Dietary rosemary oil alleviates heat stress-induced structural and functional damage through lipid peroxidation in the testes of growing Japanese quail

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ContentslistsavailableatScienceDirect

Animal

Reproduction

Science

j o ur na l h o me p a g e:ww w . e l s e v i e r . c o m / l o c a t e / a n i re p r o s c i

Dietary

rosemary

oil

alleviates

heat

stress-induced

structural

and

functional

damage

through

lipid

peroxidation

in

the

testes

of

growing

Japanese

quail

Gaffari

Türk

a,∗

_,

_Ali

_O.

_C¸

_eribas¸

_ı

b

_,

_Ülkü

_G.

_S¸

_ims¸

_ek

c

_,

_Songül

_C¸

_eribas¸

_ı

b

_,

Mehmet

Güvenc¸

d

_,

_S¸

_eyma

_Özer

_Kaya

a

_,

_Mehmet

_C¸

_iftc¸

_i

e

_,

_Mustafa

_Sönmez

a

_,

Abdurrauf

Yüce

d

_,

_Ali

_Bayrakdar

f

_,

_Mine

_Yaman

f

_,

_Fadime

_Tonbak

g

a_Department_of_Reproduction_and_Artiﬁcial_{Insemination,}_Faculty_of_Veterinary_Medicine,_Fırat_University,₂₃₁₁₉_Elazı˘g_Turkey

b_Department_of_Pathology,_Faculty_of_Veterinary_Medicine,_Fırat_University,₂₃₁₁₉_Elazı˘g_Turkey

c_Department_of_Animal_Science,_Faculty_of_Veterinary_Medicine,_Fırat_University,₂₃₁₁₉_Elazı˘g_Turkey

d_Department_of_Physiology,_Faculty_of_Veterinary_Medicine,_Fırat_University,₂₃₁₁₉_Elazı˘g_Turkey

e_Department_of_Animal_Nutrition_and_Nutritional_Diseases,_Faculty_of_Veterinary_Medicine,_Fırat_University,₂₃₁₁₉_Elazı˘g_Turkey

f_Department_of_Histology_and_Embryology,_Faculty_of_Veterinary_Medicine,_Fırat_University,₂₃₁₁₉_Elazı˘g_Turkey

g_Veterinary_Control_Institute,_Elazı˘g_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6August2015

Receivedinrevisedform

19November2015

Accepted25November2015

Availableonline2December2015

Keywords: Rosemaryoil Heatstress Spermatogeniccell Testis Quail

a

b

s

t

r

a

c

t

Supplementationofnaturalantioxidantstodietsofmalepoultryhasbeenreportedtobe effectiveinreducingorcompletelyeliminatingheatstress(HS)-inducedreproductive fail-ures.Inthisstudy,theaimistoinvestigatewhetherrosemaryoil(RO)hasaprotective effectonHS-induceddamageinspermatozoaproduction,testicularhistologicstructures, apoptosis,andandrogenicreceptor(AR)throughlipidperoxidationmechanismsingrowing Japanesequail.Malechicks(n=90)at15-daysofagewereassignedtotwogroups.Theﬁrst group(n=45)waskeptinathermo-neutral(TN)roomat22◦_C_for₂₄_h/d._The_second_group

(n=45)waskeptinaroomwithagreaterambienttemperatureof34◦_C_for₈_h/d_(from

9:00AMto5:00PM)and22◦Cfor16h/d.Animalsineachofthesetwogroupswere ran-domlyassignedtothreesubgroups(ROgroups:0,125,250ppm),consistingof15chicks(six treatmentgroupsin2×3factorialdesign).Eachofsubgroupswasreplicatedthreetimes witheachreplicateincludingfivechicks.TheHStreatmentsignificantlyreducedthe tes-ticularspermatogeniccellcounts,amountoftesticularBcl-2(anti-apoptoticmarker)and amountofAR.Inaddition,itsignificantlyincreasedtesticularlipidperoxidation,Bax (apop-toticmarker)immunopositivestaining,andtheBax/Bcl-2ratioinconjunctionwithsome histopathologicdamage.DietarysupplementationofROtodietsofquailwheretheHS treat-mentwasimposedalleviatedHS-inducedalmostallnegativechangessuchasincreased testicularlipidperoxidation,decreasednumbersofspermatogeniccells,anddecreased amountsofBcl-2andAR,increasedratioofBax/Bcl-2andsometesticularhistopathologic lesion.Inconclusion,dietarysupplementationofROforgrowingmaleJapanesequailreared inHSenvironmentalconditionsalleviatestheHS-inducedstructuralandfunctionaldamage byprovidingadecreaseinlipidperoxidation.

∗ Correspondingauthor.Tel.:+904242370000.

E-mailaddresses:gturk@ﬁrat.edu.tr,gaffariturk@hotmail.com(G.Türk).

http://dx.doi.org/10.1016/j.anireprosci.2015.11.021

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1. Introduction

Environmentalfactorsoftenaffectphysiological

func-tionsofeachorganinthebodyinalmostallanimalspecies.

However, avian species seem to be particularly

sensi-tiveto environmental challenges, especially heat stress

(HS)becauseoftheincreasedproductionofbodyheatby

modernpoultrygenotypesdue tothegreater metabolic

activityassociatedwiththisincreasedproductivity(Deeb

and Cahaner, 2002). Exposure to HS has a deleterious

effectonfertilityandisconsideredtobeoneofthe

sig-niﬁcantriskfactorscausing infertilityin males.In most

homoeothermicbirdsand mammals,includinghumans,

testicularfunctionisinﬂuencedbyambienttemperature.

