259 Original Articles Original articles · (Received: November 21, 2016; accepted: June 6, 2017) doi: 10.17533/udea.rccp.v30n4a01 Abstract Background: The brilliant cresyl blue (BCB)

Rev Colomb Cienc Pecu 2017; 30:259-266

Revista Colombiana de Ciencias Pecuarias

Original articles

259

Original Articles

¤ Tocitethisarticle:MaiaRC,MogolllónEM,DubeibeDF,PenaB,Dias,BurlaAJ.Effectofseasonalityonthequalityofbovineoocytesselectedbythebrilliantcresylbluemethod.RevColombCiencPecu2017;30:259-266.

* Correspondingauthor:ÁngeloJoséBurlaDias.UniversidadeEstadualdoNorteFluminenseDarcyRibeiro,AvenidaAlbertoLamego,2000–ParqueCalifórnia,CCTA,LRMGA,28035-200,CamposdosGoytacazes,RiodeJaneiro,RJ.E-mail:[email protected].

Effect of seasonality on the quality of bovine oocytes selected by the brilliant cresyl blue method¤

Efecto de la estacionalidad en la calidad de los ovocitos seleccionados por el método de azul cresil brillante

Efeito da sazonalidade na qualidade dos ovócitos bovinos selecionados pelo método azul cresil brilhante

RogerCMaia1,MV,MSc;EdgarMMogollónWaltero2,MV,PhD;DiegoFDubeibeMarin2,MV,MSc;BrunoPenaCarvalho3,MSc,DrSc;ÁngeloJBurlaDias1*,MV,PhD.

1Laboratório de Reprodução e Melhoramento Genético Animal, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brasil.

2Laboratorio de Biotecnología de la Reproducción Animal, Universidad Cooperativa de Colombia, Bucaramanga, Colombia.

3Setor de Implementação da Programação de Transferência de Tecnologia, Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Rio Branco, Brasil.

(Received: November 21, 2016; accepted: June 6, 2017)

doi: 10.17533/udea.rccp.v30n4a01

Abstract

Background:Thebrilliant cresyl blue (BCB) staining is a non-invasive test to select thebest-suitedoocytesforembryonicdevelopment.Thismakesitausefultooltoselectbest-qualityoocytesatthetimesoftheyearwhenthereisforagerestriction.Objective: ToevaluatetheeffectofseasonalityonthenuclearmaturationandqualityofoocytesselectedbytheBCBtest.Methods: Thecumulus-oophoruscomplexes(COCs)wereobtainedinsummerandwinterof2010and2011.SelectedCOCsweremaintainedfor90minat38.5°CinaCO2incubator,inTCM199mediumcontaining10%fetalbovineserumandantibiotics,andsupplementedwith26µMbrilliantcresylblue.Afterwards,theyweredividedaccordingtotheooplasmstaining(BCB+—blue;BCB−—unstained).Subsequently,COCswerematured for22h.Nuclearmaturationwasevaluatedat22hofculture.Results: TheproportionofBCB−oocyteswashigherinthewinterof2010,buttherewasnoincreaseinthisgroupinthewinterof2011.ThepercentageofoocytesthatreachedmetaphaseIIwashigherincontrolandBCB+groupsinrelationtooocytesfromBCB−group.Conclusion:Theseasonoftheyearinfluencesthepercentageofoocytesbestsuitedforembryonicproductioninsituationsinwhich

260


Maia RC et al. Season and quality of bovine oocytes

oocytedonors receivepasture-based feeding, since themethodwaseffective indetermining theeffectofseasonalityonthecompetenceofbovineoocytestoreachnuclearmaturation.

Keywords:colorimetric test, cumulus-oophoruscomplexes, in vitro maturation,pasture,seasons.

