Reprod Dom Anim. 2020;55:29–37. wileyonlinelibrary.com/journal/rda  | 29© 2019 Blackwell Verlag GmbH

Received:11July2019  |  Accepted:13October2019DOI:10.1111/rda.13577

O R I G I N A L A R T I C L E

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze‐drying rabbit (Oryctolagus cuniculus) spermatozoa

Francesca Mercati1 | Paula Domingo2 | Rolando Pasquariello3  | Cecilia Dall'Aglio1 | Alessandro Di Michele4 | Katia Forti5 | Paolo Cocci6 | Cristiano Boiti1 | Lidia Gil2 | Massimo Zerani1  | Margherita Maranesi1

1DipartimentodiMedicinaVeterinaria,UniversitàdiPerugia,Perugia,Italy2DepartamentodePatologìaAnimal,UniversidaddeZaragoza,Zaragoza,Spain3DipartimentodiScienzeAgrarieeAmbientali‐Produzione,Territorio,Agroenergia,UniversitàdiMilano,Milano,Italy4DipartimentodiFisicaeGeologia,UniversitàdiPerugia,Perugia,Italy5IstitutoZooprofilatticoSperimentaleUmbriaeMarche‘TogoRosati’Perugia,Perugia,Italy6ScuoladiBioscienzeeMedicinaVeterinaria,UniversitàdiCamerino,Camerino,Italy

CorrespondenceRolandoPasquariello,DipartimentodiScienzeAgrarieeAmbientali‐Produzione,Territorio,Agroenergia,UniversitàdiMilano,Milano,Italy.Email:rolando.pasquariello@unimi.it

MassimoZerani,DipartimentodiMedicinaVeterinaria,UniversitàdiPerugia,Perugia,Italy.Email:massimo.zerani@unipg.it

AbstractFreeze‐drying(FD)hasbeenexhaustivelytriedinseveralmammalianspeciesasanalternativetechniquetospermcryopreservation,butfewstudieshavebeendoneinrabbits(Oryctolagus cuniculus).ThemainobjectiveofthisstudywastocomparetheprotectiveeffectofvariousantioxidantsaddedtoEDTAmediumonstructuralandfunctionalcomponentsofFDrabbitspermatozoaandontheirstatusofglobalDNAmethylation.FDmediausedwerecomposedofbasicFDmedium(10mMTris‐HClbufferand50mMNaCl)supplementedwitheither50mMEDTAalone (EDTA)oraddedwith105µMofrosmarinicacid(RA,EDTA‐RA)or10µMofmelatonin(MLT,EDTA‐MLT).Theeffectofeachmediumon thepreservationofFDspermatozoonstructurewasevaluatedwithlightandscanningelectronmicroscopy(SEM).GlobalDNAmethylationwasquantifiedinallFDspermsamplesaswellasinfreshsperma‐tozoa.Morphologically,fracturepointswereevidencedintheneck,midandprinci‐palpieceofthespermatozoontail.NodifferencesinspermatozoonfracturepointswereevidencedamongFDtreatments:intactspermatozoawerethelargest(p < .01)category,whereasthemostfrequent(p < .01)injurywastheneckfracture,resultingintaillessheads.AtSEM,theheadofspermatozoashowedawell‐conservedshapeandintactmembraneinalltreatments.DNAmethylationstatuswasthesameinallFDtreatments. Inconclusion,supplementationofEDTA,EDTA‐RAandEDTA‐MLTduring FDpreserved rabbit spermmorphological integrity andmethylation statusaswell.Therefore,thedifficultyofgettingviableoffspringusingFDsemenislikelyunrelatedtotheimpactofthelyophilizationprocessonDNAmethylationandmor‐phologyoflyophilizedspermatozoa.

K E Y W O R D S

EDTA,lyophilisation,melatonin,rabbitsperm,rosmarinicacid,scanningelectronmicroscopy

1  | INTRODUC TION

Artificialinseminationwasthefirstassistedreproductivetechnologyused in domestic animals. Since the first successful experimented

in 18th century by Lazzaro Spallanzani,many steps forwardwereachieved inthefieldofassistedreproduction: invitrofertilization,invivoembryotransfer,embryosexing,cloningand intracytoplas‐micsperminjection(Galli&Lazzari,2008).Thesetechniqueshave

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positivelyaffectedgeneticmeritandproductionoflivestock,aswellastreatmentofhumaninfertility.