Temperaturesoutsideoftheanimal’sthermo-neutral(TN)

zoneareoftennotoptimalfortesticularfunctionandcan

potentially disrupt spermatogenesis (Durairajanayagam

et al., 2014). Reductions have been reported for testis

weight(McDanieletal.,2004),semenvolume,numbersof

normal-shapedspermatozoa(Joshietal.,1980),

spermato-zoacount,spermatozoamotility,andthereisanincreasein

numberofdeadspermatozoa(McDanieletal.,2004;Ebeid,

2012)aswellastesticulardegenerativedisorders(Terim

Kapakınetal.,2013;Türketal.,2015)indifferentpoultry

speciesinresponsetoHS.

The increased intra-testicular temperature resulting

fromincreasedbodytemperature(McDanieletal.,2004),

and in particular greater production of reactive

oxy-gen species (ROS) leading to lipid peroxidation due to

theincreasedmetabolicactivityunderstressconditions

(Ebeid, 2012), have been considered to be responsible

mechanismsfortheHS-inducedreproductivefailuresin

poultry. The plasma membrane of avian spermatozoon

contains abundant polyunsaturated fatty acids (PUFAs).

While PUFAs provide the ﬂuidity that is necessary for

ﬂagellar movement and fusion-related events, it also

makes the spermatozoa susceptible to lipid

peroxida-tion.Spermatozoa needadequateantioxidant capacities

because lipid peroxidation can lead male

reproduc-tivedysfunction. Therefore, avian testes need a precise

oxidant/antioxidantbalanceforregularspermatozoa

pro-ductionandsubsequentlysemenwithhighquality(Surai

etal.,2001).

Supplementation of natural antioxidants to diets of

male poultry is effective in reducing or completely

eliminating HS-induced reproductive failures (Lara and

Rostagno,2013).Variousantioxidantssuchascinnamon

bark oil (Türk et al., 2015), betaine, vitamin C, folic

acid(Ezzatetal.,2011),selenium,andvitaminE(Ebeid,

2012)havebeenaddedtothedietstopreventHS-related

reproductivedisturbancesinavianspecies.Rosemary

(Ros-marinusofﬁcinalis)isa commonhouseholdplant,which

belongstothefamilyofLamiaceaethatisgrowninmany

partsoftheworld.Rosemarycontainsactiveanti-oxidative

substancessuchasphenolicditerpenes,ﬂavonoids,

pheno-licacids(Hoetal.,2000),andvolatileoils(Begumetal.,

2013).Thevolatileoilsconsistofborneol,bornylacetate,

camphene, cineol, pinene, and camphor (Begum et al.,

2013).Rosemaryoil(RO)hasvariousbiologicalproperties

including great antioxidant and free radical

scaveng-ing activities(Adorjanand Buchbauer, 2010). However,

thereisinconsistentinformationregardingtheeffectsof

rosemaryonthemalereproductivesystem,becausethere

are studies indicating that rosemary and its different

extracts have been harmful(Nusieret al., 2007; El-Din

et al.,2012;Heidari-Valaetal.,2013)whileother

stud-ieshave foundtherewerebeneﬁcial (Lunoet al.,2014;

Motlaghetal.,2014;Uyeturketal.,2014)effectson

struc-tureandfunctionsofspermatozoa.Onestudy(Superchi

et al., 2005) was conducted that relates tothe

protec-tion of rosemary extract against spermatozoa damage

caused by HS in boars, but to the best of our

knowl-edge there is no previousresearch regarding theeffect

of RO on HS-inducedreproductive disturbance in male

quail. The present study was, therefore, conducted to

investigatewhetherROhasa preventiveor aggravating

effect on HS-induced disturbance impacting

reproduc-tiveefﬁciencyofmalequailbyexaminingthechangesin

spermatogeniccellcounts,testicularoxidant-antioxidant

markers, testicular histologic structures, and quantity

of testicular apoptotic cells and androgenic receptors

(AR).

2. Materialsandmethods

2.1. Rosemaryoilandchemicals

Rosemary oil was purchased from a local store

(Agromiks Food Additive Co., ˙Izmir, Turkey). The

com-pounds and percentages of volatilecomponents within

the RO have been reported to be 1,8 cineole (39.31%),

camphor (14.69%),␣-pinene(13.85%),␤-pinene (9.87%),

camphene(6.17%), limonene (3.17%), P-cymene (2.58%),

borneol (2.33%), ␣-terpineol (2.28%), myrcene (2.02%),

bornylacetate(1.46%),andothers(2.27%)bymanufacturer.

ROwaskeptat4◦Cuntilbeingused.Theotherchemicals

werepurchasedfromSigma-AldrichChemicalCo.(StLouis,

MO,USA).

2.2. Experimentalprotocolanddietaryregimen

TheAnimalExperimentationsLocalEthicsCommittee

ofFırat University(Elazı˘g,Turkey) approvedthe

experi-mentalprotocolofthepresentstudy.Atotalof90male

Japanese quail chicks (Coturnix coturnix japonica) at

5-daysofagewerepurchasedfromacommercialcompany

(Deva-Yum Co., Elazı˘g, Turkey). After a 10-day

adapta-tionperiodtoexperimentalconditionsofthePoultryUnit

of Fırat University, chickswereplaced in wire cages in

temperature-controlledrooms,andthestudywasinitiated.

Thechickswererandomlydividedintotwogroups.The

ﬁrstgroup(n=45)werehousedinaTNconditionat22◦C

for24h/d.Thesecondgroup(n=45)wereplacedinaroom

withhighambienttemperature(HS)at34◦Cfor8h/d(from

9:00AMto5:00PM)andat22◦Cfor16h/d.Thechicksin

thetwogroupswerethenrandomlyassignedtothree

sub-groups(ROgroups:0,125,250ppm)consistingof15chicks

(sixtreatmentgroupsin2_×3factorialdesign).Thestudy

witheachofthesubgroupswasreplicatedthreetimeswith

eachreplicatebeingwithﬁvechicks.Therelativehumidity

ofboth TNandHSroomswas60–65%.Atboth

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Table1

Ingredients,chemicalandfattyacidcompositionsofstandarddiet.

