r.
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