Resumen

Antecedentes: La tinción con azul cresil brillante (BCB) es unmétodono invasivopara seleccionarovocitosaptosparaeldesarrolloembrionario.Portanto,esunaherramientaútilparaselecionarlosovocitosdemejorcalidadentemporadasderestriccióndeforraje.Objetivo:EvaluarelefectodelaestacionalidadsobrelamaduraciónnuclearycalidaddelosovocitosseleccionadosporeltestBCB.Métodos: Loscomplejos cumulus-oophorus (CCOs) fueronobtenidosdurante el veranoy el inviernode2010y2011.LosCCOsseleccionadossemantuvierondurante90mina38,5oCenunaincubadoradeCO2enunmedioTCM199con10%desuerofetalbovinoyantibióticos,suplementadocon26µMdeazulcresilbrillante.Luegosesepararonsegúnelcolordelcitoplasma(BCB+—azulyBCB−—incoloro).Posteriormente, losCCOssemadurarondurante22h.Laevaluacióndelamaduraciónnuclearserealizóalas22hdecultivo.Resultados: LaproporcióndeovocitosBCB−fuemayorenelinviernode2010,peronohubounaumentodeesegrupoenelinviernode2011.ElporcentajedeovocitosquealcanzaronlaetapademetafaseIIfuemayorenelgrupocontrolyBCB+conrespectoalgrupoBCB−.Conclusión: Laestacióndelañoinfluyeenelporcentajedeovocitosmásaptosparalaproduccióndeembrionesensituacionesdondelasdonadorasdeovocitosrecibenalimentaciónabasedepastos,yaqueestemétodofueeficazparadeterminarelefectodelaestacionalidadenlacompetenciadeovocitosbovinosenalcanzarlamaduraciónnuclear.

Palabras clave: complejos de cumulus-oophorus, estaciones,maduración in vitro, pastura, test colorimétrico.

Resumo

Antecedentes:Ométododoazulcresilbrilhante(BCB)nãoéinvasivoeselecionaovócitosmaisaptosaodesenvolvimentoembrionário.Portantoéferramentaútilparaselecionarovócitosdemelhorqualidadeemépocasdoanoqueocorrerestriçãodepastagem.Objetivo: AvaliaroefeitodasazonalidadesobreamaturaçãonucleareaqualidadedosovócitosselecionadospelotesteBCB.Métodos:Os complexos cumulus oophorus(CCOs)foramobtidosnoverãoeinvernode2010e2011.OsCCOsselecionadosforammantidospor90min,a38,5°C,emincubadoradeCO2,emmeioTCM199contendo10%desorofetalbovinoeantibióticos,esuplementadocom26µMdeazulcresilbrilhante.Emseguida,estes foramdivididosdeacordocomacoloraçãodocitoplasma(BCB+—azuiseBCB−—incolores).EntãoosCCOsforammaturadosdurante22h.Aavaliaçãodamaturaçãonuclearfoirealizadaàs22hdecultivo.Resultados: AproporçãodosovócitosBCB−foimaiornoinvernode2010,masnãohouveaumentodessegruponoinvernode2011.OpercentualdeovócitosqueatingiuoestágiodemetáfaseIIfoimaiornocontroleenogrupoBCB+emrelaçãoaogrupoBCB−.Conclusão:Aestaçãodoanoinfluenciaopercentualdeovócitosmaisaptosaprodução deembriões,emsituaçõesondeasdoadorasdeovócitosrecebemalimentaçãobaseadaempastagens,jáqueométodosemostrouefetivoparadeterminaçãodoefeitodasazonalidadesobreacompetênciadeovócitosbovinosematingiremamaturaçãonuclear.

Palavras chave: complexos de cumulus-oophorus,estações do ano, maturação in vitro, pastagem, teste colorimétrico.

Introduction

Bovine in vitro embryo production (IVP) hascontributed to develop the Brazilian cattle herd(Rodrigues, 2014). However, the percentage ofoocytes that reach theblastocyst stage is low (35-45%).Evidence suggests the oocyte quality is themainfactorforblastocystyield,thusthebestoocytesshouldbeselected(LonerganandFair,2016).

The criteria for cumulus-oophorus complexes(COCs)selectionhavebeenbasedonmorphology,notonthecompleteevaluationofoocytecompetence(Van Den Hurk and Zhao, 2004). Therefore,several studies have been looking formethods todemonstrateoocytecompetence inanon-invasivemanner. Currently, only invasivemethods thatdamagetheoocytecanpredictitsquality,precludingitsuseforIVP.

