6th International Workshop on the Biology of Fish Gametes · 6th international Workshop on the...

6th International Workshop on the Biology of Fish Gametes

September 4th–7th, 2017Vodňany, Czech Republic

Jihočeská univerzita

University of South Bohemiain České Budějovice

Fakulta rybářství

6th International Workshop on the Biology of Fish GametesEdited by: Martin Pšenička, Michaela Fučíková, Zuzana DvořákováPublished by: University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Zátiší 728/II, 389 25 Vodňany, Czech RepublicGraphic Design & Technical realisation: Profitisk GroupPrinting: 120 pcs.Edition: 1st

ISBN 978-80-7514-056-2© University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters

Scientific Committee

External Scientific Committee:Rüdiger Schulz – University of Utrecht, Faculty of Science, the Netherlands

Pascal Fontaine – University of Lorraine, FranceJulien Bobe – INRA, France

Jean-Jacques Lareyre – INRA, FranceLuiz Renato de Franca – National Institute of Amazonian Research, Brazil

Igor Babiaka – Nord University, Faculty of Biosciences and Aquaculture, Norway

Catherine Labbe – INRA, FranceJuan F. Asturiano – Universidad Politécnica de Valencia, Spain

Ákos Horváth – Szent Istvan University, Faculty of Agricultural and Environmental Sciences, Hungary

Oliana Carnevali – Universita Politecnica delle Marche, Dipartimento di Scienze della Vita e dell’Ambiente, Italy

Arai Katsutoshi – Hokkaido University, JapanHamid Habibi – University of Calgary, Canada

Internal Scientific Committee:Otomar LinhartMartin Pšenička

Jacky CossonBorys Dzyuba

Sergii BoryshpoletsHana Kocour-Kroupová

Taiju Saito

Organizing Committee:Martin Pšenička – workshop convener

Klára Nachlingerová, Eva Prášková, Zuzana Dvořáková, Michaela Fučíková, Vojtěch Kašpar, Lukáš Vlk, Marek Rodina, Roman Franěk, Hilal Guralp,

Viktoriia Iegorova, Effrosyni Fatira, Rasheed Baloch, Xuan Xie

ContactUniversity of South Bohemia in České Budějovice

Faculty of Fisheries and Protection of WatersZátiší 728/II, 389 25 Vodňany, Czech Republic

Telephone: +420 387 774 601Fax: +420 387 774 634

e-mail: [email protected]

WELCOME AT the 6th International Workshop on the Biology of Fish Gametes

Dear friends and colleagues,

as a continuity of the tradition of a series of workshops gathering the community of fish gametes biologists, we are very glad to welcome you on the 2017 event that is held in České Budějovice and Vodňany (Czech Republic).

This event is the 10 years anniversary of the first meeting previously held in Vodňany in 2007, followed by the workshops organized in 2009 (Valencia, Spain), 2011 (Buda-pest, Hungary), 2013 (Faro, Portugal) and 2015 (Ancona, Italy).

The topics follow the previous tasks of the workshop addressing all aspects of fish reproductive biology. Particularly sperm and eggs development and quality; germ cells related biotechnological approaches; gametes omics from genomics, transcriptomics to proteomics; and influence of anthropogenic contaminants on fish gametes.

We’ve received exactly 100 registrations from 23 countries requested 58 oral and 60 poster presentations. The organizers adapted the program to satisfy all participants.

Thanks are due to organizers, scientific committee and all participants of the 6th IW-BFG for enthusiastic preparation of this event and to South Bohemia University in Čes-ké Budějovice and South Bohemian Region for their financial and promoting support.

We wish you a nice time spent in South Bohemia and we hope that the workshop will be a real opportunity for an in-depth exchange of experience, for meeting old friends, as well as to establish new professional contacts.

Martin Pšenička and Otomar Linhart

PROGRAMME AT A GLANCE

Sunday, September 3rd

17:00 – 20:00 Registration

Monday, September 4th (in hotel Clarion České Budějovice)

08:00 – 09:00 Registration09:00 – 09:10 Welcome speech09:10 – 11:10 Oral presentations, session I. Spermatogenesis and sperm quality11:10 – 11:30 Coffee break11:30 – 13:10 Oral presentations, session I. Spermatogenesis and sperm quality13:10 – 14:10 Lunch14:10 – 15:50 Oral presentations, session I. Spermatogenesis and sperm quality15:50 – 16:10 Coffee break16:10 – 17:10 Oral presentations, session I. Spermatogenesis and sperm quality17:10 – 18:50 Poster session I. 19:00 – ? Welcome beer

Tuesday, September 5th (in hotel Clarion České Budějovice)

09:00 – 11:00 Oral presentations, session II. Oogenesis and egg quality11:00 – 11:20 Coffee break11:20 – 13:40 Oral presentations, session III. Germ cells: from basic sciences to applied

biotechnologies13:40 – 14:40 Lunch14:40 – 16:20 Oral presentations, session III. Germ cells: from basic sciences to applied

biotechnologies16:20 – 16:40 Coffee break16:40 – 18:00 Oral presentations, session III. Germ cells: from basic sciences to applied

biotechnologies18:00 – 19:50 Poster session II.

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Programme

Wednesday, September 6th (in Vodňany)

08:00 DeparturefromhotelClariontoVodňanyforexcursionoflocalfacilities(bus1)10:40 DeparturefromhotelClariontoVodňany(bus2)08:45–11:20 ExcursioninVodňany11:20 – 11:40 Coffee break11:40 – 14:00 Oral presentations, session IV. Gametes „Omics“14:00 – 15:00 Lunch15:00 – 17:00 Oral presentations, session V. Gamete Storage and Preservation17:00 – 17:20 Coffee break17:20 – 19:00 Oral presentations, session V. Gamete Storage and Preservation19:00–? Dinner(shuttleservicewillbeoperatingfrom21:00untiltheend)

Thursday, September 7th (in hotel Clarion České Budějovice)

09:00–11:20 Oralpresentations,sessionVI.Anthropogeniccontaminantsandfishgametes11:20 – 11:40 Coffee break11:40 – 12:00 Closing ceremony12:30 – 13:30 Lunch13:45 Busdeparture–guidetourinČeskýKrumlovwithdinner21:00 Coming back to the hotel Clarion

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SCIENTIFIC PROGRAMME

Sunday, September 3rd (in hotel Clarion České Budějovice)

17:00 – 20:00 Registration

Monday, September 4th (in hotel Clarion České Budějovice)

08:00 – 09:00 Registration09:00 – 09:10 Welcome speech (Hotel Clarion) SESSION I. SPERMATOGENESIS AND SPERM QUALITYChairmen: Jacky Cosson, Otomar Linhart

09:10 – 09:50 Plenary lecture – Rüdiger Schulz,DiegoCrespo,DiegoSafian,RobertoMorais, Luiz Assis, Eva Andersson, Geir Lasse Taranger, Anna Wargelius, Jan Bogerd

EndOCRInE And PARACRInE REGuLATIOn Of zEBRAfISh SPERMATOGEnESIS p. 24

09:50 – 10:10 KaoruYoshida,KogikuShiba,JunpeiIkenaga,KazuoInaba,andManabu Yoshida

SPERMCHEMOTAXISISMEDIATEDBYCALCIUMEFFLUXVIAPLASMAMEMBRANE-TYPECALCIUMPUMP p.25

10:10 – 10:30 Beata I. Cejko,BeataSarosiek,SławomirKrejszeff,RadosławK.Kowalski SEASONALDIFFERENCESINSPERMQUALITYPARAMETERSOFTHE

COMMOn CARP Cyprinus Carpio L. p. 26

10:30 – 10:50 Vitaliy Kholodnyy, Jacky Cosson, Serhii Boryshpolets dOES OVARIAn fLuId AffECT ThE fERTILIzATIOn In fRESh WATER

fISh? p. 27

10:50 – 11:10 Carina Caldeira, Borys dzyuba, Boryshpolets Serhii, daznia Bompart, Jacky Cosson, Carles Soler

ISOSMOTICPRESSURETHEONLYFACTORTOINDUCESPERMMOTILITYONCOMMONCARP? p.28

11:10 – 11:30 Coffee break

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Programme

11:30 – 11:50 Víctor Gallego, J. Germán herranz-Jusdado, Christoffer Rozenfeld, david S. Peñaranda, Luz Pérez, Juan f. Asturiano

EUROPEANEEL(anguilla anguilla)MALESREPRODUCTIVEPERfORMAnCE duRInG InduCEd SEXuAL MATuRATIOn: EffECT Of dIffEREnT hORMOnAL TREATMEnTS p. 29

11:50 – 12:10 AlexandraDepincé,ChloéBertin,Catherine Labbé EPIGENETICSTABILITYINGOLDFISHSPERMUPONHORMONAL

InduCTIOn And OVER ThE BREEdInG SEASOn p. 30

12:10 – 12:30 José Beirão, Margaret A. Litt, Craig f. Purchase CHEMICALLYDISPERSEDCRUDEOILAFFECTSSPERMFERTILIZING

ABILITYBUTNOTSPERMSWIMMINGINCAPELIN(Mallotus villosus) p. 31

12:30 – 12:50 Ping Li,WeiGuo,HuameiYue,ChuangjuLi,HaoDu,XinmeiQiao,ZhigangLiu,ZhouQiongandQiweiWei,xi

PREDICTIVEBIOMARKERSOFSPERMQUALITYINSTURGEONSPERMATOzOA p. 32

12:50 – 13:10 Marco Graziano, Craig f. Purchase NON-NEWTONIANPHYSICALPROPERTIESOFOVARIANFLUIDMAY

BEIMPORTANTFORPOST-COPULATORYCRYPTICFEMALECHOICEINATLANTICSALMON(salMo salar) p.33

13:10 – 14:10 Lunch

14:10 – 14:30 Borys dzyuba, Marc Legendre, Jean françois Baroiller, Jacky Cosson SPERMMOTILITYOFTHENILETILAPIA(oreoChroMis nilotiCus):

EffECTS Of TEMPERATuRE On ThE SWIMMInG ChARACTERISTICS p. 34

14:30 – 14:50 fallah hP, Tovo neto A., Habibi HR PARACRInE COnTROL Of TESTICuLAR dEVELOPMEnT And

SPERMATOGEnESIS In ZEBRAFISH(Danio rerio) p. 35

14:50 – 15:10 François Chauvigné, Alba ferré, Roderick nigel finn, Joan Cerdà SPECIFICREGULATORYROLESOFAQUAPORINSINMARINE

TELEOST SPERMATOzOA p. 36

15:10 – 15:30 Güneş Yamaner,GökhanTunçelli,MominMomin,DevrimMemiş COMPARISONOFRAINBOWTROUT(onCorhynChus Mykiss)

SPERMMOTILITYPARAMETERSANDFERTILIZATIONRESULTBETWEENDIFFERENTCHAMBERSINCASASYSTEM p.37

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15:30 – 15:50 Víctor Gallego, Juan f. Asturiano FISHSPERMMOTILITY:AUSEFULTOOLFORMULTIDISCIPLINARY

STudIES In AQuACuLTuRE RESEARCh, A hISTORICAL APPROACh p. 38


16:10 – 16:30 Katarzyna Dziewulska,MalwinaPilarska INHIBITORYEFFECTOFK+IONSONTHESPERMATOZOAMOTILITYOF

EUROPEANBURBOT(lota lota L.) p.39

16:30 – 16:50 Pavlo Fedorov, J. Germán herranz-Jusdado, Víctor Gallego, Christoffer Rozenfeld, david S. Peñaranda, Luz Pérez, Ganna fedorova, Roman Grabic, Borys dzyuba, Juan f. Asturiano

BIOEnERGETICS Of EuROPEAn EEL SPERMATOzOA fOLLOWInG hORMOnAL TREATMEnT, POST ACTIVATIOn, And duRInG ShORT-TERM STORAGE p. 40

16:50 – 17:10 François Chauvigné, Wendy González, neil duncan, Ignacio Giménez, Joan Cerdà

uSInG RECOMBInAnT GOnAdOTROPIn-BASEd hORMOnE ThERAPIES TO IMPROVE SPERM PROduCTIOn In ThE fLATfISh solea senegalensis p. 41

17:10 – 18:50 Poster session I.

