EFFECT OF SUSTANON@ 250 ON REPRODUCTM EFFICIENCY IN

Transcription

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Basrahjournalof veterinaryresearch' v{ .]t'
collegeof veterinaryMedicine'
proceedings
of the 3d internationalScientificconference,
UniversitYof Basrah-Iraq'
250 ON REPRODUCTM
EFFECT OF SUSTANON@
EFFICIENCY
IN MALE AND FEMALE RATS
.NehayaM. Al-AubodYt
MohammadA' Al-Diwan'z
Basrah'Iraq
Collegeof Medicine,Universityof Basrah'
CollegeofVeterinaryMedicine,UniversityofBasrah,Basrah,Iraq
t
the effect of ( Sustanon 250;
investigate
to
conducted
was
study
present
The
two male and thirty two female rats were
abusingon reproductiveefficiency .Thirty
used'Theyweredividedintofourgroups(eachgroupconsistedofSmalesand8
females).Thefirstgroupservedascontrolgroupinwhichtheratsdosedby
weekly for !2 weeks ' the rest 3 treated
intramuscular injection of 10 p I olive oil
groups(Gl.G2andG3)inwhichtheratsdosedbyintramuscularinjectionof50.l00
t
weekly for 12 weeks'thenall rats
and 150mg Sustanon 250 / kg B.W. respectively
and LH levels in addition to seminal
were terminatedto determinetestosterone,FSH
level was significantly (pS 0'05)
analysis . The results reveled that testosterone
0.05) in all
significantlydecreased(ps
elevatedin Gr and G2 . The LH and FSH were
t
Sustanon 250 injection revealeda
animals .The seminal analysisin responseto
viable spenn percentageand
significant . reduction in the sperm concentration,
morphologicallynormalspermpercentage'Anothergroupof16maleand32female
efficiency , from which 4 male and 8
rats were used ,to determinethe reproductive
femaleweredosedbyintramuscularinjectionwithl0ploliveoilweeklyfor12
rveeks,theyareconsideredascontrolanimalsTherestratsweredosedby
@250 / kg B'W' weekly for 12 weeks'
intramuscularinjectionof.150 mg Sustanon
Thentheyweredividedintofourgroups:groupl(4nontreatedmaleswithSnon
8 treatedfemales)' group 3 (4 treated
treatedfemales) , group 2 (4 treatedmaleswith
4 (4 non treatedmales with 8 treated
males with 8 non treated females)and group
12 weeks, the reproductiveparameters
females) . After they were allowedto matefor
as 100% in Gl 'whereasthe
percentage
were investigatedwhich revealedthe fertility
269
Basrahjournalofveterinary research,vol. 11, 4,20L2
Proceedingsof the 3'd international Scientific Conference,College of Veterinary Medicine,
Universiry of Basrah-Iraq.
femalerats in G2 , G3 and , G4 showedno pregnancyat all which indicatedcomplete
infertility.
INTRODUCTION
250 is one of the androgenic- anabolicsteroidsthat usually abused
Sustanon@
by athletesand young femalesand malesto have a muscularbody and increasetheir
sportive performance(1). The presentstudy aimed to investigatethe adverseeffects
induced by Sustanonabuse on reproductive efficiency , that determinedthrough
including testosterone, LH and FSH ) , semen
measuresomereproductive..hormonss(
analysis,fertility percentageand other reproductiveparameters.Testosteroneis derived
from cholesterol ( 2) produced and secretedby the interstitial leydig cells ,which
constituteabout20 o/oof the massof adult testis(3). It hasa rangeof biological actions.