261



The search for non-invasive biomarkerswasreviewedinanearlierpaper(Goovaertset al.,2010).Thebrilliantcresylblue (BCB)staining isoneofthesemethods. It determinesglucose-6-phosphatedehydrogenase enzyme (G6PDH) activity,whichis increased in growing oocytes and decreased infull-grownones(Rodriguez-Gonzaleset al.,2002).Thereby, oocyteswith lower enzymatic activitydo not degradeBCB, turning blue (BCB+), andthus have greater developmental competence.Furthermore, researchers have identified genesthatcouldbeusedasbiomarkersofoocytequality(O’Shea et al., 2012).Markers of oocyte qualitywere proposed in oocytes selected byBCB,withBCB+ oocytes showing different gene expressionprofile(Ashryet al.,2015).Severalauthors(Almet al., 2005;Wu, 2007;Mirshamsi et al., 2013)have evaluated selection of oocytes viaBCB. Inthesestudies,theyconcludedthatoocytescouldbestainedbyBCBwithoutchangingtheircompetenceand favorable results are obtained in selectionof competent oocytes, when associated withmorphologicalselection.

SomeBrazilianregionshavemarkedseasonality,characterizedbytemperatureandhumidityvariationsthatimposedifferentenvironmentalandnutritionalstandards to cattle throughout the year (Nicolosoet al.,2006).Studiesrelatingseasonalitytooocytequality have focused onmorphological analysis,orusedharmfulsubstances,whichimpedeoocytefertilization (Castaneda et al., 2013). Thus, thepresentstudyadoptedBCBstainingtorelatebovineoocytematurationcapacitywithseasonalvariations.Although studies have demonstrated the effect ofseasonsonfeedrestrictionandcattlereproduction,tothebestofourknowledgethisisthefirstattemptofusingthismethodtoselectoocytesthataremorecompetentforIVPindifferentseasons,sinceBCB+oocyteshaveahighermaturationrate.

Materials and methods

Location

TheexperimentwasconductedattheLaboratóriodeReprodução eMelhoramentoGenéticoAnimalNorthernRiodeJaneiro(UENF),locatedinCamposdosGoytacazes,RJ,Brazil.

Climatic conditions

TheanimalsoriginatedfromtheNorthernregionofRiodeJaneiro,wheretheclimateistropicalwet-dry(Aw)accordingtheKöppenclassification,witharainysummerandadrywinter.Theannualaveragetemperature is 24 oC.Thedriest quarter occurs inthe June/July/August period and the rainy seasoninDecember/January/February, being the annualaveragerainfall1,055mm(Mendonçaet al.,2012).Insummer(DecembertoMarch)thedaysarelongerthanthenightsandthesolarradiationfavorstherisein temperature,precipitationand relativehumidity.Winter (June toSeptember)has the lowest rainfallindexandthelowest temperatures.Ingeneral,rainisfivetimeslowerinwintertimeandairhumidityislow(Emidio,2016).

Collection and morphological selection of COCs

TheCOCs recovered fromovaries obtained atslaughterhousesinthecityofCamposdosGoytacazes(21°45’15”S latitude,41°19’28”Wlongitude)weretransportedtothelaboratoryinthermalbottles,wheretheovarieswerewashedinsalinesolution(0,9%NaCl)withantibiotics(100IU/mLpenicillinand100µg/mLstreptomycin).Ovarianfolliclesof3to8mmindiameterwereaspiratedandthefollicularfluidwasplacedina50-mLconicaltube.Aftersedimentation,thepelletwastransferredtoaPetridish(Corning,100×20mm)with3-5mLofhandlingmedium[199withEarle’ssaltsplus25mMHepesandantibiotics (100 IU/mLpenicillinand100µg/mLstreptomycin)supplementedwith10%fetalcalfserum(FCS)]tosearch,retrieve,andclassifytheoocytes.OnlydegreeIandIIoocytes(Looset al.,1989)—withthreeormorecompactlayersofcumuluscellsanduniformcytoplasm—wereused.

The experimentwas conducted in the summerandwinterof2010and2011.In2010,thestudywascarriedoutinJanuary,February,March,June,July,andAugust,whilein2011ittookplaceinFebruary,March,June,andAugust.

Selection of COCs by BCB test

After selection, COCs were incubated inincubationsolution(199mediumsupplementedwithbicarbonatewithantibioticsand5%FBS)containing26µMofBCB(860867,Sigma-AldrichBrasilLtda,SãoPaulo,Brazil).

262



TheCOCsweremaintained for 90min in 5%CO2,at38.5°C(Manjunathaet al.,2007).OocytesincubatedwithBCBwere separated according tothecytoplasmstaining:Oocyteswithbluestaininginthecytoplasm(BCB+)andoocyteswithcolorlesscytoplasm(BCB−).TheCOCsinthecontrolgroupwerekeptunderthesameconditionsexceptfortheabsence ofBCB.Next,COCswerewashed twicein PBS and placed in the incubator for in vitro maturation.