Feedingredients %Fed Nutritionalcomposition %

Maize 29.03 Drymatter 88.25

Wheat 25.00 Crudeprotein 23.87

Soybeanmeal(44CP) 34.29 Crudeﬁber 2.55

CornGluten 4.10 Etherextract 4.75

Vegetableoil 2.92 Ash 5.45

Di-calciumphosphate 2.02 Calciuma _1.00

Groundlimestone 0.87 Availablephosphorusa _0.79

NaHCO3 0.12 Methioninea 0.40

Salt 0.28 Lysinea _1.18

DL-Methionine 0.02 ME,kcal/kga ₂₈₉₇

Vitaminmixb _0.25 _Total_saturated_fatty_acid₍

_SFA)c _13.16

Mineralmixd _0.10 _Total_{monounsaturated}_fatty_acid₍

_MUFA)e _22.00

Additivef _1.00 _Total_{polyunsaturated}_fatty_acid₍

_PUFA)g _64.84

a_Calculated.

b_Vitamin_premix_supplied_per_2.5_kg;_vitamin_A_12.000.000_IU;_vitamin_D

32.000.000IU;vitaminE35.000mg;vitaminK34.000mg;vitaminB13.000mg;

vitaminB27.000mg;niacin20.000mg;calciumD-pantotenat10.000mg;vitaminB65.000mg;vitaminB1215mg;folicacid1.000mg;D-biotin45mg;

vitaminC50.000mg;cholinechloride125.000mg;canthaxanthin2.500mg;apocarotenoicacidester500mg.

c

_SFA_consist_of_palmitic_(C16:0,_11.73%)_and_stearic_(C18:0,_1.43%)_acids.

d_Mineral_premix_supplied_per_kg;_Mn_80.000_mg;_Fe_60.000_mg;_Zn_60.000_mg;_Cu_5.000_mg;_Co₂₀₀_mg;_I_1.000_mg;_Se₁₅₀_mg.

e

_MUFA_consist_of_palmitoleic_(C16:1_␻7,_0.57%),_oleic_(C18:1_␻9,_20.88%)_and_eicosanoic_(C20:1_␻9,_0.55%)_acids.

f_Group_rosemary_oil₀₍₁₀₀₀_g_zeolite);_Group_rosemary_oil₁₂₅_(12.5_g_rosemary_oil₊_987.5_g_zeolite);_Group_rosemary_oil₂₅₀₍₂₅_g_rosemary_oil₊₉₇₅_g

zeolite).

g

_PUFA_consist_of_linoleic_(C18:2_␻6,_56.90%),_linolenic_(C18:3_␻3,_6.26%),_{eicosadienoic}_(C20:2_␻6,_0.22%),_{eicosapentaenoic}_(C20:5_␻3,_0.50%),

docosadienoic(C22:2,0.38%)anddocosapentaenoic(C22:5,0.58%)acids.

basaldietsupplementedwith125or250ppmROuntilthey

were43-daysofage(28daysexperimentalperiod).TheRO

wasmixedinacarrier(zeolite),whichwasaddedtothe

basaldiet attherateof1kgper1000kg.Dietandfresh

waterwereprovidedadlibitum.Lightwasprovided

con-tinuously(24h)throughouttheexperiment.Ingredients,

chemicalandfattyacidcompositionsofthebasaldietare

providedinTable1.

2.3. Collectionofsamples

In accordance withtheproposals ofthe Local Ethics

Committee,sixindividualsofthe15totalanimalsineach

group wererandomly selectedand decapitated (atotal

of36quail)attheendofthestudy(onday43)toavoid

slaughteringtheexcessivenumberofanimals.Testeswere

gentlyremovedfromthebodyandweighed.Mean

abso-lutetestisweightsofquailineachgroupwererecorded.

Inaddition,relativetestisweights[gonado–somatic-index

(GSI)=absoluteweightoftestes/ﬁnalbodyweight×100].

Lefttestissamplesofeachanimalwereusedforcounting

of testicular spermatogenic cells including

spermatogo-nia, spermatocytes,spermatids, and spermatozoa. Right

testissamplesweregentlydividedtotwoequalpartsby

scalpeland,onepieceoftissuewasﬁxedinBouin’ssolution

for histopathologicand immunohistochemical

examina-tions. The other piece of tissue was stored at −20◦_C

for biochemical analyses. For the biochemical analyses,

testes were taken from a −20◦_C _freezer _and

immedi-atelytransferredtothecoldglasstubes.Thetesteswere

diluted with a nine-fold volume of PBS (pH 7.4) and

minced in a glass and homogenized by a Teﬂon-glass

homogenizer for 3min in cold physiological saline on

ice.

2.4. Measurementoftesticularlipidperoxidation

concentrationandantioxidantactivity

Allanalyseswereperformedwiththeaidof a

spec-trophotometer(2R/Ultraviolet-Visible; Shimadzu,Tokyo,

Japan) by using the methods reported in the study of

Türketal.(2015).Amountoflipidperoxidationwas

mea-suredaccordingtotheconcentrationofthiobarbituricacid

reactivesubstancesandtheamountofmalondialdehyde

(MDA) produced wasused as an index of lipid

peroxi-dation.TheMDAconcentrationat532nmwasexpressed

asnmol/gprotein.Thereduced glutathione(rGSH)

con-centration at 412nm was expressedas nmol/g protein.

Glutathione–peroxidase(GSH-Px,EC1.11.1.9) activityat

340nm was expressed as IU/g protein. Catalase (CAT,

EC1.11.1.6) activitywas determined by measuring the

decompositionofhydrogenperoxide(H2O2)at240nmand

wasexpressedas k/gprotein, wherek istheﬁrst-order

rate constant. Protein concentration was also

deter-mined.

2.5. Countingoftesticularspermatogeniccells

TheTunicaalbugineaofthelefttestissampleswasgently

removed, and the testis wasminced and homogenized

in 10ml of a 0.9% NaCl solutioncontaining 0.5% Triton

X 100. A 100␮l sample of this homogenate was again

diluted(1:9)withthesamesolution;one drop(approx.