19:20 – ? Welcome beer

Tuesday, September 5th (in hotel Clarion České Budějovice)

SESSION II. OOGENESIS AND EGG QUALITYChairmen: Pascal Fontaine, Ozlem Yilmaz

9:00 – 9:40 Plenary lecture – Julien Bobe, Violette Thermes MATERNAL-EFFECTGENESANDEGGQUALITY:USUALSUSPECTS

ANDNEWPLAYERS p.44

9:40 – 10:00 Azin Mohagheghi Samarin,AzadehMohagheghiSamarin,Tone-KariKnutsdatterØstbye,ØivindAndersen,BenteRuyter,SabineSampels,Miroslav Blecha, david Gela, Tomas Policar

GENEEXPRESSIONCHANGESASSOCIATEDWITHPOST-OVULATORYAnd POST-STRIPPINGOOCYTEAGEINGINCOMMONCARP,Cyprinus Carpio p. 45

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Programme

10:00 – 10:20 Tainá Rocha de Almeida, Bérénice Schaerlinger, Aurélie Le Cam, Maud Alix,JérômeMontfort,JulienBobe,DominiqueChardard,PascalFontaine

TRANSCRIPTOMICANALYSISOFEURASIANPERCH(perCa fluviatilis)EGGSASSIGNEDTODISTINCTGROUPSOFQUALITYp.46

10:20 – 10:40 Karel Janko,JanPačes,HildeWilkinson-Herbots,RuiJCosta,JanRoslein,PavelDrozd,NataliiaIakovenko,JakubRídl,MilušeHroudová,JanKočí,RadkaReifová,VěraŠlechtová,LukášCholeva

WHYSEXTURNSTOASEXUALITY:THEINTERCONNECTIONBETWEENASEXUALITY,HYBRIDIZATIONANDSPECIATION p.47

10:40 – 11:00 Danielle Zanerato Damasceno, fábio Bittencourt, Elizabeth Romagosa ARGInInE InfLuEnCES fEMALE REPROduCTIVE PERfORMAnCE

And OffSPRInG GROWTh Of rhaMDia quelen p. 48


SESSION III. GERM CELLS: FROM BASIC SCIENCES TO APPLIED BIOTECHNOLOGIESChairmen: Taiju Saito, Jean-Jacques Lareyre

11:20 – 12:00 Plenary lecture – Luiz Renato de Franca SPERMATOGOnIAL STEM CELLS TRAnSPLAnTATIOn And

TRAnSGEnESIS In fISh p. 50

12:00 – 12:20 Anne-SophieGoupil,AhmedMaouche,AlexandraDepincé,LionelGoardon,MarjorieBideau,NicolasDechamp,EdwigeQuillet,Jean-Jacques Lareyre, FrancineKriegandFlorenceLeGac

IMPLEMEnTATIOn Of A GERM STEM CELL TRAnSPLAnTATIOn PROCEduRE fOR ThE REGEnERATIOn Of ISOGEnIC TROuT LInES p. 51

12:20 – 12:40 Jelena Lujić,ZoranMarinović,SimonaSušnikBajec,IdaDjurdjevič,AlešSnoj,BélaUrbányi,ÁkosHorváth

InTERSPECIfIC TRAnSPLAnTATIOn Of BROWn TROuT And GRAYLINGGERMCELLSINTORAINBOWTROUT:CONSERVATIONOFVALUABLEBALKANTROUTGENETICRESOURCES p.52

12:40 – 13:00 Alexandra Depincé,Pierre-YvesLeBail,CatherineLabbé EPIGEnETIC REPROGRAMMInG AfTER nuCLEAR TRAnSfER

In fISh p. 53

13:00 – 13:20 Viktoriia Iegorova, MartinPsenicka,TaijuSaito STURGEONPHENOMENON:FIRSTEVIDENCEOFPROGENYFROM

ThREE PAREnTS p. 54

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13:20 – 13:40 Diógenes Henrique de Siqueira-Silva, Lucas henrique Piva, Caio Augusto GomesGoes,TakafumiFujimoto,TaijuSaito,LetíciaVeroniDragone,JoséAugusto Senhorini, fabio Porto-foresti, José Bento Sterman ferraz, George ShiguekiYasui

TRIPLOIDORHYBRIDFISH:WHICHISTHEBESTHOSTFORSTEMGERM CELLS? p. 55

13:40 – 14:40 Lunch

14:40 – 15:00 Mitsuru Endoh,TakafumiFujimoto,EtsuroYamaha,KatsutoshiArai EMBRYONICDEVELOPMENTOFNUCLEO-CYTOPLASMICHYBRIDS

INDUCEDBYINTERSPECIFICANDROGENESISAMONGSPECIESINCYPRINIFORMES p.56

15:00 – 15:20 Taiju Saito, Rie Goto, Takahiro Matsubara dEVELOPMEnT Of PRIOMORdIAL GERM CELLS And PIGMEnT CELLS

In EASTERn LITTLE TunA, euthynnus affinis,EMBRYOS p.57

15:20 – 15:40 Ahmed Maouche, Edouard Curran, Aude Gautier, Anne-Sophie Goupil, ElisabethSambroni,ElodieDupindeBreyssat,YannGuiguen,TaijuSaito,florence Le Gac, Jean-Jacques Lareyre

EXPRESSIONPROFILINGOFNEWGERMLINEMARKERSSUGGESTSSuCCESSIVE WAVES Of dIffEREnT SPERMATOGOnIAL STEM CELL SuBPOPuLATIOnS duRInG RAInBOW TROuT OnTOGEnESIS p. 58

15:40 – 16:00 Charlène Rouillon,AlexandraDepincé,Pierre-YvesLe-BailandCatherineLabbé

MEIOSISRESUMPTIONANDEARLYMITOSISPATTERNAFTERSOMATIC CELL nuCLEAR TRAnSfER In GOLdfISh p. 59

16:00 – 16:20 KotaYokoyama,TakafumiFujimoto,Katsutoshi Arai INDUCEDPOLYSPERMYINPONDLOACH,MISGURNUS

ANGUILLICAUDATUS:CYTOLOGICALBEHEVIOROFSPERMNUCLEIInTRudEd InTO dEChORIOnATEd EGGS p. 60


16:40 – 17:00 Effrosyni Fatira, CatherineLabbe,AlexandraDepince,VictoriiaIegorova,KseniiaPocherniaieva,HilalGüralp,MilošHavelka,MartinPsenicka,TaijuSaito

SInGLE And MuLTIPLE SOMATIC CELLS nuCLEAR TRAnSfER In CRITICALLYENDANGEREDSPECIES,STURGEON p.61

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Programme

17:00 – 17:20 Ana Carina Nogueira Vasconcelos, Anna Octavera, danilo Pedro Streit Jr., GoroYoshizaki

fIRST EVIdEnCE In fISh Of ALTERnATIVE dEAd End GEnE SPLICInG p. 62

17:20 – 17:40 Laura Gribouval, Aude Gautier, Pierrïck Auvray, Pascal Sourdaine SPERMATOGOnIAL STEM CELLS TRAnSPLAnTATIOn In A LITTLE

SHARK,THEDOGFISHsCyliorhinus CaniCula L. p. 63

17:40 – 18:00 Xuan Xie, PingLi,MartinPšenička,HuanYe,KseniiaPocherniaieva,JieMa,LingbingZeng,ChuangjuLiandQiweiWei

ESTABLIShMEnT Of in vitro CuLTuRE COndITIOnS Of STuRGEOn GERM CELLS p. 64

18:00 – 19:50 Poster session II.

Wednesday, September 6th (in Vodňany)

08:00 Departure from hotel Clarion to Vodňany for excursion of local facilities (bus 1)

10:40 Departure from hotel Clarion to Vodňany (bus 2)08:45 – 11:20 Excursion


SESSION IV. GAMETE “OMICS”Chairmen: Julien Bobe, Aude Gautier

11:40 – 12:20 Plenary lecture – Igor Babiak, Teshome Tilahun Bizuayehu, Christopher Presslauer, Asan Meera Sahib, HajaMohideen

SMALLREGULATORYTRANSCRIPTOMEINFISHGONADS,GAMETESANDEARLYEMBRYOS p. 66

12:20 – 12:40 Elisabeth Sambroni, Amélie Patinote, florence Le Gac, Jean-Jacques Lareyre

ThE InVALIdATIOn Of ThE fShR GEnE In zEBRAfISh LEAdS TO AGAINOFBODYWEIGHT,ABIASEDSEX-RATIOANDADECREASEINSPERMQUALITY p.67

12:40 – 13:00 Ozlem Yilmaz, Amelie Patinote, Thaovi nguyen, Emmanuelle Com, Charles Pineau, Amaury herpin, Julien Bobe.

CRISPR/Cas9DISTURBANCEOFTHEMULTIPLEVITELLOGENIN(VTG)SYSTEM:ESSENTIALFUNCTIONSOFDIFFERENTVTGTYPESDURINGZEBRAFISHEGG,EMBRYOANDLARVALDEVELOPMENT p.68

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13:00 – 13:20 Laura Gribouval, Pascal Sourdaine, Jean-Jacques Lareyre, Johanna Bellaiche, florence Le Gac, Sylvie Mazan, Aude Gautier

duPLICATIOn Of nAnOS1 GEnE And EXPRESSIOn In dOGfISh GOnAdS p. 69

13:20 – 13:40 CarolineT.Cheung,ThaoviNguyen,AurélieLeCam,JérômeMontfort,Julien Bobe

nOVEL MATERnAL-EffECT GEnES ShEd nEW LIGhT On ThE MOLECULARMECHANISMSCONTROLLINGFISHEGGQUALITYp.70

13:40– 14:00 Cinta Zapater, Berta Crespo, Iciar Muñoz, Patricia Pinto, Silvia zanuy, Ana Gómez

InVOLVEMEnT Of ThE TRAnSCRIPTIOnAL COACTIVATOR nCOA7 duRInG InITIAL STAGES Of GAMETOGEnESIS In EuROPEAn SEA BASS (DiCentrarChus labrax) p. 71

14:00 – 15:00 Lunch

SESSION V. GAMETE STORAGE AND PRESERVATIONChairmen: Catherine Labbe, Juan F. Asturiano

15:00 – 15:40 Plenary lecture – Ákos Horváth GAMETE And GERM CELL PRESERVATIOn And STORAGE:

PERSPECTIVESANDTHEREALITY p. 74

15:40 – 16:00 J. Germán Herranz-Jusdado, Victor Gallego, david S. Peñaranda, Christoffer Rozenfeld, Luz Pérez, Juan f. Asturiano

OPTIMIzATIOn And STAndARdIzATIOn Of PROTOCOLS fOR EuROPEAn EEL SPERM ShORT-TERM STORAGE And CRYOPRESERVATIONOFLARGESPERMVOLUMES p.75

16:00 – 16:20 Miaomiao Xin,AnnaShaliutina-Kolešová,MohammadAbdulMominSiddique,JanŠtěrba,BorysDzyuba,SergiiBoryshpolets,VitaliyKholodnyy,Otomar Linhart, Li Ping

PROTECTIVE ROLE Of AnTIfREEzE PROTEInS duRInG CRYOPRESERVATIONOFSTERLET(aCipenser ruthenus)SPERMATOzOA p. 76

16:20 – 16:40 Eszter Kása, GergelyBernáth,ZoltánBokor,BélaUrbányi,KingaKatalinLefler,DušanJesenšek,ÁkosHorváth

dEVELOPMEnT Of SPERM VITRIfICATIOn PROTOCOLS fOR ENDANGEREDSALMONIDS:ADRIATICGRAYLING(thyMallus thyMallus)andMARBLETROUT(salMo MarMoratus) p. 77

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Programme

16:40 – 17:00 Patricia Diogo, Gil Martins, Ana Eufrásio, Rita nogueira, Isa Quinzico, Tomé Silva, Elsa Cabrita, Paulo Gavaia

GUIDELINESFORZEBRAFISHSPERMCRYOPRESERVATIONANDSTORAGE p. 78


17:20 – 17:40 Ákos Horváth, TímeaKollár,GergelyBernáth,BélaUrbányi,ZsoltCsenki-Bakos,BernadettFaragó,KatalinSzabó,CsillaBudai,EszterLosonczi,JuditCserepes

PTATPRECONDITIONINGOFZEBRAFISHEMBRYOSINORDERTOInCREASE ThEIR ChILLInG RESISTAnCE p. 79

17:40 – 18:00 Leandro Godoy,HenriqueDias,JôsieCaldas,NayaraCruz SPERM fROM ThE EndAnGEREd AMAzOnIAn fISh hypanCistrus

zebra CANBESUCCESSFULLYCOLLECTEDANDCRYOPRESERVED p.80

18:00 – 18:20 Gergely Bernáth, zsolt Csenki-Bakos, zoltán Bokor, Levente Várkonyi, JózsefMolnár,AlexandraKajtár,TamásSzabó,ÁdámStaszny,ÁrpádFerincz,KrisztiánSzabó,BélaUrbányi,BalázsCsorbai

ThE EffECTS Of dIffEREnT PRESERVATIOn METhOdS On IdE (leuCisCus iDus)SPERMANDTHELONGEVITYOFSPERM MOVEMEnT p. 81

18:20 – 18:40 Taís Silva Lopes, Eduardo Antonio Sanches, danilo Caneppele, Elizabeth Romagosa

FREEZINGSENSITIVITYOFSURUBIM-DO-PARAÍBA,steinDaChneriDion parahybaeOOCYTES p.82

18:40 – 19:00 İlkerYavaş,Yusuf Bozkurt, ZaferCantekin,TuğbaKorkmazYavaş EffECT Of EXTEndER SuPPLEMEnTEd WITh dIffEREnT

ANTIOXIDANTSONMOTILITY,DNADAMAGEANDFERTILIZINGABILITYOFWILDAFRICANCATFISH(Clarias gariepinus)SPERM p.83

19:00 – ? Dinner (shuttle service will be operating from 21:00 until the end)

Thursday, September 7th (in hotel Clarion České Budějovice)

09:00 – 09:20 Yevhen Horokhovatskyi, Mariola A. dietrich, Ievgen Lebeda, Sergii Boryshpolets, Pavlo fedorov, Marek Rodina, Borys dzyuba

CRYOPRESERVATIONEFFECTONSTERLETSPERMVIABILITYANDPROTEINCONTENTAFTERLIVE/DEADCELLSSEPARATIONBYPERCOLLDENSITYGRADIENT p.84

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09:20 – 09:40 Zoran Marinović,QianLi,JelenaLujić,EszterKása,YoshikoIwasaki,BélaUrbányi,ÁkosHorváth,GoroYoshizaki