Oocyte culture

TheCOCswere transferred to 100 µL dropsofmaturationmedium[199mediumwithEarle’sSalts (M-5017),plus20mMsodiumbicarbonatesolution (S6297), 5.0 mg/mL LH (Lutropin-V,Bioniche, Beleville, Canada), 0.5 µg/mL FSH(Folltropin-V,Bioniche,Belevile,Canada),0.2mMpyruvate(P4562),100IU/mLpenicillin(P3032),and 100 µg/mL streptomycin (S1277). Twentyoocytesperdropremainedundermineraloil, for22h,at38.5°C,in5%CO2.

Evaluation of oocyte maturation

Todeterminetheeffectofseasonalityonnuclearcompetence of oocytes, the meiosis stage wasevaluatedat22hofmaturation.AfterIVM,COCswere denudedmechanically by pipetting in PBSsolution (0.01g/mLPVA), subsequentlyplacedonslideandcoverslipsandfixedfor72hinaceticacidandethanol(1:3,v:v).Afterwards,thematerialwasstainedwith2%aceticorcein.Oocyteswereevaluatedbylightmicroscopy.Thestagesofnuclearmaturationwere defined according to Hewitt and England(1997),basedonthemorphologyofchromatin,suchasgerminalvesicle(GV,prophaseIstage),germinalvesiclebreakdown(GVBD),metaphaseI(MI),andmetaphaseII(MII).

Experimental design

Experiment one. TheCOCswere incubated intheBCBsolutionandthendividedintotwogroupsaccording to the stainingof cytoplasm.TheCOCsfromcomplexesfromcontrolgroupunderwent thesametreatmentastheothers,exceptfortheabsenceoftheBCBintheincubationmedium.Thosewithblue

cytoplasmwereclassifiedasBCB+,andthosewithcolorlesscytoplasmwereclassifiedasBCB−.Thus,theproportionofthesetwogroupswasdeterminedindifferentseasonsof2010and2011.

Experiment two.TheexposureofCOCstoBCBandthetreatmentofcontrolgroupwereperformedasdescribedinExperimentI.AfterselectionofCOCsbythecytoplasmstaining(BCB+,BCB−,andcontrol)theywere set for in vitromaturation for 22 h andsubsequentlyevaluatedfornuclearmaturationstage.

Statistical analysis

The rates of oocytes matured in differenttreatmentsandtheproportionsofBCB+andBCB−oocytes in summerandwinterwerecomparedbytheChi-squaretestat95%confidencelevel.AlldatawereanalyzedusingBioEstat5.0software(2007).Four replicateswereused ineachseasonof2010and2011 toevaluate theproportionofBCB+ andBCB−oocytes, andfive replicates to evaluate thenuclearmaturationrate.

Results

Proportion of BCB+ and BCB− oocytes

Results showed a higher percentage (p<0.05)ofBCB+ oocytes obtained in the summerof 2010comparedwith thoseobtained in thewinter of thesame year.Additionally, the percentage ofBCB−oocyteswaslower(p<0.05)inthesummercomparedwiththewinter(Table1).

Table 1. Percentage of BCB+ and BCB− oocytes obtained in summer and winter of 2010.

BCB+ n (%) BCB− n (%)

Summer 142/250 (56.8)a 108/250 (43.2)a

Winter 109/240 (45.4)b 131/240 (54.6)b

Values followed by different superscripts in the same column differ significantly by the chi-square test (p<0.05).

In the year 2011, there were no differences(p>0.05)betweenthepercentagesofBCB+orBCB−oocytesobtainedinsummerandwinter(Table2).

263



Table 2. Percentages of BCB+ and BCB− oocytes obtained in summer and winter of 2011.

BCB+ n (%) BCB− n (%)

Summer 122/207 (58.9) 85/207 (41.1)

Winter 112/219 (51.1) 107/219 (48.9)

Data were analyzed by the chi-square test at the 5% significance level.

Evaluation of nuclear maturation in different seasons

WealsoevaluatedtheeffectofseasonalityontheabilityofoocytesselectedbytheBCBmethodtoreachMIIafter22hofin vitromaturation.Datarelatedtonuclearmaturationofdifferentexperimentalgroupsin summer andwinter of 2010 and2011werenotsignificantlydifferent(Tables3and4).

Table 3. Percentages of BCB+ bovine oocytes matured in vitro that reached metaphase II during the summer and winter of 2010 and 2011.