In recent years, the development of an alternativemethod tosperm cryopreservation, such as freeze‐drying (FD) or lyophiliza‐tion,hasfacilitatedthestorageandmaintenanceofgeneticmeritofseveralspecies,includingrabbits(Keskintepe&Eroglu,2015;Liuetal.,2004),andbio‐bankingofendangeredspecies (Anzaloneatal.,2018).FDisamethodinwhichfrozenmaterialisdriedbyicesub‐limation,therebyinvolvingadirecttransitionfromasolid(ice)toavapour(gas)phase(Keskintepe&Eroglu,2015).ThepurposeofFDistheremovalofwaterthatdoesnotallowthegrowthofpathogensorcontaminants(Bielanski,Nadin‐Davis,Sapp,&Lutze‐Wallace,2000),ensuringthepreservationofthesamples.Oneofthemainadvan‐tages of lyophilized semen is its conservation and management,whichismuchsimplerandcheaper.Infact,FDsemencanbestoredtemporarilyatroomtemperature(Kaneko,2015),therebyreducingstorage,facilityandshippingcosts.

However, current FD techniquesmake spermatozoa non‐viableandmotionlessat rehydration (Keskintepe&Eroglu,2015),but thehandicapofthelostmotilitywaseasilyovercomebytheirdirectin‐jectionintotheoocytes(Salamone,Canel,&Rodríguez,2017).Thosefindingsdemonstratedthatnuclearandcellularviabilityarenotequiv‐alent:thelyophilizedspermatozoaaredead,butstillcapableofinter‐actingwithoocytesandgiving rise tonormaland liveoffspring.Attheendofthe50s,Yushchenko(1957)reported,forthefirsttime,thebirthofarabbitfollowingartificialinseminationwithFDsperm.Usingintracytoplasmic lyophilized sperm injection,normaloffspringwereobtainedinrabbits(Liuetal.,2004)andinothermammals:sheep(Loi,Iuso,Czernik,Zacchini,&Ptak,2013),pigs(Das,Gupta,Uhm,&Lee,2010),horses(Choi,Varner,Love,Hartman,&Hinrichs,2011),ham‐sters(Muneto&Horiuchi,2011),mice(Ono,Mizutani,Li,&Wakayama,2008;Wakayama&Yanagimachi,1998)andrats(Hirabayashi,Kato,Ito,&Hochi,2005).Inotherspecies,embryonicdevelopmentdidnotgofurtherfromblastocyst(Saragusty&Loi,2019).

However, FD method may affect negatively spermatozoa notonly by altering their morphology membrane and acrosome, butalsobycausingmoleculardamagesonDNAandcentrosome,duetosnap‐freezinganddryingstresses(Liuetal.,2004).Amongallthesesperm aberrations, DNA fragmentation of spermatozoa followingFD is one of the main causes of embryonic development failure(Palazzese,Gosálvez,Anzalone,Loi,&Saragusty,2018).Inparticu‐lar,endonucleasesmayinduceoxidativestressduetothereleaseofreactiveoxygenspecies(ROS)(Kusakabe,Szczygiel,Whittingham,&Yanagimachi,2001).ThechelationofcalciumseemstobeimportantforinhibitionofendonucleasesthatareinvolvedinDNAintegrity;inparticular,chelatingagentspreventDNaseactivation,asobservedinmousespermatozoa,whoseDNaseisactivatedbythepresenceofCa2+,resultinginDNAinjury(Kaneko&Nakagata,2003).

Calciumchelators,suchasEDTAorEGTA,areusedindifferentspeciestopreservespermfromthe lyophilizationprocessandob‐tainvitaloffspring (Kaneko&Nakagata,2006). In fact, spermato‐zoaarehighlysensitivetoseveraltypesofchemical/physicalstress,due to reactive oxygen species, osmotic changes and different

temperatures applied during FD procedure for freezing, warmingandthawing(Koshimoto&Mazur,2002).