CRYOPRESERVATIONOFZEBRAFISHSPERMATOGONIA:SLOW-RATEfREEzInG VS VITRIfICATIOn p. 85

SESSION VI. ANTHROPOGENIC CONTAMINANTS AND FISH GAMETESChairmen:HamidHabibi,PatrickKestemont

09:40 – 10:20 Plenary lecture – Oliana Carnevali, Stefania Santangeli, Isabel forner Piquer, francesca Maradonna

EndOCRInE dISRuPTInG ChEMICALS In AQuATIC EnVIROnMEnTS: EffECTS On fISh GAMETES p. 88

10:20 – 10:40 Ina Wagenaar and Irene Barnhoorn hISTOPAThOLOGICAL ASSESSMEnT Of GOnAdAL TISSuE

Of fISh EXPOSEd TO AnThROPOGEnIC COnTAMInAnTS, SOuTh AfRICA p. 89

10:40 – 11:00 Hana Kocour Kroupová, PavelŠauer,JitkaTumová,ChristophSteinbach,OksanaGolovko,JanaMáchová,HansKomen,VítProfant,RomanGrabic

SIMULTANEOUSEXPOSURETOENVIRONMENTALLYRELEVANTCOnCEnTRATIOnS Of dROSPIREnOnE And GESTOdEnE CAuSEd InTERSEX In COMMOn CARP, Cyprinus Carpio L. p. 90

11:00 – 11:20 Tímea Kollár, EszterKása,ÁrpádFerincz,BalázsCsorbai,BélaUrbányi,ZsoltCsenki-Bakos,ÁkosHorváth

INVITROTOXICOLOGYTESTSYSTEMBASEDONCOMMONCARP(Cyprinus Carpio)SPERMANALYSIS p.91


11:40 – 12:00 Closing ceremony

12:30 – 13:30 Lunch

13:45 Bus departure – guide tour in Český Krumlov with dinner

21:00 Coming back to the hotel Clarion

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Programme

POSTER PRESENTATIONSSESSION I. SPERMATOGENESIS AND SPERM QUALITY

Zoltán Bokor, József Molnár, Árpád Ferincz, Levente Várkonyi, Zsolt Szári, Ferenc Fodor, Tibor Tulipán, Gábor Nagy, Béla Urbányi , Gergely BernáthINVESTIGATION OF MALE MATURATION OUT-OF- AND DURING THE SPAWNING SEASON IN SICHEL (PELECUS CULTRATUS) p. 94

Olga Bondarenko, I. López-González, A. DarszonSPERMATOGENIC CELL T-TYPE CA2+ CURRENTS ARE POTENTIALLY REGULATED BY ARACHIDONIC ACID (AA) p. 95

Volodymyr Bondarenko, Miroslav Blecha, Tomas PolicarCHANGES OF SPERM MORPHOLOGY, VOLUME, DENSITY AND MOTILITY PARAMETERS IN NORTHERN PIKE DURING THE SPAWNING PERIOD p. 96

Ian Anthony Ernest Butts, Galina Prokopchuk, Vojtěch Kašpar, Jacky Cosson, Trevor Edgar PitcherOVARIAN FLUID IMPACTS FLAGELLA BEATING AND BIOMECHANICAL METRICS OF SPERM BETWEEN ALTERNATIVE REPRODUCTIVE TACTICS p. 97

Beata I. Cejko, Ákos Horváth, Radosław K. Kowalski OPTIMIZATION OF SODIUM AND POTASSIUM CONTENT AND PH IN COMMON CARP CYPRINUS CARPIO L. ARTIFICIAL SEMINAL PLASMA p. 98

Hadiseh Dadras, Sabine Sample, Tomas Policar, Miroslav Blecha, Borys DzyubaCHANGES OF SPERMATOZOA LIPID COMPOSITION IN RELATION TO THERMO-ACTIVATION PROCESS IN BURBOT, LOTA LOTA p. 99

Borys Dzyuba, Sabine Sampels, Viktoriya Dzyuba, Alexandre Ninhaus Silveira, Martin Kahanec, Rosicleire Veríssimo Silveira, Vitaliy Kholodnyy, Marek Rodina, Sergii Boryshpolets SPERMATOZOON STRUCTURE, LIPID COMPOSITION AND MOTILITY IN RELATION TO INTERNAL FERTILIZATION IN FRESHWATER STINGRAY POTAMOTRYGON MOTORO p. 100

Viktoriya Dzyuba, Alexandre Ninhaus Silveira, Martin Kahanec, Rosicleire Veríssimo Silveira, Jan Sterba, Marek Rodina and Borys DzyubaEVIDENCE OF SPERM MATURATION IN FRESHWATER STINGRAYS POTAMOTRYGON MOTORO p. 101

İlkerKeskin, Sude Atmaca, Aygül EkiciTHE EFFECTS OF GINGER ON ZEBRAFISH SPERM MOTILITY p. 102

Amin Golpour, Martin Psenička, Hamid NiksiratULTRASTRUCTURAL DISTRIBUTION OF CALCIUM DURING SPERMATOGENESIS OF ZEBRAFISH, DANIO RERIO p. 103

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Jiri Kristan, Daniel Zarski, Miroslav Blecha, Tomas Policar, Oleksandr Malinovskyi, Azin Mohagheghi Samarin, Katarzyna Palinska-Zarska, Joanna Nowosad, Slawomir Krejszeff, Dariusz KucharczykFERTILIZING ABILITY OF GAMETES AT DIFFERENT POST-ACTIVATION TIMES AND THE SPERM-EGG-RATIO IN THE ARTIFICIAL REPRODUCTION OF PIKEPERCH SANDER LUCIOPERCA p. 104

Levente Várkonyi, Zoltán Bokor, Balázs Csorbai, Zsolt Szári, István Ittzés, Zoltán Szabó, Daniel Żarski, Béla Urbányi, Gergely BernáthTHE EFFECTS OF CHILLED STORAGE AND THE pH OF THE ACTIVATING SOLUTION ON DIFFERENT MOTILITY PARAMETERS IN BURBOT (LOTA LOTA) SPERM p. 105

Momin Momin, Devrim MemişSPERM QUALITY ANALYSIS OF NORMAL SEASON AND OUT-SEASON BY PHOTOPERIOD MANIPULATION OF MALE RAINBOW TROUT BROODSTOCK (ONCORHYNCHUS MYKISS) p. 106

Gregorio Molés, Cinta Zapater, Silvia Zanuy, Ana GomézGONADOTROPIN SIALYLATION IN EUROPEAN SEA BASS (DICENTRARCHUS LABRAX), ANOTHER WAY OF ACTIVITY REGULATION? p. 107

Gregorio Molés, Cinta Zapater, Patricia Pinto, Soledad Ibañez, Ana GomézIMPACT OF GONADAL STEROIDS ON PITUITARY GONADOTROPINS IN MALE EUROPEAN SEA BASS (DICENTRARCHUS LABRAX) p. 108

Beata Sarosiek, Katarzyna Dryl, Joanna Nowosad, Dariusz Kucharczyk THE INFLUENCE OF SPERM ENZYMES INHIBITION ON THE PERCENTAGE OF FERTILIZED CARP EGGS p. 109

Danilo Pedro Streit Jr, Éverton Luís Zardo, Daniel Antônio Rotili, Lis Santos Marques, Itamar Cossina Gomes, Marcelo Bernardi, Damião Guedes, Júlia GioraREPRODUCTIVE CYCLE AND SPERMATOGENESIS OF PIRACANJUBA (BRYCON ORBIGNYANUS) IN CAPTIVITY p. 110

Michelle Thönnes, Marlen Kotte, Katja Steinborn, Alexander Froschauer, Frank PfennigNILE TILAPIA AS A VERSATILE MODEL FOR STUDYING HORMONAL CONTROL OF SPERMATOGENESIS IN VIVO AND EX VIVO p. 111

Devrim Memiş, Gökhan Tunçelli, Güneş YamanerA RESEARCH ON FERTILIZATION SUCCESS AND GAMETE QUALTY PARAMETERS OF ENDEMIC WILD TROUT (SALMO SP.) IN TURKEY p. 112

Patricio Ulloa-Rodríguez, Pablo Contreras, Elías Figueroa, Manuel Lee-Estevez, Iván Valdebenito, Jenny Risopatrón, Jorge FaríasCHARACTERIZATION AND SHORT-TERM STORAGE OF THE PATAGONIAN BLENNY (ELEGINOPS MACLOVINUS) SPERM p. 113

Rosicleire Veríssimo-Silveira , Maira da Silva Rodrigues, Patrícia Postingel Quirino, Diógenes Henrique de Siqueira-Silva, Alexandre Ninhaus-SilveiraSPERMATOGENESIS PROCESS AND SUPPORT CAPACITY OF SERTOLI CELLS IN ASTYANAX ALTIPARANAE (CHARACIFORMES) p. 114

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Programme

Kaoru Yoshida, Manabu YoshidaTRANSMEMBRANE SIGNAL TRANSDUCTION UNDERLYING HERRING SPERM-ACTIVATING PROTEINS (HSAPS) DEPENDENT ACTIVATION OF HERRING SPERM MOTILITY p. 115

SESSION II. OOGENESIS AND EGG QUALITY

José Beirão, Einar Egeland, João Santana, Sylvie BollaSPOTTED WOLFFISH (ANARHICHAS MINOR) EGG QUALITY PARAMETERS AND BIOCHEMICHAL COMPOSITION INFLUENCE IN EGG DEVELOPMENT p. 118

Mia Berwick, Craig Pooley, Carlos Garcia de LeanizREPRODUCTIVE BIOLOGY OF THE LUMPFISH (CYCLOPTERUS LUMPUS) AND ITS APPLICATION TO AQUACULTURE p. 119

Michał Blitek, Mirosław Szczepkowski, Katarzyna DrylDETERMINATION OF THE OVARIAN MATURITY IN SIBERIAN STURGEON (ACIPENSER BAERII) AND STERLET (ACIPENSER RUTHENUS) WITH USE OF ULTRASOUND AND ITS SUSCEPTIBILITY IN HORMONALLY STIMULATED REPRODUCTION p. 120

Emilie Cardona, Jerome Bugeon, Violette Thermes, Violaine Colson, Sandrine Skiba-Cassy, Julien BobeEFFECT OF FOOD RESTRICTION ON REPRODUCTIVE PERFORMANCES AND EGG QUALITY IN THE TROUT ONCORHYNCHUS MYKISS p. 121

Beata Irena Cejko, Elżbieta BrzuskaUSING MULTIPLE REGRESSION TO PREDICT THE EFFECTS OF STIMULATED HORMONAL REPRODUCTION IN 10 BREEDING LINES OF THE COMMON CARP, CYPRINUS CARPIO p. 122

Danielle Zanerato Damasceno, Morgane Cousture, Claudiane Valotaire, Aurélie Le Cam, Thao Vi Nguyen, Julien Bobe, Violaine ColsonIMPACT OF MATERNAL EXPOSURE TO HIGH TEMPERATURE ON EGG QUALITY AND SUBSEQUENT OFFSPRING BEHAVIOR p. 123

Joanna Nynca, Mariola A. Dietrich, Mariola Słowińska, Halina Karol, Ewa Liszewska, Beata I. Cejko, Beata Sarosiek, Sylwia Judycka, Katarzyna Dryl, Radosław K. Kowalski BIOCHEMICAL CHARACTERISTICS OF OVARIAN FLUID OF SALMONIDAE FISH AND STERLET p. 124 Erfan Akbari Nargesi, Bahram Falahatkar, Mir Masoud SajjadiREPRODUCTIVE PERFORMANCE IN RAINBOW TROUT, ONCORHYNCHUS MYKISS: EXCLUSIVE STUDY OF PROBIOTIC EFFECT ON FEMALE BROODSTOCK p. 125

Alba Ferré, François Chauvigné, Cinta Zapater, Roderick Nigel Finn, Joan CerdàDOMINANT-NEGATIVE REGULATION OF OOCYTE HYDRATION IN MARINE TELEOSTS MAY BE MEDIATED BY AQUAPORIN SPLICE VARIANTS p. 126

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Maud Alix, Daniel Zarski, Yves Le Roux, Dominique Chardard, Bérénice Schaerlinger, Pascal FontaineA MULTIPARAMETRIC APPROACH TO ASSESS THE DEVELOPMENTAL SUCCESS IN TELEOST DURING EMBRYOGENESIS p. 127

Azadeh Mohagheghi Samarin, Azin Mohagheghi Samarin, Sabine Sampels, Anna Krzyśków, Tone-Kari Knutsdatter Østbyec, Miroslav Blecha, Jiri Kristan, David Gela, Tomas PolicarLIPID CLASSES AND FATTY ACID COMPOSITION PROFILE DURING IN VITRO OOCYTE AGEING IN TENCH, TINCA TINCA p. 128

Martin PšeničkaA NOVEL METHOD FOR RAPID ELIMINATION OF STURGEON EGG STICKINESS USING SODIUM HYPOCHLORITE p. 129

SESSION III. GERM CELLS: FROM BASIC SCIENCES TO APPLIED BIOTECHNOLOGIES

Sude Atmaca, Aygül Ekici GERM CELL ISOLATION OF RAINBOW TROUT USING DENSITY PERCOLL CENTRIFUGATION p. 132