10␮l)wastakenandtransferredtoacountingchamber

ofImproved Neubauerinstrument (deep1/10mm,ﬁeld

sizeof 0.0025mm2_; _LABART,_Munich,_Germany), _where

itremainedfor5min.Thespermatogonia,spermatocytes,

spermatids,andspermatozoawerevisuallycountedusing

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Table2

Effectsofrosemaryoil(RO)ontestisweight,GSI,testicularspermatogeniccellcountsinJapanesequailrearedinathermo-neutral(TN)environmentand

underheatstress(HS).

Variables HS TN Maineffectsofenvironmentalconditionsandfeed

additiveonmeasuredvariables(Accordingtothe GeneralLinearModelprocedure)

RO(ppm) RO(ppm)

0 125 250 0 125 250 s.e. HS

signiﬁcance(P-value)

RO

signiﬁcance(P-value) Absolutetestisweight

[(g),right+left/2] 2.34 3.01 3.01 2.42 2.85 3.24 0.12 0.833 0.061 Gonado–Somatic-Index (GSI) 1.32 1.61 1.67 1.35 1.62 1.72 0.06 0.985 0.068 Spermatogonium count(million/per testis) 13.83b _19.66a _20.40a _28.66 _35.50 _37.50 _1.75 _0.0001*** _0.022* Spermatocyte count(million/per testis) 57.66b _87.00a _97.80a _111.66 _110.83 _122.50 _5.97 _0.005** _0.046* Spermatidcount (million/pertestis) 107.16b _242.20a _258.50a _274.00 _317.66 _345.50 _16.89 _0.0001*** _0.001** Sperm count(million/per testis) 11.50b _20.40a _22.00a _22.50 _23.33 _26.00 _0.85 _0.0001*** _0.0001***

Dataareexpressedasmeanandpooledstandarderrors(s.e.)

a,b_Mean_values_having_different_superscripts_in_the_same_row_within_the_HS_groups_are_different_from_each_other.

* _P_<_0.05.

** _P_<_0.01.

***_P_<_0.001.

spermatogonia,spermatocytes,spermatids,and

spermato-zoawerecalculated andexpressedas millionper testis

(Groteetal.,2008).

2.6. Histopathologicevaluation

TestistissueswereﬁxedinBouin’ssolutionfor48hand

weredehydratedtransferringthroughgraded

concentra-tionsofethanol,embeddedinparafﬁnwax,sectionedat

5␮mthicknessesandstainedwithMayer’shematoxylin

andeosin.Seminiferoustubules(ST,n=25)wererandomly

examinedpersectionand,thediametersandgerminalcell

layerthickness(GCLT;fromthebasalmembranetowards

thelumenofthetubule)weremeasuredusinganocular

micrometerinalightmicroscope,andthemeansizeofST

andGCLTwerecalculated.

2.7. Immunohistochemicalevaluation

Theavidin–biotin–peroxidasecomplexprocedurewas

usedforimmunohistochemicalstainingoftestistissues.

Forthisprocedure,thecommercialimmunoperoxidasekits

(UltravisionDetectionSystem,Antipolyvalent,HRP/DAB,

ThermoScientiﬁc,CatNo:TP-015-HD)wereusedand all

procedures were done according to the manufacturer’s

instructions.ThestainingintensitiesofBax,Bcl-2,andAR

forimmunopositiveproteinswereevaluatedunderalight

microscopeandrecordedasapercentagebasedon

previ-ouslydescribedprocedures(KandiCos¸kunandC¸obano˘glu,

2005).Score0:Negativestainedcells;Score1:<25%

pos-itivestainedcells;Score2:26–50%positivestainedcells;

Score3:51–75%positivestainedcells;andScore4:>75%

positivestainedcells.

2.8. Dataanalysis

The GeneralLinearModel (GLM)wasused to

deter-minethemaineffectofHSandfeedsupplementation(RO)

onallthevariablesmeasuredinthepresentstudy.Mean

differences weredeterminedwiththeone-wayanalysis

of variance(ANOVA)and posthocTukey-HSDtest. Data

are presentedasmean and pooledstandard error(s.e.).

ThevalueofP<0.05wasconsideredassigniﬁcant.Allthe

analyseswereconductedusingtheSPSSsoftwareprogram

(Version22.0;Chicago,IL,USA).

3. Results

3.1. ChangesintestisweightandGSI

Changes in the values of testis weight and GSI are

demonstrated in Table 2. Although the imposition of

HS resulted in a numerical decrease and

supplemen-tationof dietswithROresultedina numericalincrease

in the values of absolute testis weight and GSI, any

changesforthesevariableswerenotstatistically

signiﬁ-cant.

3.2. Changesintesticularlipidperoxidation

concentrationandantioxidantmarkers

ThemeanvaluesofMDA,by-productoflipid

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Table3

Effectsofrosemaryoil(RO)ontesticularmalondialdehyde(MDA),reducedglutathione(rGSH)concentrationsandglutathione-peroxidase(GSH-Px)and

catalase(CAT)activitiesintesticulartissueofJapanesequailrearedinathermo-neutral(TN)environmentandunderheatstress(HS).

RO(ppm) RO(ppm)

0 125 250 0 125 250 s.e. HS

RO

MDA(nmol/gprotein) 7.71a _4.90b _4.04b _4.00A _3.98A _2.80B _0.31 _0.001** _0.001**

rGSH(nmol/gprotein) 2.51b _9.86a _10.79a _3.13B _9.98A _10.92A _0.66 _0.679 _0.001**

GSH-Px(IU/gprotein) 1.32 1.38 2.46 1.78 1.79 3.59 0.25 0.356 0.192

CAT(k/gprotein) 18.64b _52.34a _64.79a _23.46 _42.20 _44.79 _4.56 _0.459 _0.041*

a,b_Mean_values_having_different_superscripts_in_the_same_row_within_the_HS_groups_are_different_from_each_other.