2010 n (%) 2011 n (%)

Summer 96/123 (78.0) 85/107 (79.4)

Winter 56/76 (73.7) 75/94 (79.8)


Table 4. Percentages of BCB− bovine oocytes matured in vitro that reached metaphase II during the summer and winter of 2010 and 2011.

2010 n (%) 2011 n (%)

Summer 38/80 (47.5) 35/75 (46.6)

Winter 54/113 (47.8) 45/96 (46.8)


Evaluation of nuclear maturation in BCB+ and BCB− oocytes

ThepercentageofmetaphaseIIinBCB−oocyteswaslower(p<0.05)thanthoseofcontrolandBCB+

groups(Tables5and6).

Table 5. Meiotic configuration of bovine oocytes matured in vitro obtained during the summer and winter of 2010.

Summer - MII n (%) Winter - MII n (%)

Control 71/102 (69.6)a 69/104 (66.3)a

BCB+ 96/123 (78)a 56/76 (73.8)a

BCB− 38/80 (47.5)b 54/113 (47.8)b

1MII: Metaphase II. Values followed by different superscripts in the same column differ significantly by the chi-square test (p<0.05).

Table 6. Meiotic configuration of bovine oocytes matured in vitro obtained during the summer and winter of 2011.

Summer - MII n (%) Winter - MII n (%)

Control 79/98 (80.6)a 69/98 (70.4)a

BCB+ 85/107 (79.4)a 75/94 (79.8)a

BCB− 35/75 (46.6)b 45/96 (46.8)b

1MII: Metaphase II. Values followed by different superscripts in the same column differ significantly by the chi-square test (p<0.05).

Discussion

Successinin vitrobovineembryoproductionisdirectly related to thequalityof thegametesused,whichislargelyinfluencedbynutrition(Saleset al.,2015).Ingeneral,oocytedonorsreceiveacontrolleddietwithout significant changes in quantity andcomposition throughout the year,whichmakes ita negligible factor in the variation of the process.However, in vitro embryoproduction fromovariesobtained from slaughterhouses is dependent upontheforagesupply,whichmaybeinfluencedbytheeffectsofseasonalityonforageproduction.(SartoriandGuardieiro,2010).

Due to its extension, Brazil has pronouncedclimaticvariabilityacrossregions.Theregionsnearestto the equator have lower temperature variationsthroughouttheyear,butseasonalityismainlyduetothevolumeanddistributionofrainfallduringtheyear(Sansigoloet al.,2010).Therefore,duetothehighspatial variability in the rainfall regime these datashouldbevalidated in other regions and countries(Alvareset al.,2013).

To evaluate the effect of seasonality on thequalityofbovineCOCs,weusedBCB,avitalstain,substrate fromglucose-6-phosphatedehydrogenase(G6PD).TheBCBhasbeenusedasanoninvasivemethodfortheselectionofoocytesthatreachastageofdevelopmentconsistentwith theabilityof thesegametestobefertilizedandreachembryonicandfetaldevelopment.Glucose-6-phosphate dehydrogenaseisactiveingrowingoocytes,buthaslowactivityinthoseoocytesthathavecompletedthisphase.Sincethe latter have a lowG6PD activity, they do notdegradetheBCBandturnblue(BCB+;Almet al.,2005).OocytesconsideredBCB−,bycontrast,haveahigherG6PDactivityandthusdegradethestain,

264



remaining colorless.These oocytes have a lowerabilitytocompletematurationandtobefertilizedin vitrosincetheyarestillgrowingandhaveasmallerpool ofmRNA,proteins, lipids, and sugars (Motaet al.,2009).

Oocytematurationisacrucialeventcomprisingtheprogressionofmeioticcycle.ThemajorparametertoknowoocytehasachievednuclearmaturationisameioticconfigurationofMII(Fulkaet al.,1998).In this stage oocytes canbe fertilized and supportembryonicdevelopment(Smitzet al.,2004).

Inthisstudy,valuesfoundduringthesummerofthetwoyearsassessedaresimilartothosedescribedin 2005byAlmet al. (57.9%BCB+) and in 2009byMotaet al.(60.37%BCB+),whousedtheBCBmethodtoselectmorecompetentoocytesforIVM.The total annual rainfall in themunicipality ofCamposdosGoytacazesin2010and2011was597and739mm(EvapotranspirationStationatCCTA/UENF—unpublished data), respectively.Thesevolumeswerelowerthanthehistoricalannualaveragerainfallvolume(1,055mm).