Effrosyni Fatira, Martin Pšenička, Taiju SaitoMETHODS OF SPERM MICRO-INJECTION IN UNFERTILIZED EGGS: COULD BE USED AS ASSISTED REPRODUCTION TECHNIQUES IN STURGEONS? p. 133

Roman Franěk, Katsutoshi Arai, Vojtěch Kašpar, Martin PšeničkaCOLD SHOCK ANDROGENESIS IN COMMON CARP p. 134

Ákos Horváth, Jelean Lujić, Zoran Marinović, György Hoitsy, Nataša Cvetković, Jovana Lovren, Béla UrbányiEXPLORING THE POSSIBILITY OF USING TIGER TROUT (SALMO TRUTTA × SALVELINUS FONTINALIS) AS A RECIPIENT FOR GERM CELL TRANSPLANTATION p. 135

Mario Günscht, Cornelia Wetzker, Klaus Reinhardt, Alexander Froschauer, Frank PfennigMULTI-PHoToN AUToFLUoRESCENCE LIFETIME IMAGING MICRoSCoPY (MP-FLIM) FoR NoN-INVASIVE AND LIVE METABoLIC CHARACTERIZATIoN oF TYPE A SPERMAToGoNIA IN MEDAKA TESTIS p. 136

Martin Psenicka, Hilal Guralp, Kseniia Pocherniaieva, Zuzana Linhartova, Viktoriia Iegorova, Taiju SaitoGENERATION OF GERMLINE CHIMERA IN STURGEON p. 137

Abdul Rasheed, Roman Franěk, Martin PšeničkaTARGETING dnd1 IN STERLETS (ACIPENSER RUTHENUS) BY CRISPR/Cas9 GENERATES PHENOTYPIC ABNORMALITIES p. 138

Alena Zikmundová, Martin Pšenička, Roman Franěk, Lukáš Choleva, Jan Röslein, Karel JankoSTERILIZATION OF LOACHES (GENUS COBITIS) BY USING ANTISENSE MORPHOLINO OLIGONUCLEOTIDE p. 139

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Programme

SESSION IV. GAMETES „OMICS“

Cinta Zapater, Ana Rocha, Gregorio Molés, Soledad Ibáñez, Silvia Zanuy, Ana GómezEUROPEAN SEA BASS (Dicentrarchus labrax) ANTI-MÜLLERIAN HORMONE: PRODUCTION IN A YEAST SYSTEM AND FUNCTIONAL STUDIES p. 142

SESSION V. GAMETE STORAGE AND PRESERVATION

Yusuf Bozkurt, İlker Yavaş, Fikret KaracaEFFECT OF DIFFERENT STRAW VOLUMES, CRYOPROTECTANTS AND AVIAN EGG YOLK TYPES ON CRYOPRESERVATION SUCCESS OF NILE TILAPIA (OREOCHROMİS NILOTICUS) SPERM p. 144

Roman Franěk, Zoran Marinović, Jelena Lujic, Vojtěch Kašpar, Ákos Horváth, Martin PšeničkaCRYOPRESERVATION AND TRANSPLANTATION OF COMMON CARP SPERMATOGONIA p. 145

Zoran Marinović, Nataša Radojković, Tijana Veličković, Jelena Lujić, Ákos Horváth, Vladica SimićSUBPOPULATION STRUCTURE OF THE DANUBE BARBEL BARBUS BALCANICUS SPERM BEFORE AND AFTER CRYOPRESERVATION p. 146

Alexandre Ninhaus-Silveira, Raphael S. Costa, Fabricio Marçal S. de Souza, José A. Senhorini, Cristiane Bashiyo-Silva, Diógenes H. S. Silva, Rosicleire Veríssimo-Silveira, Cristiele S. RibeiroFATTY ACID INFLUENCE ON PROCHILODUS LINEATUS (CHARACIFORMES, PROCHILODONTIDAE) EMBRYO CRYOPRESERVATION PARAMETERS p. 147

Eduardo Antônio Sanches, Danilo Caneppele, Tais da Silva Lopes, Elizabeth RomagosaTHE USE OF DIFFERENT SEMEN POOLS DOES NOT GUARANTEE THE SAME SPERM QUALITY AFTER CRYOPRESERVATION IN STEINDACHNERIDION PARAHYBAE (SILURIFORMES: PIMELODIDAE) p. 148

Sahana Shivaramu, Rupam Sharma, Shashank Ogale, Gopal KrishnaEFFECT OF DIFFERENT CRYOPROTECTANTS ON MOTILITY AND FERTILITY OF MAHSEER (TOR KHUDREE AND TOR MUSSULLAH) SPERMATOZOA p. 149

SESSION VI. ANTHROPOGENIC CONTAMINANTS AND FISH GAMETES

Jamie Das Neves, Helene Coetzee, Irene Barnhoorn, Ina WagenaarTHE POTENTIAL EFFECTS OF THE ORGANOCHLORINE PESTICIDES ALDRIN AND METHOXYCHLOR ON THE REPRODUCTIVE HEALTH OF THE MALE AFRICAN SHARPTOOTH CATFISH CLARIAS GARIEPINUS p. 152

SESSION I. SPERMATOGENESIS AND SPERM QUALITY

Miroslav Blecha, Petr Svačina, Borys Dzyuba, Sergii Boryshpolets, Yevhen Horokhovatskyi, Hadiseh Dadras Asaybar, Oleksander Malinovskyi, Tomáš PolicarCOMPARISON OF THE SPERMATOZOA QUALITY PARAMETERS AND FATTY ACIDS COMPOSITIONS IN CULTURED AND WILD BURBOT SPERM p. 153

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Víctor Gallego, J. Germán Herranz-Jusdado, Christoffer Rozenfeld, Simone Pulsoni, Luz Pérez, Juan F. AsturianoA COMPARISON OF SUBJECTIVE AND OBJECTIVE ASSESSMENT OF FISH SPERM MOTILITY p. 154

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Programme

SeSSiOn i. SPERMATOGENESIS

AND SPERM quALITy

ORAL PRESENTATIONS

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PLENARy PRESENTATION

ENDOCRINE AND PARACRINE REGuLATION OF ZEBRAFISH SPERMATOGENESIS

Rüdiger Schulz1,2*, Diego Crespo1, Diego Safian1, Roberto Morais1, Luiz Assis1, Eva An-dersson2, Geir Lasse Taranger2, Anna Wargelius2, Jan Bogerd1

1University of Utrecht, Faculty of Science, Department of Biology, Institute of Biodynamics and Biocomplexity, Division of Developmental Biology, Reproductive Biology Group, Padualaan 8, NL-3584 CH Utrecht, The Netherlands; [email protected] 2Institute of Marine Research, Research Group Reproduction & Developmental Biology, PB 1870 Nordnes, 5817 Bergen, Norway

We start by examining the histology and cellular composition of the testis. This will show us that during spermatogenesis, germ cells proceed through three main phases of development: (i) the geometric expansion of germ cell numbers during successive mitotic cell cycles; (ii) the recombination of maternal and paternal genetic information and its reduction to a haploid set of genes during the two meiotic divisions; (iii) the spermiogenesis phase when round spermatids metamorphose into flagellated sper-matozoa while proliferation is inhibited. During all three phases, germ cells depend on the constant nutritional, physical and regulatory support provided by Sertoli cells. Spermatogenesis starts with the spermatogonial stem cells and the testicular stem cell niche concept will be introduced. After briefly discussing microscopical techniques to measure/quantify spermatogenesis, we turn to the use of these methods to study the regulation of spermatogenesis. Follicle-stimulating hormone (Fsh) from the pituitary plays a major role in this context, since it directly stimulates Leydig cell steroidogenesis and Sertoli cell activities, such as the production of growth factors modulating germ cell proliferation and differentiation behaviour. RNA sequencing experiments and fol-low up studies showed that the steroid-independent actions of Fsh included suppress-ing inhibitors (e.g. anti-Müllerian hormone) and stimulating activators (e.g. insulin-like growth factor 3) of spermatogenesis. Comparing zebrafish and salmon showed that selected growth factors responded in a similar manner in testis tissue of the two spe-cies. Using the salmon model, we also started exploring puberty-associated changes in testicular gene expression in context with endogenous increases in androgen levels. We found in part overlapping (e.g. Tgf beta signalling), in part additional (e.g. Wnt signalling) pathways that were differentially expressed in salmon testis tissue during the initiation of spermatogenesis. In summary, sperm production can be modulated quantitatively by regulating the production of spermatogenic cysts that subsequently enter the spermatogenic process, which is an important regulatory domain of Fsh. This pituitary signal is converted in the testis into local signals, such as androgens and growth factors, overall resulting in a decreased tone of inhibitors and an increased tone of activators of spermatogenesis.

Keywords: Spermatogenesis, follicle-stimulating hormone, growth factors, androgens

Acknowledgements: The study was supported by the following grants: LiFeCYCLe (eU FP7-222719-1) and SALMOSTeRiLe (Research Council of norway Project 221648/O30).

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Oral presentation, Session I. SpermatogeneSiS and Sperm quality

SPERM CHEMOTAXIS IS MEDIATED By CALCIuM EFFLuX VIA PLASMA MEMBRANE-TyPE CALCIuM PuMP

Kaoru Yoshida1, Kogiku Shiba2,4, Junpei Ikenaga2, Kazuo Inaba4, Manabu yoshida2,3*

1Toin University of Yokohama, Faculty of Biomedical Engineering, Yokohama, Kanagawa 225-8503, Japan2the University of Tokyo, Misaki Marine Biological Station, School of Science, and 3 Center for Marine Biology, Miura, Kanagawa 238-0225, Japan; [email protected] of Tsukuba, Shimoda Marine Research Center, Shimoda 415-0025, Japan

Spermatozoa are activated their motility and attracted toward an egg in response to certain factors released from the eggs or female reproductive organs. These phenome-na constitute the first communication event between the gametes during fertilization to prevent crossbreeding among different species. Thus, the sperm motility activation and chemotaxis may act as safety process for authentication between conspecific egg and sperm, and help to prevent of crossbreeding. When a spermatozoon shows che-motactic behavior, transient [Ca2+]i increases in the spermatozoon are induced in a che-moattractant gradient. The [Ca2+]i transient triggers a series of stereotypic responses of flagellar beatings that comprises turning and straight-swimming, however the mo-lecular mechanism of [Ca2+]i modulation controlled by the chemoattractant are little known.

We previously identified the sperm attractant released from the eggs of the ascidian Ciona intestinalis as (25S)-3α,4β,7α,26-tetrahydroxy-5α-cholestane-3,26-disulfate and was designated as the sperm-activating and -attracting factor (SAAF). In this study, we examined receptive mechanism of sperm for SAAF, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF receptor. One of the PMCA splice variants, Atp2b_var.b specifically expressed on the sperm flagella, and binds with SAAF by basic amino acids located in the 2nd and 3rd extracellular loops. ATPase activity of PMCA was accelerated by SAAF, and PMCA is responsible to mediate the chemotactic behavior. On the other hand, chemotactic behavior of the sperm was disordered not only low-Ca2+ but also in high-Ca2+ condition. Thus, the direct control of Ca2+ efflux is the fundamental mecha-nism to regulate the chemotactic responses in the ascidian sperm.

Keywords: Sperm chemotaxis, plasma membrane Ca2+-ATPase, chemoattractant, AT-Pase activity, ascidian

Acknowledgements: The study was supported from project number 123. We would like to thank to Martin Kocour providing us with his abstract example.

This work was supported by JSPS KAKenHi to MY (grant number 15H04398). We thank the national Bio-Resource Project of the AMeD, Japan for suppling materials.

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SEASONAL DIFFERENCES IN SPERM quALITy PARAMETERS OF THE COMMON CARP CYPRinUS CARPiO L.

Beata I. Cejko1*, Beata Sarosiek1, Sławomir Krejszeff2, Radosław K. Kowalski1

1Polish Academy of Science, Department of Gamete and Embryo Biology, Institute of Ani-mal Reproduction and Food Research, Olsztyn, Poland; [email protected]

2University of Warmia and Mazury, Department of Lake and River Fisheries, Faculty of Environmental Sciences, Olsztyn, Poland

Sperm quality varies during the reproductive period in cyprinids such as the common carp Cyprinus carpio L. There are many factors and biomarkers of semen quality that directly influence sperm maturation and fertilization ability. The most important include sperm motility and sperm concentration. On the other hand, the composition of seminal plasma (ions, lipids, proteins, sugar) as well as enzymatic and proteolytic activity might also influence sperm quality. Moreover, flow cytometry has become an established me-thod to obtain sperm diagnostic information. The aim of the presented study was to determine the effects of different phases i.e., early (June 1), middle (June 12) and late (June 19) of the reproductive period of the common carp on sperm quantity (semen volume/sperm count) and quality (sperm motility/sperm viability). The percentage of live, dead, and apoptotic sperm was determined using flow cytometry. The enzymatic and proteolytic activities of seminal plasma samples of sperm collected during the early, middle and late phases of the reproductive period were also measured. The lowest vo-lumes of semen and sperm counts as well as sperm velocity (VCL and VSL) and sperm viability were noted in the early phase (June 1) of sperm maturation in this species. At this time, seminal plasma contained the highest values of lactate dehydrogenase and ß-N acetylglucosaminidase activity. On the other hand, the highest volume of semen and sperm count was observed in the middle phase (June 12) of the reproductive period of common carp. At this time of semen collection the lowest pH and osmolalality of seminal plasma were also noted. At the late phase of reproductive period (June 19) of common carp the highest values of sperm motility and velocity were found. At this time the hig-hest percentage of live sperm was also noted. Results of our study shown the specific pattern of the common carp sperm maturation process. This process is characterized by low sperm quality at the beginning of the spawning season, which is associated with high values of cell damage indicators such as necrosis and apoptosis values and high sperm-derived enzymatic activity. According to our data, common carp sperm maturati-on most probably occurs in the sperm duct and is influenced by the specific conditions such as pH and osmolality. Moreover, the successful maturation of the sperm depended on their duration in the seminal plasma and the accumulation of the appropriate amount of nutrients/ions in the seminal plasma. The results showed that although the highest sperm quantity could be obtained in the middle of the spawning season, their highest quality was observed at the end of the spawning period.