A,B_Mean_values_having_different_superscripts_in_the_same_row_within_the_TN_groups_are_different_from_each_other.

*_P_<_0.05.

**_P_<_0.01.

Although the imposition of HS resulted in an increase

(P<0.01) intheMDAconcentrationin comparisonwith

the quail reared in TN conditions, it didnot affect the

rGSH, GSH-Px, and CATantioxidant markers. However,

dietarysupplementationof125and250ppmROdecreased

(P<0.01) theHS-induced increment in MDA

concentra-tion compared with the quail that had no RO dietary

supplementation.AlthoughtheimpositionofHShadno

signiﬁcanteffectsontherGSHconcentration,GSH-Px

activ-ityandCATactivity,ROsupplementationinamountsof125

and 250ppm tothefeedunderHSconditionsincreased

(P<0.01)therGSHconcentrationandincreased(P<0.05)

CAT activity. For birds with the TN treatment, dietary

supplementation of 250ppm ROresulted in a decrease

(P<0.01)inMDAconcentrationandbothamounts(125and

250ppm)ofdietarysupplementationsincreased(P<0.01)

therGSHconcentrations.

3.3. Changesintesticularspermatogeniccellcount

The mean values of spermatogenic cell counts in

the left testis samples are given in Table 2.

Signif-icant reductions were determined in the counts of

all the spermatogenic cells including spermatogonia

(P<0.001),spermatocytes(P<0.01),spermatids(P<0.001),

andspermatozoa(P<0.001)ofquailwheretheHS

treat-mentwasimposed.However,supplementationofboth125

and 250ppm ROto diets of animals, in which HS was

imposed, prevented theHS-induced reductionsin

sper-matogonium(P<0.05),spermatocyte(P<0.05),spermatid

(P<0.01),andspermatozoon(P<0.001)counts.

3.4. Changesintesticularhistologicstructure

When assessed microscopically, the testes of quail

had normal histology with 0 (Fig. 1A), 125 (Fig. 1C)

and 250 (Fig. 1E) ppm of RO dietary supplementation

for animalshousedintheTNenvironmentalconditions.

The histopathologic changes were mostly observed in

thequail withnoROdietarysupplementation (Fig. 1B)

andhistopathologicdisorderswerelessinquailthathad

dietarysupplementationsof125(Fig.1D)and250(Fig.1F)

ppm ROunder theHSenvironmentalconditions.

Disor-ganization and degenerationof germinal cells (Fig. 1B)

togetherwithdilatationofseminiferoustubules(P<0.001)

andreductionsinGCLT(P<0.001)werethemostmarked

changesobservedinthetestesofquailwithoutROdietary

supplementationand rearedundertheHS

environmen-talconditions(Table4).Inadditiontothehistopathologic

lesions,thereweremoreimmaturespermatidsand

sper-matogoniawithinthetubularlumenalongwithindications

ofspermatogeniccessationandfewerspermatocyteswere

observedinsomeseminiferoustubulesofthequailwithno

ROdietarysupplementationwhenthebirdswerehoused

under the HS environmental condition. Increases were

observed in the GCLT and there wasa decrease in the

diametersofseminiferoustubules(DST;P<0.001,Table4)

anddegreeofdegenerationsintesticulartissuesofquail

withboth125(Fig.1D)and250(Fig.1F)ppmROdietary

supplementationin comparison with thequail withno

ROdietarysupplementationthatwerehousedunderthe

HSenvironmentalcondition.Supplementationof125and

250ppmROtoquaildietsrearedintheTNenvironmental

conditionincreased(P<0.001,Table4)theGCLTvalueas

comparedwithquailprovidednoROdietary

supplemen-tation.

3.5. Changesinnumbersoftesticularapoptoticand

anti-apoptoticgermcells

Thenumbersofapoptoticgermcellsasdeterminedby

Baximmunostainingofthetestesofquailrearedinboth

TNandHSenvironmentalconditionsareshowninFig.2.

Bax immunopositive staining was more intense in the

germinal cell line of the quail that had HS imposed

(Fig.2B,D,andF)comparedwiththosehousedunderthe

TNenvironmentalcondition(Fig.2A,C, andE).TheBax

immunopositivestainingoftestistissueswasmostintense

inquailthat werefeddietswithnoROdietary

supple-mentationandrearedintheHSenvironmentalcondition

(Fig.2B),whiletherewaslessBaximmunopositivestaining

inthetissuesquailwith125(Fig.2D)and250(Fig.2F)ppm

ROdietarysupplementationthatweremanagedunderthe

(6)

Fig.1.Effectsofrosemaryoil(RO)onhistopathologicstructureoftestesinJapanesequailrearedinathermoneutral(TN)environmentandunderheat

stress(HS)(hematoxylinandeosin-staining,×40magniﬁcation).FootnoteforFig.1.Normalappearanceofseminiferoustubulesoftestesinquailthatwere

supplementedwith0(A),125(C),and250(E)ppmROinTNenvironmentalconditions;Dilatationinthediametersofseminiferoustubules,decreasein

germinalcelllayerthickness,andimmaturespermatocytesinlumenoftubulesoftestesinquailwithdietarysupplementationof0ppmRO(B),andthe

decreaseinlesionsalongwithdilatationinquailsupplementedwith125(D)and250(F)ppmROinHSenvironmentalconditions.

Table4

Effectsofrosemaryoil(RO)onsometesticularhistopathologicandimmunohistochemicalmeasurementsinJapanesequailrearedinathermo-neutral(TN)

environmentandunderheatstress(HS).