Rosemary(Rosmarinus officinalis)anditsextracts,suchasrosma‐rinic acid (RA), are natural antioxidants that show antiproliferative,antiradical and protective properties against oxidant‐induced stresseffect(Ho,Wang,Wei,Huang,&Huang,2000).Inseveralmammals(Luñoetal.,2014;Olaciregui,Luño,Domingo,González,&Gil,2017;Olaciregui,Luño,González,etal.,2017;Yenietal.,2018),includingrab‐bits(Amidi,Pazhohan,ShabaniNashtaei,Khodarahmian,&Nekoonam,2016;Domingo,Olaciregui,González,DeBlas,&Gil,2018),additionofRAinFDorpost‐thawingmedium(Motlaghetal.,2014)protectedspermatozoaagainstoxidativestressand improvedspermpreserva‐tion.Incontrast,inasperminvitrostudy,Lvetal.(2018)foundade‐creasedintracellularCa2+concentrationcompromisingspermfunctionaftertheadditionofRAinadose‐dependentmanner.

Melatonin(MLT) isakeyregulatorofthecircadianwake–sleepcycle and reproduction in animals characterized by photoperiod(Chemineauetal.,2008); ithasalsoa relevant role in spermbiol‐ogy(Cebrián‐Pérezetal.,2014).Infact,MLTcontrastsavarietyofROSspeciesduetoitswellestablishedantioxidantactivity(Jangetal.,2010).Accordingtovariousstudies,melatoninimprovessemencharacteristics of goats (Ramadan, Taha, Samak,&Hassan, 2009),rats (Sönmez,Yüce,&Türk, 2007), boars (Janget al., 2010), rams(Ashrafi, Kohram,Naijian, Bahreini, & Poorhamdollah, 2011),mice(Sarabia,Maurer,&Bustos‐Obregon.,E.,2009)andmen(Ortizetal.,2011).Byconverse,nostudyontheroleofMLThasbeencarriedoutinrabbitspermatozoa.Inaddition,tothebestofourknowledge,nostudiesevaluatedthecombinedeffectsofRAandMLTaddedtoEDTAonrabbitspermduringFD.

SemenconservationmethodscanaffectDNAmethylation,thusinfluencinggeneexpressionandanimal fertility (Aurich,Schreiner,Ille,Alvarenga,&Scarlet,2016;Vermaetal.,2014).AlterationofDNAmethylationhasbeen associatedwith several pathologies, leadingtoabnormalembryodevelopmentandincreasedincidenceofearlyembryonic loss (Al‐Khtib, Blachère, Guérin, & Lefèvre, 2012). Forthese reasons, assessmentofDNAmethylation represents a new,relevantapproachtoevaluatetheabilityofspermatozoatofertilizetheoocyteandleadtonormalblastocystandembryodevelopment(Benchaibetal.,2003).

TheaimofthisstudywastodeterminetheprotectivecapacityofchelatingagentEDTA,eitheraloneorwiththeadditionofRAorMLTasantioxidants,throughoutthespermFDprocessintherabbit(Oryctolagus cuniculus).Therefore,weevaluatedthemorphologicalstructureoflyophilizedspermatozoabylightandscanningelectronmicroscopy(SEM)aswelltheDNAmethylationstatus.

2  | MATERIAL S AND METHODS

2.1 | Reagents and media

Unlessnotedotherwise,allchemicalswerefromSigma‐AldrichCo.(Alcobendas). Freeze‐dryingmediumusedwas composed of basicmedium (10mMTris‐HCl buffer and 50mMNaCl) supplemented

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with50mMEDTA.TwomoreexperimentalFDmediawerepreparedbyadding105µMRAtoEDTA(EDTA‐RA)and10µMMLTtoEDTA(EDTA‐MLT).ThefinalpHofthesolutionswasadjustedbetween8.2and8.5(Kanekoetal.,2003).

2.2 | Animals

ThisstudywasapprovedbytheVeterinaryEthicalCommitteeoftheUniversityofZaragoza.Thecareanduseofanimalsweredoneinac‐cordancewiththeSpanishPolicyforAnimalProtectionRD1201/05,whichmeetstheEuropeanUnionDirective86/609ontheprotec‐tionofanimalsusedforexperimentalandotherscientificpurposes.

All semen samples were collected from five sexually ma‐tureNewZealandwhitemalerabbits (4.5–5kgbodyweightand5–8 mo of age), raised in premises owned by the University ofZaragoza.Malesweremaintained in individual cagesunder lightcycleof12hr light/darkat a room temperatureof22–24°Candarelativehumidityof55%–60%.Allanimalswerefedacommer‐cialpelletdietaccordingtotheirreproductivecondition,andfreshwaterwasgivenadlibitum.