Keywords: Common carp, Sperm, CASA, LDH, Flow cytometry

Acknowledgements: The presented study was supported by national Science Centre Grant nn 311 351239 and funds appropriated to the institute of Animal Reproduction and Food Research, of the Polish Academy of Sciences, Olsztyn, Poland.

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DOES OVARIAN FLuID AFFECT THE FERTILIZATION IN FRESH WATER FISH?

Vitaliy Kholodnyy*, Jacky Cosson, Serhii Boryshpolets

University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]

In externally fertilizing fish, the eggs and sperm are released into water and thereafter fertilization usually occurs without any participation of parents. The aqueous environ-ment is adverse and deleterious for gametes, therefore the lifespan of activated sperm is quite short, especially in freshwater, where the fish male gametes should reach their target, the female gamete, as soon as possible because spermatozoa become damaged within several minutes due to osmotic shock. Under such conditions, the reproductive success is chronically limited to the ability of spermatozoa to find the egg and reach the fertilization site (micropyle). Nowadays, the common belief that fertilization occurs ran-domly, through release and simple dispersion of a large number of spermatozoa, is being replaced by the guidance hypothesis. The ability of sperm cells to sense and react to changes of the environment, from the fluid viscosity and background flows to the pH, ion concentration and even temperature, makes the simplistic view of random fertilization to be unlikely occurring during sperm navigation. There are some empirical evidences of possible chemotactic response (one of the guidance mechanisms) from experiments in several marine fishes. Nevertheless, there are no studies to date on the direct demon-stration of any type of mechanism for guidance of freshwater fish spermatozoa towards the egg. Ovarian fluid, which coats fish eggs during spawning, is the best candidate to provide guidance for male gametes, its composition in ions, proteins, amino acids, sugars etc. is ideal for supporting and protecting eggs and sperm against deleterious effect of fresh water. It was shown in some fish species that factors that are part of ovarian fluid or released by the eggs could significantly affect the behavior of male gametes and, in such a way, influence the outcome of fertilization. This could result from the support of sperm motility traits on certain level, attraction or repulsion of gametes with some pre-defined qualitative characteristics and targeted promotion of sperm with proper genetic material to encounter the egg. In externally fertilizing fish species, the specific mechanisms by which females could perform this selection are still unclear. Our preliminary experiments in rainbow trout and common carp confirmed several effects of ovarian fluid on sperm motility traits, in particular velocity, linearity and longevity, e.g. rainbow trout sperma-tozoa changed the pattern of motility from circular to straight forward and significantly increased the duration of motility. In experiments where ovarian fluid was micro-injected into activation media, a kind of chemotactic/trapping behavior of spermatozoa was also found, especially in carp where this was more strongly expressed. As a result, we con-clude that an effect of ovarian fluid does exist in fresh water fish species and could be one of the factors supporting the process of fertilization.

Keywords: Ovarian fluid, fertilization, fresh water fish, rainbow trout, common carp

Acknowledgements: This study was financially supported by the Ministry of education, Youth and Sports of the Czech Republic – projects „CenAKVA“ (no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii“ (no. LO1205 under the nPU i program) and COST (no. LD14119) and by the Grant Agency of the University of South Bohemia (no. 125/2016/Z).

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IS OSMOTIC PRESSuRE THE ONLy FACTOR TO INDuCE SPERM MOTILITy ON COMMON CARP?

Carina Caldeira1,2*, Borys Dzyuba3, Serhii Boryshpolets3, Daznia Bompart1, Jacky Cosson3, Carles Soler1,2

1PROISER R+D, Av. Catedrático Agustín Escardino, 9, Building 3 (CUE), Floor 1, 46980 Paterna, Spain; [email protected] of Valencia, Faculty of Biological Sciences, Campus de Burjassot, C/ Dr. Moliner 50, 46100 Burjassot, Valencia, Spain3University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic

In fish species with external fertilization, the released spermatozoa are inactive, and motility activation occurs at contact with the external environment (marine- or fresh-wa-ter) mainly due to osmotic changes. However, the exact mechanism of sperm motility activation is still unknown and it is important to understand how the changes in environ-ment may be involved in activation of sperm motility. Therefore, the aim of the present work was to study, for the first time, the effects of pressure and temperature on the spermatozoa motility activation of Common carp (Cyprinus carpio). Carp sperm samples from 6 males were suspended either in seminal fluid (SF) or in physiological solution (PS). Three controls were prepared on a glass slide with coverslip: sperm supplemented with SF without activator, or with tap water as activator; the third control was sperm samples diluted in PS. Percentage of motility were analysed on control samples for 10 to 60 s post activation every 10 s, using ISAS® v1 (CASA system). Subsequently, a pressure of 6 kPa using the TruMorph® was applied to the sperm samples and the sperm motility was immediately observed and recorded over the motility period. Spermatozoa motility was also observed in sperm samples suspended in their own SF, using thermal shock (temperature rise from 20 to 50 °C). Alternatively, sperm samples suspended in SF or PS were stored at 4 °C, and sperm motility was analysed 24 hours later, following the same procedure. Among the three controls, sperm motility was observed only for diluted sperm after activation in tap water. Regarding controls submitted to pressure, sperm motility was observed only in samples suspended in SF, but with a lower percentage. In sperm samples exposed to thermal shock, no motility was observed. Twenty-four hours later, sperm suspended in SF showed about 10% of sperm motility, but this value in-creased after application of a 6 kPa pressure shock to the cells. Suggesting that during storage some changes in spermatozoa occurs, which make them more sensitive for the signals of motility activation and allow the part of spermatozoa to activate motility even in SF. In conclusion: seminal fluid could have some components that helps cells to save potential motility for at least 24 hours; during time of storage in SF some spermatozoa changing their sensitivity to external signals activating motility; application of physical pressure only may activate motility in the part of carp spermatozoa.

Keywords: Fish sperm activation, pressure, temperature, motility, Common carp

Acknowledgements: This project was supported from the european Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie project iMPReSS (GA no 642893); Ministry of education, Youth and Sports of the Czech Republic – projects „Ce-nAKVA“ (no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii“ (no. LO1205 under the nPU i program).

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EuROPEAN EEL (AnguillA AnguillA) MALES REPRODuCTIVE PERFORMANCE DuRING INDuCED SEXuAL MATuRATION: EFFECT OF DIFFERENT HORMONAL TREATMENTS

Víctor Gallego, J. Germán Herranz-Jusdado, Christoffer Rozenfeld, David S. Peñaranda, Luz Pérez, Juan F. Asturiano*

Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Po-litècnica de València, Camino de Vera s⁄n, 46022 Valencia, Spain; [email protected]

Over the last decades, the European eel has suffered a sharp reduction in its popu-lation and breeding in captivity is considered an alternative to save this species. The aim of this trial was to assess the effect of different hormonal treatments on the males reproductive performance.

A preliminary experiment was carried out comparing three doses of OVI (a recombi-nant α-choriogonadotropin marketed as Ovitrelle; Merck S.L., Madrid), and 10 males/treatment were weekly injected with 1.5, 0.75 or 0.25 IU/g fish of this hormone during 12 weeks. In a second experiment, 20 eel males were submitted to two hormonal treatments: OVI and VET (purified human chorionic gonadotropin marketed as VETER-IN CORION; Divasa-Farmavic S.A., Barcelona), with a weekly dose of 1.5 IU/g fish during 20 weeks. Percentage of spermiating males and sperm volume were immediately ap-praised, while sperm density and sperm kinetic parameters (total motility, TM; pro-gressive motility, PM; curvilinear velocity, VCL) were assessed by CASA software (ISAS; Proiser R+D, S.L.; Spain) within the next 2 h.

Preliminary experiment indicated that the highest OVI dose (1.5 IU/g fish) produced the best results in most of the sperm parameters from 8th to 12th week, reaching max-imum values of around 70% of TM, 45% of PM, and 135 µm/s of VCL. Sperm volume showed an inverse relationship with OVI doses, and the highest sperm volumes were reached using the lowest OVI doses. Results from the second experiment indicate that the type of hormone used affects significantly the onset and progression of spermia-tion. OVI treatment produced the best results in most of the sperm parameters during the first weeks of spermiation (6th to 10th), reaching maximum values of around 70% of TM, 40% of PM, and 150 µm/s of VCL. In addition, OVI males also produced significantly higher amounts of sperm (more than 3.5 mL /100 g fish at 9th week) than VET males (barely 1 mL), while no differences were found in sperm density. However, later sper-miation weeks showed a different pattern: while OVI males showed a clear decrease in sperm kinetic parameters from 11th week, VET-treated males showed a continuous in-crease just from this week, reaching maximum values of around 80% of TM, 50% of PM, and 170 µm/s of VCL. The economic performance of each hormonal treatment in terms of production of good-quality sperm will be evaluated at the end of the experiment.

Our results demonstrate that simultaneous treatments using different hormones be-come as an effective method for increasing the number of weeks in which eel males produce a suitable volume of high quality sperm, increasing the fertilization chances during hatchery operations.

Keywords: eel, hormone, maturation, sperm, motility

Acknowledgements: Funded by the european Union’s Horizon 2020 research and in-novation program under the Marie Skłodowska-Curie grant agreement no 642893 (iM-PReSS) and the COST Office (COST Action FA1205: AQUAGAMeTe). VG has a grant from the UPV (PAiD-10-16).

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EPIGENETIC STABILITy IN GOLDFISH SPERM uPON HORMONAL INDuCTION AND OVER THE BREEDING SEASON

Alexandra Depincé1, Chloé Bertin1, Catherine Labbé1*

1INRA, Fish Physiology and Genomics, UR 1037, Campus de Beaulieu, F-35000 Rennes, France; *[email protected]

Over the last few years, it has been proposed that the spermatozoon is not only a DNA bag. Indeed, several genes involved in early embryonic development already dis-play permissive epigenetic marks in the spermatozoon, as if these genes were poised for early transcription after fertilization and upon the embryo genome activation. Thus, the sperm chromatin likely bears epigenetic marks that are important for embryo de-velopment, but little is known about the stability of these marks when sperm is ex-posed to various reproductive biotechnologies. Among those technologies, hormon-al induction of domesticated broodstock, in order to trigger gamete final maturation and release, is common practice. The objective of the present work was to investigate whether male hormonal induction would change some of the sperm epigenetic marks. We focused the study on DNA methylation, the most well know epigenetic actor during the differentiation of the gametes, during the reprogramming after fertilization and during the ultimate gene silencing after tissue differentiation.

Six goldfish males used as model fish were studied for 2 months during their breed-ing season. They were induced once a week by a single injection of Ovaprim (Syndel, Canada) at 0.5 mL/kg. Sperm was collected twice a week, 16 h and 4 days after induc-tion. Sperm DNA was extracted and the global DNA methylation level was assessed using LUMA (Luminometric Methylation Assay) which is based on DNA cleavage by methylation-sensitive restriction enzymes, MspI and HpaII, followed by a polymerase extension assay by pyrosequencing. We demonstrated that every week, the DNA meth-ylation level of the spermatozoa collected just after induction was significantly higher than the one 4 days after induction (Rank test). This indicates that the sperm popu-lation released just after the artificial induction of final maturation is epigenetically different from the sperm population which was given more time prior to collection. In this experiment, we could also show that at a given day post induction, the sperm DNA methylation level was very stable over the 8 weeks of the experiment (Friedman test). This means that the DNA methylation level of spermatozoa produced by one male over a long period is fairly stable. This emphasizes even more the significance of the variations observed in response to hormonal induction. It remains to be deciphered whether the genomic regions affected by hormonal induction play a role in the progeny quality and development ability.

Keywords: DnA methylation, chromatin, sperm quality, spermatogenesis

Acknowledgements: This work is funded by the French CRB Anim project «investisse-ments d’avenir», ANR-11-INBS-0003. U3E inra is acknowledged for providing the gold-fish. The inRA LPGP fish facility took care of the breeders‘ rearing.