RO(ppm) RO(ppm) 0 125 250 0 125 250 s.e. HS signiﬁcance(P-value) RO signiﬁcance(P-value) DST(␮m) 246.80a _229.30b _227.70b _206.20 _209.50 _208.88 _1.68 _0.0001*** _0.0001*** GCLT(␮m) 52.10c _58.20b _63.80a _59.80C _64.70B _74.00A _0.75 _0.0001*** _0.0001*** ARpositivity(0–4) 1.16b _1.26b _1.47a _1.66B _1.72B _2.00A _0.03 _0.0001*** _0.0001*** Baxpositivity(0–4) 1.66 1.50 1.33 0.66 0.50 0.50 0.04 0.0001*** _0.505 Bcl-2positivity(0–4) 0.50b _0.66ab _0.87a _1.00B _1.33A _1.33A _0.03 _0.0001*** _0.0001*** Bax/Bcl-2ratio 3.32b _2.27ab _1.53a _0.66B _0.38A _0.38A _0.05 _0.0001*** _0.0001***

DST:Diameterofseminiferoustubules,GCLT:Germinalcelllayerthickness,AR:Androgenicreceptor.

a,b,c_Mean_values_having_different_superscripts_in_the_same_row_within_the_HS_groups_are_different_from_each_other.

A,B,C_Mean_values_having_different_superscripts_in_the_same_row_within_the_TN_groups_are_different_from_each_other.

(7)

Fig.2. Effectsofrosemaryoil(RO)onapoptoticgermcellintensity,demonstratedbyBaximmunopositivestaininginthetestesofJapanesequailrearedin

athermoneutral(TN)environmentandunderheatstress(HS)(Mayer’shematoxylin,×200magniﬁcation).FootnoteforFig.2.Baximmunopositivestaining

(atspeciﬁcratios)inthespermatogeniccellsofquailthatweresupplementedwith0(A),125(C),and250(E)ppmROinTNconditions;Therewasmore

Baximmunopositivestaininginthespermatogeniccellsofquailthatweresupplementedwith0ppmRO(B),andtheslightbutaninsigniﬁcantdecrease

inthestainingintensityofspermatogeniccellsofquailthatweresupplementedwith1250(D)and2500(F)ppmROinHSenvironmentalconditions.

of Bax immunopositive germinal cell stainingintensity,

althoughtheimpositionoftheHSenvironmentalcondition

resultedinanincrease(P<0.001)whencomparedtothe

cellsfromtheTNgroup,dietarysupplementationofboth

125and250ppmROtothedietsofquailwhere

imposi-tionofHSoccurreddidnotresultindecreasesingerminal

cellsstainedwithBaximmunopositivestainingwhen

com-paredwiththegroupwithnoROdietarysupplementation

that washousedunder theHSenvironmentalcondition

(Table4).

Anti-apoptoticgermcellBcl-2immunopositive

stain-ingintensityresultsareshowninFig.3forthetestesof

quailrearedunderbothTNandHSconditions.Withboth

HS(Fig.3B,D,F)andTNenvironments(Fig.4A,C,andE),the

Bcl-2immunopositivestainingintensityoftestesofquail

providedboth125(Fig.3C)and250(Fig.3E)ppmofRO

wasmorepronouncedthanforquailwithnoROdietary

supplementation(Fig.3A).Withrespecttonumericalvalue

of Bcl-2 immunpositive staining intensity, HS caused a

decrease(P<0.001) whencompared withtheTNgroup.

However,dietarysupplementationof250ppmROtoquail

withHSimpositionresultedinanincrease(P<0.001)when

compared tothegroupwithnoROdietary

supplemen-tationthatwasrearedinHSconditions.Similarincreases

wereobservedinquailwithboth125(P<0.01)and250

(P<0.01)ppmROdietarysupplementationascomparedto

animalswithnoROsupplementationthat werehoused

undertheTNcondition.WhileHSincreased(P<0.001)the

Bax/Bcl-2ratio,dietarysupplementationof250ppmROin

quailimposedwithHSdecreased(P<0.001)Bax/Bcl-2ratio

(Table4).

3.6. ChangesintesticularAR

TheamountofARimmunopositivestainingwasgreater

in round and elongated spermatids than

spermatogo-nia, primary and secondary spermatocytes, Sertoli and

Leydig cells in the testes of all groups of quail reared

in both TN and HS conditions. While there was some

(8)

Fig.3. Effectsofrosemaryoil(RO)onanti-apoptoticgermcellintensity,demonstratedbyBcl-2immunopositivestaining,inthetestesofJapanesequail

rearedinathermoneutral(TN)environmentandunderheatstress(HS)(Mayer’shematoxylin,×200magniﬁcation).FootnoteforFig.3.NormalBcl-2

immunopositivestaininginthespermatogeniccellsofquailthatweresupplementedwithnoadditive(A),andthegreaterBcl-2immunopositivestaining

inthespermatogeniccellsofquailsupplementedwith125(C)and2500(E)ppmROinTNenvironmentalconditions;Bcl-2immunopositivestaining(at

speciﬁcratios)inthespermatogeniccellsofquailsupplementedwith0ppmRO(B)andtheincreaseinstainingintensityofspermatogeniccellsofquail

supplementedwith125(D)and250(F)ppmROinHSenvironmentalconditions.

noROdietary supplementationthat were reared in HS

environment (Fig. 4B, Table 4), greater staining was

observedinthetestesofquailwith250ppmROdietary

supplementationandrearedinTNenvironment(Fig.4E,

Table4).In germinal cells of quail raisedin TN

condi-tions, theintensity of AR immunopositive staining was

greater in the RO-treated groups (Fig. 4C, and E and

Table4)particularlythosewithdietarysupplementation

of250ppmROascomparedwiththenon-supplemented

group(Fig.4A, Table4). Althoughboth amounts of RO

supplementation(Fig.4DandF)todietsofbirdsreared

under HS imposed conditions resulted in an increase

intheintensityof ARimmunopositive stainingin

com-parisonwiththequailwithnodietarysupplementation

(Fig.4B)thatwerehousedintheHSimposedcondition,

theincreaseobservedwith250ppmsupplementationin

theRO-treatedgroupwastheonlyvalueofstatistical

sig-niﬁcance(Table4).