2.3 | Sperm collection and processing

Rabbitspermsampleswerecollectedbyartificialvaginapreheatedat55°Candacollectorpreviouslytemperedat37°C.Aftersemencollection, any gel plugwas removed. Immediately, each ejaculatewas diluted (1:3) in pre‐warmed (37°C) phosphate‐buffered saline(PBS) solution. A macroscopic analysis was performed to assesscolourandvolumeofeachejaculate;themotilitywasevaluatedbycomputer‐assisted semen analysis (CASA, PROISER R + D). Onlyejaculateswithwhitecolour,>0.2mlandgoodwavemotion(atleast85%ofmotility)wereusedfortheresearch.

Eachspermsamplewasdividedintofourfalcontubes:onetubewasusedascontrol(freshsperm)andtheotherthreetubesforthreefreeze‐dryingprocedures(EDTA,EDTA‐RA,EDTA‐MLT).Afterwards,the sampleswere centrifuged twice to remove seminal plasma asmuchaspossible.Thefirstcentrifugationwasperformedat500×gfor 10min; thereafter, seminal plasmawas removed, and samplesweredilutedwith1mlofPBS.Straightaway,asecondcentrifugationwascarriedoutat500gfor5minandthesupernatantwasagainre‐movedleavingadropofittosuspendthepelletedspermatozoainto200µloffinalvolume.Then,1.800µlofPBSwasaddedtothesam‐plesandmixedslowly.ThenumberofspermatozoaofeachsamplewascountedusingaNeubauerchamber,andtheconcentrationwasadjustedto106spermatozoapersamplebyaddingPBS.Lastly,1mlofspermsolutionwasplacedintoanEppendorftubeandcentrifugedat500gfor5min;thesupernatantwasthendiscarded.

2.4 | Fresh sperm samples processing

Fresh sperm samplesweredilutedwithDNA/RNAshield solution(Zymo Research) to a final volume of 200 µl and then frozen at−20°C.

2.5 | Freeze‐drying and rehydration procedure

EachsamplewassuspendedinthethreedifferentFDmedia:EDTA,EDTA‐RAandEDTA‐MLT tohave150µl of final volume. Freeze‐drying procedure was performed as reported byWakayama andYanagimachi (1998). Sperm suspensions from each group wereplaced into1ml glass cryovials (LabconNorth) and thenplungedintoliquidnitrogen(LN2)for5min.Immediately,thefrozensamplesweretransferredontotheshelf(−50°C)ofaprogrammablefreeze‐drier(MillrockTechnology).Twodryingswereperformedforfreeze‐dryingthesamples:afirstdryingat0.053mbarofpressureandat−68°Candaseconddryingat0.018mbarofpressureandat−20°Cof temperature.After theFDprocess, cryovialswere sealedwithrubbercupsandparafilm.

The rehydration of FD spermatozoawas performedby adding300µlof0.1MPBS(pH7.2).Rehydratedspermatozoawerecentri‐fugedonceat1,000gfor2min,andthesupernatantwasremoved.

2.6 | Light and SEM microscopy

Afterrinsing,spermatozoawerefixedfor1hratroomtemperaturein 2.5%glutaraldehyde in 0.1Mphosphate buffer (pH7.2). Then,theywerewashedfor10mininthesamebufferanddehydratedinan increasingconcentration seriesofethanol solutionsuntil abso‐lute.Finally,adropofsolutionwasgentlydroppedontopoly‐L‐ly‐sine‐coatedslidestospreadthespermatozoa.

Atotalof300cells/animalwereanalysedforeachtreatmentatlightmicroscopy.Thespermatozoawereclassifiedaccordingtotheirintegrityandtheregionwherethedamagewasdetected:(a)intactspermatozoa(IS)(b)head,middletailandpartialprincipaltail(HMP),(c)headandmiddletail(HM)and(d)headonly(H).

Toevaluatespermultrastructureintegrity,SEMwasperformedonsamples(30cells/animal)fromalltreatments.Spermatozoa,fixed,dehydrated and layered on slides as above described, were sput‐ter‐coatedwithgoldandexaminedwithaFieldEmissionScanningElectronMicroscopy(FEG‐LEO1525ZEISS;CarlZeiss)atanaccel‐eratingvoltageof2kV.Thespermatozoawereclassifiedasforlightmicroscopy:(a)intactspermatozoa(IS)(b)head,middletailandpar‐tialprincipaltail (HMP), (c)headandmiddletail (HM)and (d)headonly(H).