Despitethelowvolumeofrainfallinthetwoyearsunderstudy,in2011,therainfallwasslightlyhigherthan in thepreviousyear.This situationmayhaveprovidedagreatersupplyofforagetoanimalsinthedryseason,whichmayhaveincreasedthepercentageofBCB+oocytesobtainedinthewinter.

Ovarieswereobtainedfromcowsfromthenorthregion ofRio de Janeiro State, inwhich the useof pastures for cattle feedingpredominates.Thesepastures are subject to seasonal variations,withmaximumyield occurring in the rainy season anda significant decline in production during the dryseason.(Euclideset al.,2007;Teixeiraet al.,2011).

Thus, cattle are not feed uniformly during theyear, and this affects reproductive characteristics,since folliculargrowth,maintenanceofpregnancy,andestrouscyclicityarelow-priorityactivitiesonanutrient-distribution scale andonlyworknormallywhenfunctionslikegrowthandnutrientreservesaremaintained(Zeronet al.,2001).

In thiswork, BCB− oocytes showed a lowernuclearmaturationratecomparedwithcontroland

BCB+groups(Tables5and6).AstheprogressionofoocytemeiosistometaphaseIIisarequirementforfertilizationandsubsequentembryonicdevelopment,thesedatasupportthehypothesisthatBCB−oocytesarelessabletodevelop,reinforcingthefactthattheBCBmethodisanimportanttooltoselectoocytesforin vitroembryoproduction.WefoundnodifferenceinmaturationratebetweenBCB+andcontroloocytes,proving the lack of deleterious effect fromBCBexposureandreinforcingthefactthatBCBisavitaldye(Rodríguez-Gonzálezet al.,2002).

In this regard,Ashryet al. (2015)demonstratedthat abundanceofoocytegene transcripts related tomaturation and proper development (such as JY-1andBMP15)washigher inBCB+oocytescomparedwithBCB−.Ontheotherhand,BCB−COCsshowedincreased expression of cathepsins B, S, and Zofcumulus cells.Thus, the authors correlated theabundanceoftranscriptsofthesegeneswithBCB+andBCB−oocytestoprovethattheBCBmethodcanbeusedtoacquiremorecompetentoocytes(Ashryet al.,2015).Moreover,theuseofthiscolorimetricmethodinoocytesandzygotesincreasesthepossibilityofpredictingtheearlyembryonicdevelopmentpotential,sinceoocyteswithbluecytoplasmandunstainedzygotessignificantlyincreaseembryoproduction(Mirshamsiet al.,2013).

ThesedataconfirmtheefficacyoftheBCBtestin selecting oocytes able to undergomaturation.Oocytesobtainedfromprepubertalgoats(Rodriguez-Gonzalezet al.,2002)andfromswine(Wongsrikeaoet al.,2006)haveshown increasedmaturationandfertilizationratesofBCB+oocytesinrelationtoBCB−andcontrol.However,Almet al.(2005)foundsimilarnuclearmaturation rates betweenBCB+ oocytesandthecontrolgroupincattle.Nevertheless,BCB+oocytes resulted in a significantly higher (p<0.05)blastocyst rate (34.1%) thanBCB− (3.9%)and thecontrol(18.3%)groups.

DatafromTables3and4indicatethatthenuclearmaturationratesofBCB+andBCB−oocyteswerenotaffectedbyseasonality,buttheeffectofseasonalityon theproportionof theseoocytes obtained in thesummerandwinterwasremarkable.Adamiaket al.(2005),demonstratedthatfeedingdonorstwicetheirnutritionalrequirementsformaintenanceincreasesthenumberoffolliclesfrom4to8mmandthenumber

265



ofembryosproduced.Bycontrast,malnutritionduetoseasonal issueshasnegative implicationson theresumptionofmeiosis,numberoffollicles,andoocytequality(Santoset al.,2008).

In thiswork,mainly cattle ofBos primigenius indicus breeds such asNellorewere used.Theseanimals aremore resistant to tropical conditions(Wolfensonet al.,2000) likehightemperatureandhumiditythanbreedsevolvedinEurope(e.g.AngusandHolstein;Hansen, 2004; Paula-Lopes et al.,2003).TheovarieswerecollectedatslaughterhousesinCampos dosGoytacazes (RJ,Brazil)where thetropical conditions are intense.Thus, the effect ofseasonality influenced by nutrition becomesmoreevidentthanthethermalstressduringsummer.