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CHEMICALLy DISPERSED CRuDE OIL AFFECTS SPERM FERTILIZING ABILITy BuT NOT SPERM SWIMMING IN CAPELIN (MAllOTuS VillOSuS)

José Beirão1,2*, Margaret A. Litt2, Craig F. Purchase2

1Nord University, Faculty of Biosciences and Aquaculture, NO-8049 Bodø, Norway; [email protected] University of Newfoundland, Fish Evolutionary Ecology Research Group, St. John’s, N.L., A1B 3X9, Canada

Offshore oil activities have increased concerns about accidental oil spills and the potential acute effects on aquatic organisms. In the event of major oil spills, disper-sants are often used in large amounts to disperse floating oil and increase microbial degradation. Because of their mode of action, dispersants can cause the disruption of biological membranes and their interaction with oil releases greater concentrations of toxic components in the water, causing higher toxicity than the untreated oil on early life stages. For the first time in any teleost, we tested sperm performance of capelin (Mallotus villosus), under direct exposure to realistic concentrations of WAF (water accommodated fraction) and CEWAF (chemically enhanced water accommodated frac-tion) in an oil spill event. None of the measured sperm motility parameters (percent-age of motile sperm, sperm velocity or sperm linearity) were affected by the different concentrations of both WAF and CEWAF or the dispersant alone. Nonetheless, there was a significant effect of the treatments on sperm fertilizing ability. The percentage of fertilized eggs was significantly impaired when the sperm were activated either in CEWAF at 10% corresponding to 16.1 mg/L TPH and 0.048 mg/L PAH. The mechanism responsible for this lower fertilizing ability are not clear but it could be related with the mode of action of the dispersants that contain surfactants that act as detergents and can disrupt cell membranes.

Keywords: Oil spill, dispersant, capelin, sperm performance

Acknowledgements: This research was supported by the national Contaminants Ad-visory Group of Fisheries and Oceans Canada, the Canada Foundation for innovation, the Research and Development Corporation of newfoundland and Labrador, and the natural Sciences and engineering Research Council of Canada via grants to CFP.

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PREDICTIVE BIOMARKERS OF SPERM quALITy IN STuRGEON SPERMATOZOA

Ping Li1,2, Wei Guo2, Huamei Yue1, Chuangju Li1, Hao Du1, Xinmei Qiao1, Zhigang Liu1, Zhou Qiong1, Qiwei Wei1*

1Chinese Academy of Fishery Sciences, Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Wuhan 430223, China; *Corresponding author: [email protected] of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Rese-arch Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic

Conventional sperm analysis (i.e., motility and fertility) has been used to evalua-te sperm quality. Understanding the quality of sperm on the molecular level in the sturgeons, Acipenser baerii and A. schrenckii, is essential for the improvement of the conservation of genetic resources and farming performance. In this study, we used the iTRAQ proteomics approach to perform proteomic profiling of spermatozoa associated with sperm quality in sturgeons. The results showed 291 and 359 differentially expre-ssed proteins in A. baerii and A. schrenckii, respectively, of which 72 were common to both species and all were upregulated in high quality compared with low quality sam-ples. The differentially expressed proteins were mainly categorized into the generation of precursor metabolites and energy and oxidation, and they were localized to the mitochondria. Three distinguishing pathways, Arginine and proline metabolism, Pyruva-te metabolism and the Citrate cycle (TCA cycle) were found to play an important role in energy metabolism, and some substrates could be used in the sperm medium for storage and cryopreservation. The expression levels of two proteins, CKMT1 and LDHB, were verified by western blot analysis. Moreover, other potential biomarkers involved in oxidation reduction, ubiquitin-proteasome-dependent proteolysis, chaperones and binding activity were also discussed. Our study is the first to use the iTRAQ-based proteomics approach to analyse the sturgeon spermatozoa proteome, and the results that we obtained are valuable for the prediction of sperm quality and reproduction management in these threatened species.

Keywords: iTRAQ, proteomics, spermatozoa, sturgeon, biomarker

Acknowledgements: This study was financially supported by the national natural Sci-ence Foundation of China (grant number 31402301), the Special Scientific Research Fund for Central non-profit institutes, Chinese Academy of Fishery Sciences (grant number 2015B02YQ01), the Ministry of education, Youth and Sports of the Czech Republic-projects “CenAKVA” (grant number CZ.1.05/2.1.00/01.0024), “CenAKVA ii” (grant number LO1205 under the nPU i program), and the Czech Science Foundation (grant number 16-02407Y).

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NON-NEWTONIAN PHySICAL PROPERTIES OF OVARIAN FLuID MAy BE IMPORTANT FOR POST-COPuLATORy CRyPTIC FEMALE CHOICE IN ATLANTIC SALMON (SAlMO SAlAR)

Marco Graziano 1*, Craig F. Purchase 2

1, 2Memorial University, Department of Biology, Purchase research group, Evolutionary Ecology of Fishes,230 Elizabeth Ave, St. John’s, NL A1B 3X9, Newfoundland, Canada; [email protected]

Cryptic female choice is considered to be one the most relevant mechanisms of post-copulatory sexual selection. In internal fertilizers, females can favour certain part-ners over others both before, and after mating. In external fertilizers such as Salmonids, the lack of internal genital traits, theoretically introduce relevant constraints to mate choice. Despite pre-copulatory selection, external fertilizers are generally assumed to be unable to determine parenthood; the female cannot influence fertilization because this process occurs outside of her body. However, it has recently been shown, that in some teleosteans, cryptic female choice can be mediated by the ovarian fluid (OF), and by gamete-recognition proteins thus influencing the fertilization outcome. In fish, the OF is a viscous substance that is released with the eggs, and comprises a relevant part of the total volume of the spawned egg mass.

At fertilization, spermatozoans face an increasing OF concentration as they approach to the egg to fertilize it. The positive relation between OF and sperm behaviour has been well documented. Several works have focused on the importance of OF chemical composition and have addressed its importance as a post-copulatory selective mechanism in fish. Re-cently, it has been found that OF shows non-newtonian properties, therefore its rheology needs to be clarified to understand the potential influence on the reproductive outcome.

For this reason, we analyzed the rheologic properties of the OF in order to establish if they are involved in post-copulatory sexual selective mechanisms. In this study, OF from spawning Atlantic salmon (Salmo salar) females (N=20) were analysed using a MCR rhe-ometer looking for non-newtonian characteristics of the fluid’s viscosity when subjected to increasing shear rates ranging from 0.001 to 500 s-1, and when subjected to variable angular frequencies (0.01 to 500 rad x s-1) comparable to speeds and angular frequen-cies exerted by swimming spermatozoans moving toward the eggs trough the OF.

Our results indicate that Atlantic salmon OF has non-newtonian shear thinning proper-ties at shear rates, angular frequencies, and temperature that occur during the fertiliza-tion process in this species. Importantly, these results address that the physical proper-ties of the OF itself have been evolutionary shaped to favour the selection of the fastest spermatozoans. A faster sperm will likely swim through a less viscous medium in its jour-ney toward the egg, contrary to a slower sperm that will instead encounter more physical resistance. These findings open a novel field of research that can have important implica-tions in understanding the evolution of sexual traits and in exploring the usually underes-timated role of the physical properties of biological matter in biological processes.

Keywords: Cryptic female choice, ovarian fluid, rheology, sperm velocity, sexual selection

Acknowledgements: The study was done in collaboration with the soft matter lab in the Physics Department of Memorial University of newfoundalnd. For this reason, we would like to thank Prof. Anand Yethiraj and Dr. Swomitra Palit for the essential tech-nical and intellectual support.

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SPERM MOTILITy OF THE NILE TILAPIA (OREOCHROMiS nilOTiCuS): EFFECTS OF TEMPERATuRE ON THE SWIMMING CHARACTERISTICS

Borys Dzyuba1, Marc Legendre2, Jean François Baroiller2, Jacky Cosson1*

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]é de Montpellier, Institut des Sciences de l’Evolution (ISE-M), CNRS, IRD, EPHE, Mont-pellier, France

In teleost fish species exhibiting external fertilization, sperm is generally quiescent in the testis and sperm movement is initiated after contact with the external milieu, either hyper- or hypo-osmotic depending on species (marine or fresh-water). Previous studies on differ-ent fish species, mostly from temperate or cold water habitat, reported an inverse relation-ship between environmental temperature and duration of sperm motility, which is simply explained by expenditure speed of limited energy stores available for motility. However, the broad variety of temperature at which some fish species reproduce (Nile tilapia as example) raises the question of the sperm swimming behavior of tropical species in extreme tem-perature conditions. The current study aims a better appraisal of the effects of environmen-tal temperature on sperm motility of tilapia (Oreochromis niloticus), a tropical fish species selected because of its aquaculture importance and ability to reproduce in a broad range of water temperature, ranging from 20 °C to 40 °C depending on climatic variations and fish populations. Effects of environmental temperature on spermatozoa motility, in controlled conditions of osmolality and ionic composition of activation media, were studied in rela-tion to sperm cells motility characteristics by video-microscopy and CASA analysis, using a temperature-controlled microscope stage. Over a temperature range from 5 to 55 °C, we observed a large variation of curvilinear velocity (VCL, mean values range 31–76 µm/s) and linear velocity (VSL, mean values range 12–53 µm/s) at 30 s post–activation, while the du-ration of the motility period was also greatly affected. The motility duration decreased grad-ually from about 15 min at 5 °C to less than one minute at 55 °C. In the low temperature range (5–10 °C), the percentage of motile cells exhibited a very large variability between males. An abrupt increase in the linearity index (VSL/VCL) was observed between 15 °C and 20 °C suggesting a physiological threshold in sperm movements below 20 °C.

Therefore, it is concluded that Nile tilapia sperm exhibits a specific ability to express high velocity and linearity characteristics in the 30–45 °C temperature range but this occurs to the expense of the motility duration. All together, these results clearly show the importance of considering ambient temperature when trying to characterize sperm motility parameters and optimal fertilization conditions in fish.

Keywords: Sperm motility velocity and linearity, sperm motility duration, temperature, nile tilapia, reproduction

Acknowledgements: The study was supported by the Ministry of education, Youth and Sports of the Czech Republic (Project “CenAKVA” no. CZ.1.05/2.1.00/01.0024 and Proj-ect “CenAKVA ii” no. LO1205 under the nPU i program) and COST (no. LD14119), by the Grant agency of the University of South Bohemia (no. 125/2016/Z), and by AnR program CLiMSeX “Water warming: lessons from tilapia’s adaptation” (AnR-15-Ce02-0012).

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PARACRINE CONTROL OF TESTICuLAR DEVELOPMENT AND SPERMATOGENESIS IN ZEBRAFISH (DAniO RERiO)

Fallah HP1, Tovo Neto A1,2, Habibi HR1

1University of Calgary, Department of Biological Sciences, Drive NW, Calgary, Alberta, Canada T2N 1N42Aquaculture Program, São Paulo State University (Unesp), Jaboticabal, São Paulo, Brazil

It is now established that control of testicular development is multifactorial, and result from complex interaction among gonadal hormones with hypophysial and pe-ripheral hormones. Here we investigated the potential role of local testicular peptides (GnRH & GnIH), and their paracrine effects in the control of spermatogenesis in ze-brafish. These peptides are among local factors involved in paracrine control of testicu-lar development and influce steroidogenesis and spermatogenesis. The present study provides nove on the direcl information on the role of gonadal peptides on basal and gonadotropin (GtH)-induced control of testicular development and function in zebraf-ish. Our results provide information on the mechanisms underlying paracrine control of testicular development and function. We analysed development of spermatozoz from spaermatoginia stem cells and investigated how GnRH and GnIH influence mitotic and meiotic proliferation of germ cells, using cell cycle and morphological analysis as well as measuring production of gonadal testosterone. The result of this study demonstrat-ed direct actions of gonadal peptides on spermatogonial proliferation and differenti-ation after treatment for 7 days, in vitro. These peptides were found to significantly alter production of postmeiotic haploid cell populations directly and by interacting with pituitary gonadotropins. The overall findings illustrates a complex multifactorial mechanisms involving pituitary hormones, gonadal steroids and peptides working in an orchestrraed manner to regulate gonadal development and function. Funded by NSERC grants to HRH.

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SPECIFIC REGuLATORy ROLES OF AquAPORINS IN MARINE TELEOST SPERMATOZOA

François Chauvigné1*, Alba Ferré1, Roderick Nigel Finn2, Joan Cerdà1*

1Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain; [email protected] of Bergen Department of Biology, Bergen High Technology Centre, Bergen, Norway

Recent studies of aquaporins in marine teleosts, using gilthead seabream (Sparus aurata) as the model organism, have shown that several paralogs may play distinct roles in water and solute transport during the induction of sperm motility in seawater (SW). By assessing the motility parameters of activated sperm in the presence of Aqp1aa, -1ab, -7 and -8bb antisera, we found that the water efflux from ejaculated spermatozoa exposed to hyperosmotic SW is predominantly mediated by Aqp1aa, which is distributed along the flagellum. Immunological inhibition of Aqp1aa reduces the rise of intracellular Ca2+ that normally occurs upon activation, resulting in the suppression of sperm motility. In turn, Aqp1ab and -7, which are expressed in the anterior tail and head of spermatozoa, might be involved in the control of the trajectory and swimming pattern. By contrast, during SW-activation, Aqp8bb is rapidly transported to the mitochondrion, where it acts as a peroxiporin facilitating the efflux of reactive oxygen species (ROS), such as H

2O

2. When Aqp8bb trafficking and function are inhibited, ROS levels accumulate,

resulting in mitochondrial membrane depolarisation, a reduction of ATP production, and the arrest of sperm motility. The targeting of Aqp8bb to the inner mitochondrial membrane involves its phosphorylation, but the upstream regulatory mechanisms are yet unknown. Incubation of non-activated sperm with a Ca2+ ionophore in the presence of external Ca2+ can induce the phosphorylation of Aqp8bb and its accumulation in the mitochondrion, a process that can be prevented with an inhibitor of protein kinase C. In addition, both incubation of immotile spermatozoa with xanthine-xanthine oxidase, which generates ROS in vitro, and the SW-activation of sperm in the presence of a Ca2+ chelator, also promote Aqp8bb trafficking. These results suggest that both Ca2+ and ROS are independent primary signals that together regulate the Aqp8bb-mediated detoxification mechanism of marine teleost spermatotozoa. The specific signaling pathways involved in this mechanism are under investigation.