2.7 | DNA methylation analysis

GenomicDNAwasisolatedusingQuick‐DNATMMicroPrepaccord‐ingtothemanufacturer'sinstructions(ZymoResearch).TheDNAconcentrationfromwhitebloodcells, freshspermandFDsperm(EDTA,EDTA‐RA,EDTA‐MT)wasquantifiedusing2000NanoDropspectrophotometer. Furthermore, integrity of DNA sampleswasassessed using agarose gel electrophoresis. Afterwards, quan‐tification of global DNA methylation was examined using thecolorimetric MethylFlashTM Methylated DNA Quantification Kit(EpigentekGroupInc.)followingthemanufacturer'sinstructions.Atotalof100ngDNApersamplewasanalysedinduplicate.Briefly,

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allsampleswereaddedto96‐wellplatesusinganinputDNAcon‐centrationof100ng.TheglobalDNAmethylationwasdetectedbyspecificallymeasuringlevelsof5‐methylcytosine(5‐mC)usingcapture and detection antibodies. ThemethylatedDNA fractionwasquantifiedusingcolorimetricanalysisbyreadingtheabsorb‐ance(450nm)inamicroplatespectrophotometerusingastandardreferencecurve.Thequantificationofthemethylationstatuswasexpressedaspercentageofmethylatedcytosines(5‐mC)usingtheformulaprovidedbythemanufactureranddescribedbyCoccietal.(2018).

2.8 | Statistical analysis

Shapiro–Wilktestscertifiedthedistributionnormalityofdatathatwere analysed by two‐way ANOVA (treatments vs. spermatozoaintegrity,TableS1)followedbyStudent–Newman–Keulsposthoct test.

3  | RESULTS

3.1 | Light microscopy evaluation

Spermatozoa were clearly detectable allowing the assessment oftheirintegrity.SpermsamplesfromEDTAtreatmentwereeasilydi‐lutedinPBS.Incontrast,thosefromEDTA‐RAandEDTA‐MLTtreat‐mentsweresomewhatagglutinatedanddifficulttodissolve.

Thethreemedia(EDTA,EDTA‐RAandEDTA‐MLT)affectedsim‐ilarlyspermatozoa integrity (Figure1).Thepercentageof ISgroupwashigher(p<.01)comparedwiththeotherthreegroups;Hgroupwashigher(p<.01)withrespecttoHMPandHMones(Figure1).

3.2 | Scanning electronic microscopy

Spermatozoa heads were 7–8 μm long and 4 μm wide, whiletailswere 0.35μm thick in the principal piece (Figure 2a). In all

F I G U R E 1  LightmicroscopyanalysisofFDrabbitspermatozoa.Upperfigure:intactspermatozoa(IS);tailprincipalpiecefracturepointcomprisinghead,middletailandpartialprincipalpiecetail(HMP);tailmiddlepiecefracturepointcomprisingheadandtailmiddlepiece(HM);andheadonly(H).Lowerfigure:lesionpercentageofrabbitspermatozoaafterFD.Spermatozoawerecollectedfromfiverabbits.Experimentalgroups:EDTAalone;EDTA‐RA,EDTAsupplementedwithrosmarinicacid;EDTA‐MLT,EDTAsupplementedwithmelatonin.Valuesarethemeans±SDoffivereplicates.Differentlettersabovethebarsindicatesignificantlydifferentvalues(p < .01):Greeklettersamongexperimentalgroups;Latinlettersinthesameexperimentalgroup

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treatments, the head of spermatozoa showed a well‐conservedpaddle‐shaped form with an intact membrane (Figure 2b),while membrane lesions were occasionally observed in the tail.Membrane acrosomeand acrosomal andpost‐acrosomal regionsseparatedbyaslightlycurvedridgewereidentified;serrationsintheanteriorborderofthepost‐acrosomalregionwereusuallyob‐servedinthehead(Figure2b)suchasacharacteristicswollenattheendofthetail(Figure2b).TheresultsofSEM,confirmedlightmicroscopyone, in fact, the threeusedmedia (EDTA,EDTA‐RA,EDTA‐MLT) affected similarly spermatozoon integrity (Figure 3).Whereas,asregardthepercentageofintegrity,theISgroupwashigher (p< .01)with respect to theother threegroups,Hgroupwashigher(p<.01)withrespecttoHMPandHMones(Figure3).

3.3 | DNA methylation of lyophilized sperm

Genome integritywasnotaffectedafterFDandgenomicDNAofEDTA, EDTA‐RA and EDTA‐MLT samples were similar to that offreshsemenafteragarosegelelectrophoresis(Figure4).