4. Discussion

4.1. DetrimentaleffectsofHS

Small amounts of ROS have an important

physio-logical role in modulating gene and protein activities,

necessary for spermatozoa proliferation, differentiation,

maturation and fertilizing ability. However, the

patho-logical effects ofROS occurwhenthese compoundsare

producedinexcessofthetypicalphysiologicalamounts.

Thecellmembranephospholipidsareverysusceptibleto

thedetrimentalimpactsofROSthatinitiateandenhance

lipidperoxidation(Sharmaetal.,2012).Avianspermatozoa

havelargeamountsofPUFAsthatincreasesusceptibility

to the detrimental actions of ROS and lipid

peroxida-tion.Enzymatic[superoxidedismutase(SOD),GSH-Pxand

CAT]andnon-enzymatic(vitaminsA,C,E,GSH,selenium,

(9)

Fig.4.Effectsofrosemaryoil(RO)ontheintensityofandrogenicreceptor(AR)immunopositivestaininginthetestesofJapanesequailrearedina

thermoneutral(TN)environmentandunderheatstress(HS)(Mayer’shematoxylin,×200magniﬁcation).FootnoteforFig.4.NormalARimmunopositive

staininginthespermatogeniccellsofquailthatweresupplementedwith0(A)and125(C)ppmROand,thegreaterARimmunopositivestainingin

thespermatogeniccellsofquailsupplementedwith250(E)ppmROinTNenvironmentalconditions;ThelesserARimmunopositivestaininginthe

spermatogeniccellsofquailsupplementedwith0(B)and125(D)ppmROand,theincreaseinthestainingintensityofspermatogeniccellsofquailthat

weresupplementedwith250(F)ppmROinHSenvironmentalconditions.

unsaturatedchemicalbondsofmembranephospholipids

againstlipidperoxidationbydecreasingthedetrimental

impactsofROS.Whentheoxidant-antioxidantsystemis

notoptimalinitsequilibriumasaresultofincreased

pro-ductionof oxidants,there areimpairments intesticular

histologic structure, spermatogenesis and spermatozoa

functionsduetotheincreasedlipidperoxidationandthere

isanensuingreductioninfertility(Suraietal.,2001).In

thepresentstudy,HSincreasedtesticularMDA

concentra-tions,aby-productoflipidperoxidation,incomparisonto

whatoccurredwiththeTNconditions,whichisin

agree-ment withﬁndings from a previous study(Türk et al.,

2015).TheHShad noeffectontesticulartissue

antiox-idant markers including rGSH,GSH-Px, and CAT in the

presentstudy.Thisresultisconsistentwiththeﬁndings

ofTürketal.(2015)butnot thoseofAltanetal.(2003)

where there were increasesin blood SOD, GSH-Px and

CATactivitiesofbroilerchickenshousedinHSconditions.

Thereasonforthisinconsistencyinﬁndingsmaybethe

useofdifferentspecies ofanimalsand differentsample

assessmentsbetween thepresent and previous studies.

Reductionsinspermatozoacount,spermatozoamotility,

andincreasednumbersofdeadspermatozoaofchickens

(McDanieletal.,2004;Ebeid,2012)havebeenreportedto

bedetrimentaleffectsofHSonsemenquality.Inaddition,

inthepreviousstudy(Türketal.,2015),HSreducedboth

spermatidandtesticularspermatozoacounts.Consistent

withtheseﬁndingsinthepreviousstudywasthe

signiﬁ-cantreductionsinthenumbersofallspermatogeniccells

includingspermatogonium,spermatocyte,spermatid,and

spermatozooninthetesticulartissueofthequailhoused

underHSconditions.Thereasonfor theincrease in

tes-ticularlipidperoxidationanddecreaseinspermatogenic

cellnumbersobservedin thepresentstudymaybethe

increasedconcentrationofROSthatdevelopedinresponse

toHS.

Loss of spermatogenic cells, germ cell degeneration

inseminiferoustubules,cessationofspermatogenesisin

broilers(TerimKapakınetal.,2013)andquail(Türketal.,

(10)

alongwithgermcelldisorganizationinquail(Türketal.,

2015)havebeenreportedtobeHS-relatedoutcomesalong

withanincreaseinsometesticularhistopathologiclesions.

Similarhistopathologiclesionsincludingtheincreaseinthe

DST,reduction intheGCLT,disorganization,and

degen-eration in germinal cells, cessation of spermatogenesis,

existenceofimmaturespermatidsandspermatogoniain

thelumenofsomeseminiferoustubulesweredetectedin

thetestesof quailwhere HSwasimposedonthebirds

inthepresentstudy.Apoptosisisprogrammedcelldeath

andisabiologicalprocessthatprotectstissueby

eliminat-ingthecellswithabnormalfunction,therebypreventing

the increase in numbers of aberrant cells. All types of

spermatogenic cells undergo apoptosis, suggesting that

programmedcell death maybe an important regulator

ofspermatogenesis(Sharmaetal.,2012).Whenthe

pro-ductionof ROS is greater than optimal leading tolipid

peroxidation, apoptosis of germ cells can be enhanced

(Maheshwariet al.,2009).Theactionswhenthereisan

elevatedamountof anti-apoptotic(Bcl-2)protein,there

is an extended survival of cells and increases of

pro-apoptotic(Bax)geneexpressionthatinturnleadstoan

accelerationofcelldeathinthetissues(SinhaHikimand

Swerdloff,1999).In thepresentstudy,HScaused

signif-icantincreases intesticularBaximmunostainingand in

Bax/Bcl-2ratio,andsigniﬁcantdecreasesintesticular

Bcl-2immunostaininginquailascomparedwithﬁndingsin

birdsunderTNconditions.HS-inducedlipidperoxidation

causesincreasedtesticularapoptosisinapreviousstudy

(Türket al.,2015)in developingquail,which is

consis-tent withthe ﬁndings of the present study. Androgens

andtheAR haveimportant rolesin male

spermatogen-esisandfertility.Theactionsofandrogensaremediated

bytheAR (Wang etal., 2009).In an invitrostudy, the

numbersofARinmonkeySertolicellswerereducedafter

HS(Chenetal.,2008).Consistentwiththeﬁndingsofa

previousstudy(Türketal.,2015),therewasasigniﬁcant

reductioninARimmunopositivestainingintesticular

tis-sueofquailwhereHSwasimposedinthepresentstudy.