Keywords: Sperm, aquaporin, mitochondria, motility, Ca2+, ROS

Acknowledgements: The study was supported by the Spanish Ministry of economy and Competitivity (MineCO) (Grant no. AGL2016-76802-R to JC), and the Research Council of norway (Project 254872/e40 to RnF). Participation of FC and AF was funded, respectively, by a “Ramon y Cajal” contract and a predoctoral fellowship from Spanish MineCO.

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COMPARISON OF RAINBOW TROuT (Oncorhynchus mykiss) SPERM MOTILITy PARAMETERS AND FERTILIZATION RESuLT BETWEEN DIFFERENT CHAMBERS IN CASA SySTEM

Güneş yamaner*, Gökhan Tunçelli, Momin Momin, Devrim Memiş

İstanbul University Fisheries, Faculty Aquaculture Department Ordu street, No:200, Laleli, İstan-bul, Turkey; [email protected]

Sperm analysis is the most common and informative laboratory technique for the investigation of male gamete quality in many fish species. The analysis of motility and kinematic parameteres which are main key for fertilization by computer assisted sperm analysis (CASA) prevents the loss of effort in fertilization studies. However, there are many factors that affect the results of the CASA system, and one of the most impor-tant variables is the chamber used for the analysis. In this study, the effect of the chamber used for the automated analysis of sperm motility and sperm kinematics parameters by CASA was evaluated of Rainbow trout (Oncorhynchus mykiss) sperms. The assesment of motility parameters was carried out using CEROS II (Hamilton-Thor-ne, Beverly, MA, USA) connected to CX41 microscope (Olympus, Japan) at room tem-perature. Sperm samples were collected from five adult males by abdominal massage during the reproduction season and analysed with two different chambers as follows specialty: Leja 2 cell chambered with 20 µl deep (Leja Products, Netherlands) and Ma-kler chamber, round shape with 10 µl deep (Sefi-Medical Instrument, Haifa, Israel). Eeach sperm samples analyzed at three times. In order to understand the relationship between fertilization rate and total motility parameters depending on the different chamber applications of CASA system, progressive motility results were discarded. To-tal sperm motility (Mot,%), and kinematic parameteres were measured. For fertilization test, eggs from one female (550 g approximately 7500 eggs) were separated in equal five parts. Each part of eggs (approximately 1500 eggs) were fertilized with each ana-lyzed sperm (5ml). Fertilization, incubation prosedure and calculation of fertilization rates have been kept as used routinly for rainbowtrout culture prosedure. The CASA results were compared with regards to the fertilization rates.

The fertilization rates were found ˃80% for all used males. The motility precentange of samples analyzed by Leja has been found higher 90% while by makler changed between 26–45%. The different chambers used in this study did significantly affect the motility percentange. The motility percentange of samples obtained with each cham-ber were quite similar, however, regardless with the fertilization rate, the high stabili-ty results was detected in Leja 2-chamber. Statistical study with motility percentage showed a significant difference between Leja and Makler chambers (p<0.05).

As a result of the study, altough the Makler chamber is preferred due to the econo-mical specialty, the using of Leja resulted in more accurately according to fertilization results.

Keywords: CASA, Leja, makler, motility parameters, rainbow trout

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FISH SPERM MOTILITy: A uSEFuL TOOL FOR MuLTIDISCIPLINARy STuDIES IN AquACuLTuRE RESEARCH, A HISTORICAL APPROACH

Víctor Gallego, Juan F. Asturiano*

Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Po-litècnica de València, Camino de Vera s⁄n, 46022 Valencia, Spain; [email protected]

The first scientific reports focused on fish sperm motility date from a century ago. From this date, new articles about this topic were published sporadically until 60s and were more continuous but still scarce until 80s. Nevertheless, a marked and con-tinuous increase of scientific contributions were detected from 90s to present, and more than 1500 SCI papers can be found using fish sperm motility (assessed by sub-jective or objective ways) as a tool for research in a wide range of topics, from ecology to molecular issues.

Within these topics, sperm storage has been the most investigated field, involving both the use of chilled-storage protocols for short-term periods, and sperm cryopreser-vation techniques for long-term storage. Over 400 manuscripts reporting spermatozoa kinetic parameters have contributed to discover and improve sperm storage protocols in a large number of fish species. Nowadays, sperm-storage techniques have a high number of potential applications, ranging from ecology goals (cryobanking, etc.) to aquaculture purposes (breeder’s synchronization, genetic improvement programs, etc.).

Sperm physiology has been another of the most investigated fields using the sper-matozoa motion as a research tool. In fact, first physiology studies using fish sperm motility were carried out at the beginning of the last century. Since then, this research field has shown a continuous increase over the years, and more than 300 physiolo-gy-articles focused mainly on the fish sperm activation process and the whole propul-sion machinery of the sperm cells have been published.

Finally, broodstock management has been the other topic widely studied by sperm motility analysis, with around 300 manuscripts published in JCR journals. In this cont-ext, several aspects such us i) rearing conditions, ii) dietary requirements, iii) hormonal induction treatments, iv) gamete collection techniques, or v) biotechnology and ge-netic engineering have been faced improving a large number of issues for undergoing successfully reproductive maturation and fertilization processes.

To sum up, sperm motion parameters from 340 fish species belonging to different fa-milies have been already studied to date. However, only a few of these species (about 30) represent more than 50% of published papers, of which salmonids, cyprinids and sturgeons are the most studied groups. In this context, scientists from more than 70 countries and belonging to 150 universities or research centers have devoted much more time to study freshwater than seawater species, what explain that the volume of information is five-fold bigger in freshwater fish. Fish sperm motility represents, there-fore, a useful tool for multidisciplinary studies in aquaculture research.

Keywords: Motility, fish, aquaculture, CASA, research

Acknowledgements: Funded by the european Union’s Horizon 2020 research and in-novation program under the Marie Skłodowska-Curie grant agreement no 642893 (iM-PReSS) and the COST Office (COST Action FA1205: AQUAGAMeTe). VG has a grant from the UPV (PAiD-10-16).

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INHIBITORy EFFECT OF K+ IONS ON THE SPERMATOZOA MOTILITy OF EuROPEAN BuRBOT (lOTA lOTA L.)

Katarzyna Dziewulska*, Malwina Pilarska

University of Szczecin, Faculty of Biology, Department of General Zoology, Felczaka 3c, 71412 Szczecin, Poland; [email protected]

External fertilization occurs in most teleostean fish. In the reproductive tract, gametes remain in an inactive state until they are ejaculated into the water, where they activate to swim and find eggs. In freshwater fish, two main startup mechanisms of the path of blocking or activating the spermatozoan motility apparatus are known. In fish belonging to Scorpaeniformes (Cottidae), Cypriniformes (Cyprinidae), Esociformes (Esocidae), Perciformes (Percidae, Eleotridae, Cichlidae), and Siluriformes (Clariidae) osmotic pressure of the external environment is a factor that influences spermatozoa activation and swimming. Spermatozoa of the second group, represented only by Salmoniformes (Salmonidae), Osmeriformes (Osmeridae, Plecoglossidae) and Acipenseriformes (Acipenseridae, Polyodontidae), are K+ ion-sensitive, while osmolality is considered as a secondary factor.

In previously conducted studies of European burbot and North American burbot contradictory findings regarding factors influencing the onset of spermatozoa motility were reported. These findings were the basis for assumptions leading to the creation of two diverged phylogenetic groups. The objective of the current study was to determine the effect of potassium and osmolality on the European burbot (Lota lota L.) spermatozoa motility. Moreover, the influence of pH, as well as sodium and calcium ion concentrations was investigated. Composition of seminal plasma was determined. Seven parameters characterising motility: VCL – curvilinear velocity, VAP – average path velocity, VSL – straightline velocity, LIN – linearity, ALH – amplitude of lateral head displacement, and BCF – beat cross frequency, as well as duration of motility in prepared buffered solutions were traced by means of computer-assisted sperm analysis (CASA).

The spermatozoa of European burbot are K+ ion-sensitive. KCl concentration over 12 mM ceased spermatozoa movement. Increasing the concentration of K+ ions in the prepared solution mainly caused a decrease in percentage of motile spermatozoa. Sucrose, Na+ and Ca2+ solutions inhibited spermatozoa movement only at concentrations far exceeding the physiological osmolality of seminal plasma. The European burbot spermatozoa were motile over a wide range of pH values. A decrease in some motility parameters occurred below pH 7 and over pH 12.

Conclusion It was demonstrated that the spermatozoa of a Lotidae representative are inhibited by K+ ions. It is likely that Lotidae is the another family with ion-sensitive spermatozoa.

Keywords: CASA, burbot, motility, potassium, sodium, calcium, pH, osmolality

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BIOENERGETICS OF EuROPEAN EEL SPERMATOZOA FOLLOWING HORMONAL TREATMENT, POST ACTIVATION, AND DuRING SHORT-TERM STORAGE

Pavlo Fedorov1*, J. Germán Herranz-Jusdado2, Víctor Gallego2, Christoffer Rozenfeld2, David S. Peñaranda2, Luz Pérez2, Ganna Fedorova1, Roman Grabic1, Borys Dzyuba1, Juan F. Asturiano2

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, Vodňany 389 25, Czech Republic; [email protected] Politècnica de València, Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Camino de Vera s⁄n, 46022 Valencia, Spain.

It was shown in some fish species (e.g. sturgeon) that spermatozoa undergo maturation to get the ability to be activated and this process is associated with bioenergetic state of the cells. It is known that sperm of European eel also needs maturation for initiation of motility, and this can be triggered by hormonal treatment, but nothing is known about bioenergetic substrates content in these conditions. In addition, there are no data about effect of short-term storage conditions on bioenergetics of the sperm cells, which can be essential for eel aquaculture.

This study showed that European eel spermatozoa were virtually unable to be activated by dilution into seawater (SW) at the beginning of the artificially induced spermiation period (6th week of hormonal treatment with hCGrec). After 10 weeks of hormonal treatment, they acquired the ability of being activated into SW. This was associated with higher adenylate energy charge (AEC, the ratio of AMP, ADP, and ATP) on the 10th week of treatment in comparison to the 6th week. Hormonal treatment seems to induce a final maturation process, changing bioenergetic state, that in turn is important for acquiring motility by spermatozoa.

A significant increase of creatine phosphate (CP) and cyclic-AMP (cAMP) content in spermatozoa of the 10 weeks-treated males was observed prior to and post-activation if compared with cells from 6 weeks-treated fish. Current study represents the first successful quantification of cAMP in fish spermatozoa prior to and post activation using liquid chromatography coupled with high-resolution product scan (LC/HRPS).

Moreover, temperature and duration of short-term storage affected the content of creatine, CP, AMP, ADP, and ATP in European eel spermatozoa. Short-term storage (up to 7 days) at 4 °C resulted in higher macroergic phosphates content and higher motility traits compared to 20 °C. Obtained results could be used for future improvement of spermiation induction technique and methods for short-term storage of European eel sperm.

Keywords: Macroergic phosphates, sperm maturation, sperm bioenergetics, sperm storage

Acknowledgements: Supported by the european Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement nº 642893 (iMPReSS), the Ministry of education, Youth and Sports of the Czech Republic: projects “CenAKVA” (no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii” (no. LO1205 under the nPU i program); COST (no. LD14119), COST Office (COST Action FA1205: AQUAGAMeTe), the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z). VG has a postdoc grant from the UPV (PAiD-10-16).

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uSING RECOMBINANT GONADOTROPIN-BASED HORMONE THERAPIES TO IMPROVE SPERM PRODuCTION IN THE FLATFISH SOlEA SEnEgAlEnSiS

François Chauvigné1*, Wendy González2, Neil Duncan2, Ignacio Giménez3*, Joan Cerdà1*

1Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain; [email protected]; [email protected] 2IRTA, 43540 Sant Carles de la Rapita, Spain 3 Rara Avis Biotec, S. L., 46002 Valencia, Spain; [email protected]

The flatfish Senegalese sole, Solea senegalensis, is one of the target species for aquaculture diversification in Southern Europe. However, the development of artificial fertilization methods and of selective breeding programs in this species is hampered by the very low amounts of sperm produced by the first generation (F1) of males reared in captivity. In recent years, we have investigated the endocrine regulation of spermatogenesis in Senegalese sole, focusing on the function of the pituitary gonadotropins, the follicle-stimulating (Fsh) and luteinizing (Lh) hormones. By using homologous single-chain recombinant gonadotropins (rFsh and rLh), produced in a mammalian host system, we previously showed that both hormones can induce the production of androgens in vivo and in vitro and regulate common and distinct genes involved in spermatogenesis. Also, we found that rLh activation of the Lh receptor in mature spermatids directly triggers spermatozoa differentiation. In this study, we tested whether combined treatments with rFsh and rLh could stimulate spermatogenesis and sperm production in F1 males. The rFsh and rLh produced in Chinese hamster ovary cells showed a half-life of ~5 and ~3 days, respectively. Weekly intramuscular injections of sole males with rFsh over 9 weeks increased gonad weight, and stimulated steroidogenesis and germ cell proliferation and meiosis, whereas a subsequent single rLh injection potentiated spermatozoa differentiation. Also, repeated rLh injection of males previously treated with rFsh for 4 to 10 weeks could enhance sperm production up to 7 times. These data suggest that in vivo application of rFsh and rLh is effective at stimulating spermatogenesis and sperm production in Senegalese sole F1 males, setting the basis for the future establishment of recombinant gonadotropin-based hormone therapies to ameliorate reproductive dysfunctions of cultured males.