the classicaleffectsof testosteroneare induction and maintenanceof secondarysexual
characteristicand preservationof libido ,senseof well being ,lean mass ,bone
density(4)and stimulationof protein synthesis(5). Testosteroneis found in the body in
three forms as free , Sex Hormone - Binding Globulin (SIIBG) and thesethat are
impotently boundedto albumin or cortisol binding globulin (6).Circulatingbound and
free testosteroneare collectively referredto as total testosterone(7).LH and FSH are
glycoprotein secretedfrom the anterior lobe of pituitary gland as a responseto the
releaseof gonadotropicreleasinghormone(GnRtI) from the hypothalamus(8).(FSH)
has been implicated in Leydig cell function through its action on Sertoli cells (9) to
(11). LH
producethe spermcells (10).It is essentialfor initiation of spermatogenesis
had been called interstitial cell - stimulatinghormone (2) , it stimulatesLeydig cell
productionof testosterone
with FSH (12).
, It actssynergistically
MATERIALS AND METHODS
Testosterone ,foilicle
stimulating hormone (FSH)
and
lutenizing
hormones(LH)were assayedby using enzyme- linked immunosorbentassay(ELISA)
kits specific for rat hormones,manufacturedby USCN Life ScinceInc. in China. The
spermsconcentrationand spenn viability were countedaccordingto method of Evan
and Maxwell (13) by using Neubauerhemocytometerchamberwhich use for RBC and
were countedin the sameslide that was used
WBC count . The abnormalspermatozoa
270
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Medicine'
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of the3'dinternational
Proceedings
of Basrah-Iraq'
Universiry
using account200 spermsunder the light microscope
to measurethe deadsperrnatozoa
using 100X power accordingto Sakamotoand Hashimoto ( 14)'
high
The reproductiveeffects causedby weekly intramuscularinjection of
@
( 16 male
doseof sustanon 250 (150 mg / kg body weight ) for male and femalerats
rats and 32 femalerats) divided into 2 groups
ptl
1- Control group ( 8 malesand 16 femalesrats ) injectedweekly with only 10
olive oil
kg body
2- Treatedgroup (8 males and 16 femalesrats ) injected with 150 mg /
t
weight sustanon 250
4
At the end of treatmentperiod (12 weeks ) the animals fuither divided into
groupsas follows : Group 1 consistsof 4 non treatedmaleswith 8 non treatedfemales'
r
e Group2 consistsof 4 treatedmaleswith 8 treatedfemales.
r
r
Group 3 consistsof 4 treatedmaleswith 8 non treatedfemales.
Group 4 consistsof 4 non treatedmaleswith 8 treatedfemales.
The animalsleft for 12 weeks for mating before measuringthe parametersto
percent,
determinethe reproductiveeffrciency (numberof pregnantfemales ' fertility
number of
number of life birth , weight of life birth , abnormalitiesof life birth ,
corporalutea and numberof implantingsites
groups
The results were expressedas mean * SD. The comparisonsbetween
sPSS
were performed with analysis of variance (ANOVA) by using computerized
lest
program(StatisticalProgramfor Social Sciences).P<0.05was consideredto be the
to test
limit of significance.Least significant difference test (LSD) was calculated
differencebetweenmeans(groups)for (ANOVA) SPSS(1998)'
RESULTS
Injection on SomeHormones
Effect of Sustanon@250
In Male Rats.
/kg
50 and 100mg
The testosteronelevel in male rats treatedwith (sustanon@250;
B.W were significantly increased(p S O.OS)comparedwith testosteronelevel of
seem
control males (table 1) whereasthe group treatedwith 150 mg /kg B'W doesnot
27L
Basrahjournalofveterinary research,vol. 11, 4,20L2
Proceedingsof the 3'd international Scientific Conference,Collegeof Veterinary Medicine,
University of Basrah-Iraq.
injection comparedwith control group and other
to be affected by sustanon@250
treatedgroups.
significantly(p S 0.05)inall injectedmale
The LH and FSH levelsdecreased
rats with (sustanon@250
) 50,100and 150 mg /kg B.W comparedwith control male
rats. No significant difference (p > 0.05) in LH and FSH level among treatedmale
groupswere recorded.