TheHS-inducedincrease inlipidperoxidationmaylead

toimpairmentsintesticularhistologicstructure,increases

intesticularapoptoticgermcells,aswellasdecreasesin

numbersofAR.Additionally,therelaxationofperitubular

myoidcellsinresponsetoHS(Türketal.,2015)maybe

thereasonfortheincreasedDSTobservedinthepresent

study.

4.2. AlleviatingeffectsofRO

Naturalantioxidantproductsareincreasinglyusedto

treatvarious pathologic conditionsrelating tooxidative

stress and the resulting pathogenesis (Raskovic et al.,

2014).Antioxidantsareaddedtothedietasafeed

supple-mentationfor improvingperformance, productivityand

reproductiveoutcomesinthepoultryindustry.Rosemary

containsactiveanti-oxidativesubstancessuchasphenolic

diterpenes,ﬂavonoids,phenolicacids(Hoetal.,2000)and

volatileoils(Begumetal.,2013).TheROhasbeenused

pre-viouslyasapreservativeinthehumanfoodindustrydueto

itsantioxidantandantimicrobialactivities(Raskovicetal.,

2014).DifferentamountsofsupplementationofRO

pre-ventsHS-induceddecreasesingrowthperformance and

carcass traits of Japanese quail(C¸iftc¸iet al., 2013) and,

alsoimprovesgrowthperformance,eggtraits,egg

oxida-tivestability,andmeatqualityofPharaohquailrearedin

TNenvironmentalconditions(Yesilbagetal.,2013).

Dif-ferentconstituents(terpenoids,ﬂavonoids,phenolicacids,

volatileoils)ofrosemaryhavebeenreportedtofunction

bydecreasingoxidativestressindifferenttissuesof

mam-malsthatisinducedbydifferentchemicals(Tanyıldızıetal.,

2009;Singhetal.,2012;Raskovicetal.,2014)orthatis

producedasaresultofexposuretoelectromagneticﬁeld

(Hajhosseinietal.,2013).RO(Melusovaetal.,2014),

ros-marinicacid(Hajhosseinietal.,2013),andterpenes(Singh

etal.,2012)haveDNAprotectiveandanti-apoptotic

fea-tures.Inaddition,Lin(2014)demonstratedthattriterpenes

fromAlismaorientalisfunctionasanARreceptoragonist.

However, there are inconsistenciesin ﬁndings between

the studies concerning the effects of rosemary on the

reproductive system of male mammals. While ﬁndings

insomestudiesindicatethatrosemaryhasa

contracep-tive(Nusieretal.,2007)anddeterioratingeffects(El-Din et al., 2012; Heidari-Vala etal., 2013)on testicular

tis-sues,ﬁndingsinotherstudiesindicatethatrosemaryand

itsdifferentextractsare abletopreventboth functional

spermatozoa damage that is induced by

cryopreserva-tion(Lunoetal.,2014;Motlaghetal.,2014)andvarious

toxicant-induced testicular, hormonal and spermatozoa

damage(Hozayenetal.,2014;Uyeturketal.,2014).In

addi-tion,Superchietal.(2005)suggestedthattheantioxidant

activityofrosemaryextractlimitsthenegativeeffectsof

temperaturesthat induceHS onreproductiveefﬁciency

of boars. Tothebest of ourknowledge,there hasbeen

no evidence regarding the effect of RO on HS-induced

damage as related to reproductive variables including

spermatozoa production, testicular oxidant-antioxidant

balance, testicular histopathologic structures, and

inci-denceoftesticularapoptoticcellformationand number

ofARinquail.Therefore, thisistheﬁrstreport

evaluat-ingtheprotectionofROonHS-inducedtesticulardamage

inJapanesequail.Inthepresentstudy,supplementation

ofROtoquaildietsprovidedforsigniﬁcantreductionsin

theHS-inducedincrementsintesticularlipidperoxidation,

testicularhistopathologiclesions,andBax/Bcl-2ratio,and

decreasedthedetrimentaleffectsofHSonspermatogenic

cellcounts,GCLT,amountsofBcl-2andARinthe

reproduc-tivetissuesthatwereassessed.WhileHShadnosigniﬁcant

effects ontheantioxidantmarkers,both amountsofRO

dietary supplementationincreasedthe rGSH

concentra-tionsandCATactivityofquailunderHSimposition.With

theTNenvironmentalcondition,250ppmRO

supplemen-tation resultedina decreasein MDAconcentrationand

anincreaseinAR.BothamountsofROsupplementation

increasedtherGSHconcentration,GCLT,and amountof

Bcl-2andalsodecreasedtheBax/Bcl-2ratio.Thepossible

reasonfortheimprovementsobservedinthetestesofquail

rearedinbothHSandTNenvironmentalconditionsinthe

presentstudyinbirdswheredietsweresupplementedwith

ROisthatROhaspotentantioxidantandradicalscavenging

(11)

5. Conclusion

TheresultsofthepresentstudyclearlysuggestthatRO

additiontodietsofgrowingmalequailhousedunderHS

environmentalconditionsreducedtheHS-induceddamage

inthetestesandspermatogeniccells.Thispositiveeffectof

ROmaybeattributedtoitsanti-peroxidativeactivity.

Acknowledgment

TheauthorswishtoexpresstheirgratitudetoProf.Dr.

J.E.Kinder,DepartmentofAnimalSciences,TheOhioState

University,Wooster,OH,USA,forrevisingthelanguageof

themanuscript.

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