In viewof the above considerations, it can beconcludedthattheseasoncaninfluencethepercentageofoocytesbestsuitedforin vitroembryoproductionin situations inwhich oocyte donors receive feedexclusively on pasture.Additionally, the brilliantcresylbluemethodiseffectivetodeterminetheeffectofseasonalityonthecompetenceofbovineoocytestoachievenuclearmaturation.

Acknowledgments

The authors thank the Carlos Chagas FilhoFoundationforResearchSupportatRiodeJaneiroState(FAPERJ)forfinancialsupport.

Conflict of interest

Theauthorsdeclaretheyhavenoconflictsofinterestwithregardtotheworkpresentedinthisreport.

ReferencesAdamiakSJ,MackieK,WattRG,WebbR,SinclairKD.Impactof nutrition on oocyte quality: Cumulative effects of bodycompositionanddietleadingtohyperinsulinemiaincattle.BiolReprod2005;73:918-926.

AlmH,TornerH, LohrkeV,Ghone IM,KanitzW.Bovineblastocystdevelopment rate in vitro is influencedbyselectionofbybrilliantcresylbluestainingbeforeIVMasindicatorforglucose-6-phosphate dehydrogenase activity.Theriogenology2005;63:2194-2205.

AlvaresCA,StapeJL,SentelhasPC,GonçalvesJLM,SparovekG.Köppen’sclimateclassificationmapforBrazil.MeteorologischeZeitschrift2013;22(6):711-728.

AshryM, LeeK,MondalM,DattaTK, Folger JK,RajputSK,ZhangK,HemeidaNA, SmithG. Expression ofTGFβsuperfamilycomponentsandothermarkersofoocytequalityinoocytesselectedbybrilliantcresylbluestaining:Relevanceto early embryonic development.Mol Repro Dev 2015;82(3):251-264.

CastanedaCA,KayeP,PantaleonM,PhillipsNC,NormanS,FryR,D’occhioM.Lipidcontent,activemitochondriaandbrilliantcresyl blue staining in bovine oocytes.Theriogenology2013;79:417-422.

DeLoosF,VanVlietC,VanMaurickP,KruipTA.Morphologyofimmaturebovineoocytes.GameteRes1989;24:197-204.

EuclidesVPB,MacedoMCM, ZimmerAH,Medeiros RN,OliveiraMP.Característicasdopastodecapim-tanzâniaadubadocomnitrogênio no final do verão. PesqAgropecBras 2007;42(8):1189-1198.

EmidioZPO.EstaçõesdoAno.Ipmet2016;[Accessdate:May10th,2017].URL:https://www.ipmet.unesp.br/4estacoes/#

FerreiraAM.Nutriçãoeatividadeovarianaembovinos:Umarevisão.PesqAgropecBras1993;28:1077-1093.

Fulka JJr, First NL,Moor RM. Nuclear and cytoplasmicdeterminants involved in the regulationofmammalianoocytematuration.MolHumReprod1998;4:41-49.

GoovaertsIGF,LeroyJLMR,JorsenEPA,BolsPEJ.Noninvasivebovineoocytequalityassessment:Possibilitiesofasingleoocyteculture.Theriogenology2010;74:1509-1520.

HansenPJ.Physiologicalandcellularadaptationsofzebucattletothermalstress.AnimReprodSci2004;82:349-360.

LonerganP,FairT.Maturationofoocytein vitro.AnnuRevAnimBiosci2016.4:255-268.

ManjunathaBM,GuptaPSP,DevarajM,Ravindra JP,NandiS.SelectionofdevelopmentallycompetentbuffalooocytesbybrilliantcresylbluestainingbeforeIVM.Theriogenology2007;68:1299-1304.

MirshamsiSM,KaramishabankarehH,AhmadI-HamedaniM,SoltaniL,HajarianH,AbdolmohammadiAR.Combinationofoocyteandzygoteselectionbybrilliantcresylblue(BCB)testenhancedpredictionofdevelopmentalpotentialtotheblastocystincattle.AnimReprodSci2013;136:245-251.

MotaGB,BatistaRITP,SerapiãoRV,BoitéMC.Developmentalcompetence and expression ofMATER and ZAR1 gene inimmaturebovineoocytesselectedbybrilliantcresylblue.Zygote2009;26:1-8.