Table ( I ) Effect of Sustanon@2s0
Injection on SomeIlormones in Male Rats
means* SD
N
Control
(0.lmloliveoil)
Groupl
(50mg/kg
B.w.)
Testosterone
LH
FSH
nglml
mIU/ml
mIU/ml
4.453
2.9t7
2.626
b
a
a
it.049
10.333
J.0.492
6.375
1.803
t.737
a
b
b
*0.751
*0.753
+0.t23
1 aaa
I.JJJ
Group2
(100mglkgB.W.)
Group3
(150mg/kgB.W.)I
LSD
6.t39
b
'+0.421
a
t.484
b
+0.557
*0.t23
4.471
0.72r
1.143
b
b
b
*1 .454
+0.087
+0.237
t.6 04
1 . 11 3
0.603
N=8
Different lettersin the samecolumnrepresentsignificantdifferenceat (pS0.05).
In FemaleRats
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UniversitY
in female rats is shown to cause significant
The injection of sustanon@250
groupscomparedwith control
increase ( p s 0.05 ) in testostefonelevel of all treated
three treated
group (table 2) . There were no significantdifferences observedamong
groups
) showed a
level in female rats injected with (sustanon@250
B'W)
decrease(p 5 0.05) in treated groups (50,100 and 150 mg/kg
The LH
significant
the LH
) could decreased
comparedwith control females. It seemsthat (sustanon@250
dose 'The FSH level
level significantly (p S 0.05) according to increase the
50 100 and 150
significantlydecreased(p <'0.05) by iirjectionof (sustanon@z5o; ,
(p z 0'05)
mg/kg B.w comparedwith control females.while no significantdifferences
were observedamongall treatedgroups.
in Female Rats
Table ( 2 )Effect of Sustanon*250Injection on SomeHormones
meanst SI)
Controlgroup
(0.lmloliveoil)
Groupl
(50mg/kg
B.W.)
Group2
B.W.)
(100mg/kg
Group3
B.W.)
(150mgikg
2.694
6949
b
a
+0.699
+t.044
6.270
5.469
a
b
*0.782
r0.460
6.701
4.s93
a
b
+0.245
+0.470
6.230
4.232
a
b
+0.510
+0.806
N=8
significantdifferenceat (pS0.05)
Differentlettersin the samecolumnrepresent
273
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Basrahjournal of veterinary research' vol' 11' 4'2012
proceedingsof the 3d international Scientific Conference,Collegeof Veterinary Medicine,
UniversicY of Basrah-Iraq.
SeminalAnalYses
,
injection for 12 weekswith 3 different doses( 50
The effect of sustanon@25g
that the
100 and 150 mg / kg B.w.) on seminalanalysesas shown in table 3 revealed
percentagein
spennconcentration,viabilitypefcentageand normal spennmorphology
B.W were
male rats injected with (sustanont2sg) 50 ,100 and 150 mg /kg
. while the
significantlydecreased( p s 0.0s ) comparedwith that of control male rats
all
percentageof abnormal spermmorphology increasedsignificantly (p S 0'05 ) in
treatedgroupscomparedwith control group ( table 3)'
Injection on SeminalAnalyses
Table ( 3 )The Bff""t of Sustanon@250
means* SD
Sperm
viable
Normal
Abnormal
%
%
Yo
2902
7r,40
82.0
18.0
a
a
a
b
+ 0.653
L0.027
+0.034
+0.034
1.402
6t.24
47.29
52.71
b
b
b
a
*0.341
+0.092
+0.027
+0.027
1 .0 5 5
55.25
45.41
s4.59
b
b
b
a
+0.064
+0.102
+0.031
+0.031
1 .0 5 5
52.52
42.68
47.22
b
b
b
a
+0.221
+0.077
+0.040
+0.040
1.490
t9.21
09.56
7.r7
concentration
Xl06/mm3
Control group
(0.lmlolive oil)
Groupl
B.W )
(50mg/kg
Group2
B.W)
(100mg/kg
Group3
B.W)
(150mg/kg
LSD
N:8
Different lettersin the samecolumnrepresentsignificantdifferenceat (p50.05)
274
Basrahjournalof veterinaryresearch, vol' 11' 4'2012
Collegeof VeterinaryMedicine'
of the 3'dinternationalScientificConference,
Proceedings
Universityof Basrah-Iraq.
the
The sperm was consideredmorphologically abnormal if it had one of
detached
following features: rudimentarytail , absenceof tail, round head and / or
head.