The DNAmethylation of lyophilized rabbit spermwas not af‐fectedbysupplementationwithEDTA(1.11±0.60ngµl‐1),EDTA‐RA(0.86±0.55ngµl‐1)orEDTA‐MLT(1.28±0.62ngµl‐1)withrespecttothefreshsemen(1.16±0.43ngµl‐‐1).

4  | DISCUSSION

ThisstudyindicatesthatchelatingagentEDTA,eitheraloneorsup‐plementedwith antioxidant substances (RAorMLT) added to thelyophilizationmedium,hasbeneficialeffectsonthemorphologyandmethylationgradeoflyophilizedrabbitspermatozoa.

In previous studies, supplementation of TRIS buffer chelatingagents, such as EDTA or EGTA, to the freeze‐dryingmedium in‐creased sperm stability (Bhowmick et al., 2003). Liu et al. (2004)producedblastocystsafterintracytoplasmicsperminjection(ICSI)usingFDspermatozoausingmediumsupplementedwithEGTAatasimilarratetothatofICSIcarriedoutusingfreshsperm.Despitethisrelevantachievement,onlyaviablepuppywasbornandhencethe

F I G U R E 2  SpermatozoaatSEMafterlyophilization.(a)Anintactspermatozoon.(b)Ahead,characterizedbyatypicalpaddleshapeformshowingaswollenrimonthetopofthehead(arrow)andacurvedridge(arrowhead)delimitatingthepost‐acrosomalregion.(c)Theneckofaspermatozoonwithavisiblefracturepoint(arrow).Theasteriskpointsouttheserrationsinthepost‐acrosomalregion.(d)Aswollenendofthetail

*

(a) (b)

(c) (d)

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F I G U R E 3  ScanningelectronmicroscopyanalysisofFDrabbitsperm.Upperfigure:intactspermatozoa(IS);tailprincipalpiecefracturepointcomprisinghead,middletailandpartialprincipalpiecetail(HMP);tailmiddlepiecefracturepointcomprisingheadandtailmiddlepiece(HM);andheadonly(H).Lowerfigure:lesionpercentageofrabbitspermatozoaafterFD.Spermatozoawerecollectedfromfiverabbits.Experimentalgroups:EDTAalone;EDTA‐RA,EDTAsupplementedwithrosmarinicacid;EDTA‐MLT,EDTAsupplementedwithmelatonin.Valuesarethemeans±SDoffivereplicates.Differentlettersabovethebarsindicatesignificantlydifferentvalues(p < .01):Greeklettersamongexperimentalgroups;Latinlettersinthesameexperimentalgroup

F I G U R E 4  AssessmentofDNAintegrity.RandomsamplesofeachtreatmentweretestedforDNAintegrityusing0.8%agarosegel.Spermatozoawerecollectedfromfiverabbits.Experimentalgroups:EDTAalone;EDTA‐RA,EDTAsupplementedwithrosmarinicacid;EDTA‐MLT,EDTAsupplementedwithmelatonin

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need tostudy thepossible factors thatcompromised thesuccessofthetechnique.Alaterstudyonlyophilizedrabbitspermdemon‐stratedthatEDTApreservedDNAintegrityofspermatozoabetterthan EGTA aswell as the positive effect of RA supplementation(Domingoetal.,2018).However,theeffectofchelatingmoleculesonthemorphologyoffreeze‐driedspermisstillnotwelldefined.

In this study, we performed an accurate morphologic assess‐mentthatallowedtoevidenceanormalmorphologyofspermatozoaaccordingtoFlechon&BustosObregon (1974)andContrerasandFierro(2004).However,somefracturepointswereobservedinspe‐cificregionsofthecells,suchastheneck,middleandprincipalpiecesofthetail.Previousstudiesinpigs(Kwon,Park,&Niwa,2004),rab‐bits(Liuetal.,2004)andmice(Wakayama&Yanagimachi,1998)re‐portedadetachmentofthetailfromtheheadaftertheFDprocessinagreementwithourresults.