NicolosoRS,LanzanovaME,LovatoT.Manejodaspastagensde inverno e potencial produtivo de sistemas de integraçãolavoura-pecuárianoestadodoRioGrandedoSul.CiencRural2006;36(6):1799-1805.

266



Paula-LopesFF,ChaseCCJR,Al-KatananiYM,KriningerCE,RiveraRM,TekinS,MajewskiAC,OconOM,OlsonTA,HansenPJ.Geneticdivergenceincellularresistancetoheatshockincattle:Differencesbetweenbreedsdevelopedintemperateversushotclimatesinresponsesofpreimplantationembryos,reproductivetissuesandlymphocytestoincreaseculturetemperatures.Reprod2003;125:285-294.

Mendonça JC, FreitasRM, ShimabukuroYE,MarquesVS.AvaliaçãodeeventosdeinundaçãonaRegiãoNorteFluminense,RiodeJaneiro,utilizandoimagensdesensoresremotos.Ambi-Água2012;7:255-267.

O’SheaLC,MehtaJ,LonerganP,HenseyC,FairT.Developmentalcompetenceinoocytesandcumuluscells:Candidategenesandnetworks.SystBiolReprodMed2012,58:88-101.

Rodríguez-GonzalezE,Lopez-BejarM,VelillaE,ParamioMT.Selectionofprepuberal goatoocytesusing thebrilliant cresylbluetest.Theriogenology2002;57:1397-1409.

RodriguesCFM.Historical context of cattle embryo transfertechniqueinBrazil.AnimReprod2014;11(3):137-140.

SalesJN,IgumaLT,BatistaRI,QuintãoCC,GamaMA,FreitasC,PereiraC,CamargoLS,VianaJH,SouzaJC,BaruselliPS.Effectsofahigh-energydietonoocytequalityandin vitroembryoproductioninBos indicusandBos Tauruscows.JDairySci2015;98:3086-3099.

SansigoloCA,KayanoMT.TrendsofseasonalmaximumandminimumtemperaturesandprecipitationsinSouthernBrazilforde1913-2006period.TheorApplClimatol2010;101:209-216.

SantosJ,CerriR,SartoriR.Nutritionalmanagementofthedonorcow.Theriogenology2008;69:88-97.

SartoriR,GuardieiroMM.Fatores nutricionais associados àreproduçãodafêmeabovina.RBrasZootec2010;39:422-432.

SmitzJEJ,NogueiraD,VanhoutteMatosDG,CortvrindtRN.Oocyte: In vitromaturation. In:SuhCS,SonntagB,EricksonGF.Theovarianlifecycle:Acontemporanyview.RevEndocrMetabDisord2004;3:5-12.

TeixeiraFA,BonomoP,PiresAJV,SilvaFF,FriesDD,HoraDD.ProduçãoanualequalidadedepastagemdeBrachiaria decumbens diferidaeestratégiasdeadubaçãonitrogenada.ActaSciAnimSci2011;33:241-248.

VanDenHurkR,Zhao J. Formation ofmammalian oocytesandtheirgrowth,differentiationandmaturationwithinovarianfollicles.Theriogenology2004;63:1717-1751.

WolfensonD,RothZ,MeidanR.Impairedreproductioninheatstressedcattle:Basicandappliedaspects.AnimReprodSci2000;60:535-547.

WongsrikeaoP,OtoiT,YamasakiH,AgungB,TaniguchiM,NaoiH,ShimizuR,NagaiT.Effectsofsingleanddoubleexposuretobrilliantcresylblueontheselectionofporcineoocytesforin vitroproductionofembryos.Theriogenology2006;66:366-372.

WuY-G,LiuY,ZhouP,LanG-C,HanD,MiaoD-Q,TanJ-H.Selectionofoocytes for in vitromaturationbybrilliantcresylbluestaining:Astudyusingthemousemodel.CellRes2007;17:722-731.

ZeronY,OcheretnyA,KedarO,BorochovA,SklanD,AravA. Seasonal changes in bovine fertility:Relation to oocytedevelopmental competence,membrane properties and folliclefattyacidcomposition.Reprod2001;121:447-454.

Documents

259 Original Articles Original articles · (Received: November 21, 2016; accepted: June 6, 2017) doi: 10.17533/udea.rccp.v30n4a01 Abstract Background: The brilliant cresyl blue (BCB)