Reproductive
fficiencY
for
After l2weeks of mating period of femaleand male rats treatedpreviously
injectedweekly as shown in table (4)
12 weekswith 150 mg / kg B.W.Sustanon@250
percent to give
eight pregnancycaseswere recorded. This indicated T00%fertility
numberof
5.25 +.0.75 litters weighted5.l * 0.35 and after killing the mothersthe
*0'20'
implantingsiteswere 5.1 + 0.20andthe numberof corporaluteawere 6
of
Table (a)The Reproductive Efficiency Results of Mating Rats After L2 weeks
means* SD
intramuscular injection of 150mg / kg B.W. of Sustanon@250
ut)
4)
0
{)
boc)
E?
Hl:
.tl
z
EE
,i
z
vi
ti
q}
s>,
4ntm+8nJf
G2
4tm+8tf
G3
4tm+8ntf
G4
8
8
9?n
{)
E7
tse
t
q)
z
r00%
ij
:40
z.=
1..
e
6!
(J
q)
o=
.bn
FV
5.1
6
*
5.1
+
+
+
0,75
0.35
0.20
0.20
{?{
G1
0
tr
8
0
0%
0
0
0
0
I
0
0%
0
0
0
0
8
0
0%
0
0
0
0
4ntm+8tf
tm :treatedmale
ntm: non treatedmale
tf :treatedfemale
ntf : non treatedfemale
27s
Basrahjournalofveterinary research,vol. 11, 4,2012
Proceedingsof the 3'd international Scientific Conference,College of Veterinary Medicine,
The other 3 groups, which includeeither treatedmale with non treatedfemale
or treatedfemalewith non treatedmalesor treatedboth malesand females showedno
"
pregnancyin all females indicatingcompleteinfertility.
DISCUSSION
The spermconcentrationin male rats of all treatedgroupswere significantly
decreasedcomparedwith the sperm concentrationof control male rats(table3). This
finding agreeswith previous studieswhich found that the abuseof supraphysiologieal
dosesof anabolicandrogenicfteroids(AAS ) resultedin transientseveroligospermiain
malesbecausethe AAS inducedreversiblehypogonadotrophic
hypogonadisim(15,
16;and 17).Thelow spermcountof treatedratscouldbe dueto reductionin FSH level
induced by testosteroneinjection , becausethe FSH is essentialto .initiate the
spermatogenesisin Sertoli cells (2) and for nourishment of sperms( 12 ). The
percentagesof viable spenn and that of morphologically normal spenns were
significantlydecreasedcomparedwith control group,while the percentageof deadand
morphologicallyabnormalspermswere significantly increasedcomparedwith control
group(table3) .The findings of Torres{alleja et al. ,2001 (18) that abuseof AAS
alone not only reduced the spenn count but also impairs the percentage of
morphologicallynormal semen.The reason for the decreasingpercentageof viable
and morphologically normal spenns and inueasing the percentageof dead and
morphologicallyabnormalsperms,was due to the testosteroneinjection which led to
hormonal disturbanceand increasedROS formation as supportedby increasedMDA,
and becausethe mammalianspermatozoaaremore vulnerableto lipid peroxidationdue
to their cell membranerich with lipids especiallypoly unsaturatedfatty acids(PUFA)
are the main substratesfor lipid peroxidation, that may provoke severefunctional
disorderof sperm(19) .MDA levelswerenegativelycorrelatedto spermconcentration
andmotility which was consistentwith previousreports(20and2l) .lnother studies,it
was reported that peroxidation reaction affects membranestructure and fluidity and
causesdamageto axonemal proteins leading to a permanentimpairment in sperm
motility ( 22; 23 ; and 24).A accordingto (25; 26; and 27) the RoS, in addition to
reactingwith the polyunsaturatedfatfy acids,might also react with DNA nucleotides
(particularly 8-hydroxy-2' deoxyguanosine)
276
of
speffns leading to
base
Basrahjournalof veterinaryresearch
, vol. 11' 4,2012
Collegeof VeterinaryMedicine,
Proceedings
Universityof Basrah-Iraq.