Inalltreatments,theneckfracturewasthemostfrequentinjury;theconsequentpresenceof taillessheadsattests that theneck istheweakestpointof thegamete. Incontrast,Martins,Báo,Dode,Correa,andRumpf(2007)reportedalowdegreeofhead–tailsep‐arationinlyophilizedbovinespermatozoa,suggestingagreatersta‐bilityofthisconjunctionpointinthisspecies.Takentogether,theseconflicting results suggest that the fragilityof lyophilized sperma‐tozoaisspecies‐specific, independentlyfromthemediumusedfortheFDprocess.InagreementwithLiuetal.(2004),acertaindegreeofbrokentailswasevidenced,confirmingthatplasmamembraneishighlysusceptibletodamageduetothelossofwaterduringdehy‐dration.Finally,intactspermatozoawerethelargestcategoryinallthethreemediaevaluated,attestingthattheyadequatelyprotectedthe cell structure. Moreover, very few spermatozoa with plasmamembranedamageweredetected,suggestingthatchelatingagentsplusadditionofantioxidant treatmentspreserveDNA integrityaspreviouslyreported(Domingoetal.,2018).

Sperm from EDTA treatment was easily diluted in phosphatebuffer, whereas those from EDTA‐RA and EDTA‐MLT treatmentswerequiteagglutinatedanddifficulttodissolve,evenafterrepeatedpipetting,asobservedbyMartinsetal. (2007). Inagreementwiththeseauthors,ourresultsshowedthatEDTA‐treatedspermatozoaappearedregularlyscatteredonslidesurface,whiletheothersap‐pearedaggregatedinsmallclustersandonlyfewgameteswerefree.

DNAmethylationisanepigeneticmodificationthathasrelevantimportanceduringearlyembryogenesis.Indeed,DNAmethylationiswidely remodelled in embryonic stem cells of several species(Al‐Khtibetal.,2012).Inthegermlinedevelopment,paternalDNAmethylationtractsarecancelledandagainsetupafterwavesofdemethylationandmethylation(O'Doherty&McGettigan,2015).Inhumans,spermDNAmethylation levelhasbeenpositivelyas‐sociatedwithconventionalspermparameters, likeconcentrationandmotility,whereaschromatinfragmentationanddenaturationnegativelyinfluenceDNAmethylation(Montjeanetal.,2015).

OurresultsdemonstratedthattheFDprocessuseddidnotaf‐fecttheoverallDNAmethylationstatusofrabbitsperm.Moreover,supplementationofEDTAaswellasadditionofRAorMLTtostor‐agemediadidnotinfluencethespermatozoamethylationratewith

respecttothefreshmedium.AsreducedDNAintegrityofsperma‐tozoa isoneofthemaincausesoffailureofassistedreproductivetechnologies,thepresenceofstructurallyintactDNAinlyophilizedsamplesisparticularlyrelevant.

Inconclusion,ourstudydemonstratedthatFDofspermatozoawith two different antioxidant supplementations does not affectrabbitmalegametepreservationandcouldbebeneficial for long‐term sperm storage during clinical application research. FurtherstudiesareneededtounderstandthedifficultiesrelatedtotheuseofFDtechniqueinrabbits.

ACKNOWLEDG EMENTS

TheauthorsgratefullyacknowledgethetechnicalassistanceofDr.LindaPetrucci,Dr.GiulioCanonicoandMrsMariaGabriellaMancini.

CONFLIC T OF INTERE S T

Noneoftheauthorshasanyconflictofinteresttodeclare.

AUTHOR CONTRIBUTIONS

FM,MM,PDandLGdevelopedtheidea,designedtheresearchandanalysedtheresults.PD,RP,CD,AD,KFandPCcontributedtotheexperiments.MZcontributedtothestatisticalanalysis.FMandMMwrote themanuscript.CBandMZrevised the finalversionof themanuscript,whichwaseditedandapprovedbyallauthors.

ORCID

Rolando Pasquariello https://orcid.org/0000‐0003‐3191‐7009

Massimo Zerani https://orcid.org/0000‐0002‐2476‐5097

Margherita Maranesi https://orcid.org/0000‐0002‐1727‐4118

DATA AVAIL ABILIT Y

Thedatasetsgeneratedduringand/oranalysedduringthecurrentstudyareavailable fromthecorrespondingauthorson reasonablerequest.

R E FE R E N C E S

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How to cite this article:MercatiF,DomingoP,PasquarielloR,etal.EffectofchelatingandantioxidantagentsonmorphologyandDNAmethylationinfreeze‐dryingrabbit(Oryctolagus cuniculus)spermatozoa.Reprod Dom Anim. 2020;55:29–37. https://doi.org/10.1111/rda.13577

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