modifications(oxidativeDNA damage)and DNA fragmentationresultingin alteringthe
sperm quality and its impact on reproductiveoutcomes.After 12 weeks of mating
period of female and male rats treatedpreviously for 12 weeks with 150 mg / kg
injectedweekly to study the reproductiveefficiency abovedoseof
B.W.sustanon@250
defiantly had negativeeffectson
on rats . It seemsthat the sustanon@250
sustanon@25g
fertility percentageof treatedmales and / or females .It is known ( 2) that the FSH
receptor -hormone formation at the Sertoli cell results in the initiation of
process, thgrefore, the reductionin FSH level in treatedmale serum
spermatogenesis
may reducethe level of fertility , thus the reasonfor treatedrats infertility could be due
causedby damage of leydig cells as a result of
to impaired spermatogenesis
testosteroneinjection and therefore very low sperm count in treated rats which
supportsby seminalanalysis.The productionof abnormalquantitiesof ROS is thought
to be involved in male infertility
( 28). Sperm exposedto superoxideanions are
apparentlydysfunctionalby lipid peroxidationand alteredmembranefunction , along
with impairedmetabolism, morphologyand motility (29) . A accordingto (25;26; and
27) the ROS, in addition to reactingwith the polyunsaturatedfatty acids,might also
react with DNA nucleotides (particularly 8-hydroxy-2' deoxyguanosine)of sperms
leading to
base modifications(oxidativeDNA damage) and DNA fragmentation
resulting in alter the spenn quality and its impact on reproductiveoutcomes.Sperm
DNA damage is thought to be detrimental to all reproductive tests outcomes,
particularly to pregnancyrates(26 ;27; 30 and 31). Many hypothesesexplainedthe
origin of sperm DNA damage such as defective chromatin packaging during
(32; 25; and33) .The infertility of treatedmale in the presentstudywas
spermiogenesis
in matchwith somestudies(16 ; l7and, 34).Thetreatedfemale rats infertility may be
on ovary secretion(estradiolandprogesterone
)
attributedto the effect ofsustanon@250
and gonadohopeinhormones(LH and FSH ) due to damageof ovariantissuein which
seemsas a sort atrophy , therefoi.egranulosacells which responsiblefor expressionof
the androgenreceptorsto promotefollicle developmentand oocyte maturationwould
be affected . Thus androgenscould involve in the regulation of granulosacell death
and hence follicular atresia (35). The oxidative stress( induced by sustarion@250
)
affects natural fertility (36). Other studies.havesuggestedthat ROS are involved in
various causafive factors of female infertility as peritoneal factor otubal factor,
277
vol.11,4,20L2
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ofveterinary
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University
endometeriosisand unexplainedinfertility (37 and38 ). According to Polak et al. (39)
the antioxidantsconcentrationsin womenwith unexplainedinfertility is lessthan those
-induced lipid
in fertile women , the above authors found that increasedROS
peroxidationdamageresultingin infertility. The presentstudy revealedthat the abusing
250 cause seriousphysiological changesin the most body structures
of Sustanon@
,particularlythe reproductiveorgans;thusaffectedthe fertility .
.013..1+ll &Ul
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EFFECT OF SUSTANON@ 250 ON REPRODUCTM EFFICIENCY IN

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