Keywords: Flatfish, spermatogenesis, sperm; spermiation, recombinant hormone, Fsh, Lh

Acknowledgements: This work was partially funded by the Spanish Ministry of economy and Competitivity (MineCO) (Grant no. AGL2013-41196-R to JC), and the Spanish national institute for Agronomic Research (iniA)-european Fund for economic and Regional Development (FeDeR) (Grant no. RTA2014-00048 to nD). Participation of FC and WG was funded, respectively, by a “Ramon y Cajal” contract (MineCO) and a predoctoral grant from the national Board of Science and Technology (COnACYT, Mexico).

SeSSiOn ii. OOGENESIS AND EGG quALITy

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MATERNAL-EFFECT GENES AND EGG quALITy: uSuAL SuSPECTS AND NEW PLAyERS

Julien Bobe1, Violette Thermes1

1 INRA, UR1037 Fish Physiology and Genomics, Campus Beaulieu, Rennes, [email protected]

In fish, as in other animals, maternal-effect genes (i.e. genes expressed by the mo-ther and are critical for embryonic success) play important roles in the molecular me-chanisms that control the egg’s ability to be fertilized and subsequently develop into a normal embryo. Over the last few years, we have initiated several genomic screens to identify novel maternal effect genes that would broaden our knowledge of the mo-lecular mechanisms behind fish egg quality. Recently, this approach was broadened to incorporate non-coding RNA, and especially miRNAs. Over the last decades, micro RNAs have emerged as important regulator of many physiological and pathological pro-cesses. Their role in the regulation of reproductive functions remains however poorly investigated, especially in fish. Among the different miRNAs that we identified, miRNA 202 was of particular interest. The microRNA 202 (mir-202) is expressed in both tes-tis and ovary of many vertebrates and was reported to play an important role during spermatogenesis in adults. However, its function during oogenesis in the ovary and underlying mechanisms remain unknown. To analyze its function, we inactivated the mir-202 gene in medaka by using the CRIPSR-Cas9 system. We generated a mutant fish line in which the processing of the pri-miR-202 is impaired and the mature miR-202-5p and miR-202-3p are absent. Analysis of the progeny from adult mir-202-/- revealed a low female fecundity and a high rate of early developmental arrest of fertilized eggs, suggesting a role of mir-202 during oogenesis. For further analysis, we combined flu-orescence imaging techniques and segmentation algorithms to enumerate follicles at the different stages of oogenesis in the ovary. Results revealed a significant reduction of the number of follicles recruited throughout vitellogenesis. All together our data indicate that mir-202 is likely to be important in the ovary for oocyte development and egg quality, thus providing evidence that mir-202 is a key microRNA for female fertility, and a novel maternal-effect gene.

These novel findings stress the importance of miRNAs in the control of female fer-tility and reveal the role of non-coding RNAs in the mechanisms that control fish egg quality. They also shed new light on our knowledge of the respective roles of the dif-ferent maternal-effect genes that contribute to the egg’s ability to be fertilized and subsequently develop into a normal embryo.

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Oral presentation, Session II. OOGENESIS AND EGG QUALITY

GENE EXPRESSION CHANGES ASSOCIATED WITH POST-OVuLATORy AND POST-STRIPPING OOCyTE AGEING IN COMMON CARP, CYPRinuS CARPiO

Azin Mohagheghi Samarin1*, Azadeh Mohagheghi Samarin1, Tone-Kari Knutsdatter Øst-bye2, Øivind Andersen2, Bente Ruyter2, Sabine Sampels3,4, Miroslav Blecha1, David Gela1, Tomas Policar1

1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected] (Norwegian Institute of Food, Fisheries and Aquaculture Research), P.O. Box 210, NO-1431 Ås, Norway3University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, Husova tř. 458/102, 370 05 České Budějovice, Czech Republic4Swedish University of Agricultural Sciences, Department of Molecular Sciences, PO Box 7015, 75007 Uppsala

To identify molecular mechanisms involved in oocyte ageing in common carp Cypri-nus carpio changes in the mRNA expression of selected genes were examined by real time quantitative PCR. Oocytes from 6 females were incubated in vivo for 14 hours post ovulation (HPO) and in vitro for 10 hours post stripping (HPS) at 20 °C before fertilization. Hatching rates remained more than 65% up to 4–6 HPO and thereafter decreased linearly and finally dropped to 1% at 12–14 HPO. The hatching rates were more than 70% for the eggs stored in vitro up to 6 HPS and then decreased to 21% at 10 HPS. Genes related to oxidative injury and stress response (hsp70, cox1, sod, gpx1), cell cycling (cyclinA, cyclinB, jnkA and jnkB), apoptosis (caspase3A, caspase9, bax, bcl2, cathepsinB and cathepsinZ), reproduction and germ line speciation (vasa) and devel-opmental competence (igf2) showed the upward expression trend at both in vivo and in vitro aged oocytes. The results suggest that the genes examined are associated with both post-ovulatory and post-stripping ova ageing and probably induction of egg quality defects. These transcripts might be used as markers for predicting egg quality associated with the oocyte ageing in common carp.

Keywords: Cyprinus carpio, egg quality, gene expression, maternal transcripts, oocyte ageing

Acknowledgements: This study was financially supported by the Ministry of ed-ucation, Youth and Sports of the Czech Republic – projects „CenAKVA“(no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii“(no. LO1205 under the nPU i program), Grant Agency of the University of South Bohemia in Ceske Budejovice (no. 060/2016/Z), nF--CZ07-MOP-3-184-2015, nF-CZ07-iCP-3-185-2015, GAJU projects (no. 060/2016/Z and 085/2017/P) and nAZV projects (no. QK1710310 and QJ1510117).

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TRANSCRIPTOMIC ANALySIS OF EuRASIAN PERCH (PERCA fluViATiliS) EGGS ASSIGNED TO DISTINCT GROuPS OF quALITy

Tainá Rocha de Almeida1, Bérénice Schaerlinger1*, Aurélie Le Cam2, Maud Alix1, Jérôme Montfort2, Julien Bobe2, Dominique Chardard1, Pascal Fontaine1

1University of Lorraine, Unit of Research Animal and Functionality of Animal Products, USC 340 INRA, Vandoeuvre-les-Nancy, France; [email protected], UR1037 Fish Physiology and Genomics, Campus de Beaulieu, Rennes, France

Eggs quality improvement imposes to find early indicators allowing a reliable prediction of the developmental success. Molecular indicators seem particularly interesting as they give clues to understand the causes and consequences of developmental impair-ments. However, for practical reasons, works on the eggs quality are usually performed by comparing two categories of spawn using variable criteria depending on the studied species and authors’ scientific approach of the quality. In reality, it appears that the de-velopmental success is rather reflected by a continuum from the best ones to the worst with high early mortalities or no fertilization. The present work aims at linking groups of spawn showing different developmental performances (representing a continuum of quality) to the gene expression pattern in the correspondent eggs of Eurasian perch, Perca fluviatilis. To do so, perch spawn were classified into four clusters (groups I to IV from the best to the worst developmental success) using 12 parameters characterizing the embryonic survival, deformities occurrence and the hatching period. In a second time, a microarray analysis has been performed to compare the transcriptomic profile of spawn representing each of these groups. It reveals more than 3000 genes differ-ently expressed between the groups as a whole. The difference on genes expressions we found in this study shows a higher number of genes differently expressed between the group IV when compared with the groups I, II and III than within these 3 groups. To be more precise, the transcriptomic profile only showed few genes differently ex-pressed between the groups I (better developmental success), II and III (intermediate groups) (e.g. 13 genes between groups I and II, 53 between I and III and 70 between II and III). On the contrary, more than 2000 genes were differently expressed between the group IV (early mortalities or fertilization failures) and each of the other groups of spawn. It demonstrates that the cluster IV, showing the highest and earliest level of developmental failures, mainly presents a huge transcriptomic defects that could either be due to a lack of incorporation or a premature degradation of the RNAs. The Gene Ontology study revealed that functions as the oxidative phosphorylation and the cytokinesis are particularly affected in this group. As these functions are essential for the basic functions of the cell or the early steps of the embryogenesis, their modifica-tions easily explain the early mortality or fertilization failures, with a direct association with the quality of the egg.

Keywords: Oocyte, developmental success, microarray, egg content, genomic

Acknowledgements: This study is funded in part by AnR (Agence nationale de la Re-cherche/French national Research Agency) Maternal Legacy (AnR-13-BSV7-0015). We are also thankful for inRA PHASe department for supporting this work and for the Bra-zilian national Council for Scientific and Technological Development (CnPq) and the French ministry of research for TRA and MA PhD fellowships, respectively.

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Oral presentation, Session II. OOGENESIS AND EGG QUALITY

WHy SEX TuRNS TO ASEXuALITy: THE INTERCONNECTION BETWEEN ASEXuALITy, HyBRIDIZATION AND SPECIATION

Karel Janko, Jan Pačes, Hilde Wilkinson-Herbots, Rui J Costa, Jan Roslein, Pavel Drozd, Nataliia Iakovenko, Jakub Rídl, Miluše Hroudová, Jan Kočí, Radka Reifová, Věra Šlechto-vá, Lukáš Choleva

Sexual reproduction is one of the most ubiquitous properties of eukaryotes. Howe-ver, in the course of the evolution, the molecular machinery controlling meiosis has been repeatedly disrupted in different ways leading to the emergence of many asexual lineages which produce unreduced gametes. Despite intensive research, very little is known about causes of the switch from sexual reproduction to asexuality. Interspecific hybridisation is one of the candidate explanations but it remains unclear whether ap-parent association between hybridisation and asexuality is truly causal.

We propose the hypothesis that hybrid asexuality arises as a by-product of the gra-dual accumulation of reproductive incompatibilities and thus represents an inherent stage of the species diversification process. In other words, we suggest that hybrid asexuality represents a previously unnoticed form of postzygotic reproductive isolation mechanism.

We combined cross-breeding experiments with population genetic and phylogeno-mic approaches to reveal the history of speciation and asexuality evolution in European spined loaches (Cobitis) as a model organism. Contemporary species readily hybridize in hybrid zones, but produce infertile males and fertile but clonally reproducing fema-les that cannot mediate introgressions. However, our analysis of exome data indicates that intensive gene flow between species has occurred in the past. Crossings among species with various genetic distances showed that, while distantly related species produced asexual females and sterile males, closely related species produce sexually reproducing hybrids of both sexes.

Our results suggest that hybridization leads to sexual hybrids at the initial stages of speciation, but as the species diverge further, the gradual accumulation of repro-ductive incompatibilities between species could distort their gametogenesis towards asexuality. Interestingly, comparative analysis of published data revealed that hybrid asexuality generally evolves at lower genetic divergences than hybrid sterility or in-viability. Given that hybrid asexuality effectively restricts gene flow, it may establish a primary reproductive barrier earlier during diversification than other ‘classical’ forms of postzygotic incompatibilities. Hybrid asexuality may thus indirectly contribute to the speciation process.

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ARGININE INFLuENCES FEMALE REPRODuCTIVE PERFORMANCE AND OFFSPRING GROWTH OF RHAMDiA quElEn

Danielle Zanerato Damasceno1*, Fábio Bittencourt2, Elizabeth Romagosa3

1CAUNESP, Aquaculture Center of São Paulo State University, CEP-14884-900, Jaboticabal- SP, Brazil; [email protected], State University of Western Parana, CEP 85903-000, Toledo-PR, Brazil3APTA, São Paulo Fisheries Institute, Cep:05001-900, São Paulo-SP, Brazil

Studies have shown that arginine inclusion in the feed of some mammal and bird spe-cies results in improved reproductive parameters and higher quality and survival of the offspring. However, there are few studies evaluating this amino acid effect on fish. In order to evaluate arginine effect on Rhamdia quelen reproduction, 60 females were fed with isoproteic (35%) and isocaloric (3250 kcal.kg-1) diets containing different levels of arginine (1.37, 1.67, 1.97, 2.27 and 2.57%). After five months, fish were submitted to artificial reproduction (0.5+5.0 mg kg-1 of carp pituitary extract), oocytes were collected and an aliquot was established to measure their diameter. The remaining oocytes were fertilized with a pool of semen. After collecting oocytes, fish were anesthetized for blood sampling. Then, they were euthanized, and their ovaries and livers were collect-ed to calculate the gonadosomatic, hepatosomatic indices and the nitric oxide (NO) concentration in the ovaries. Eggs were hatched and, after larvae eclosion, larviculture was conducted for 10 days. Larvae were euthanized and counted to find survival rate, and then set to measure weight and length. The inclusion of 2.27% arginine in the female R. quelen diet increased hepatosomatic index and plasmatic vitellogenin, nitric oxide concentrations in the ovaries. Consequently, this treatment showed the largest oocytes diameter (p<0.05). Larvae from females fed diets with 2.27% arginine inclu-sion showed a larger yolk sac volume and a greater length shortly after the eclosion (p<0.05). At the end of the larviculture, higher values for survival (95,95±0,33%) and growth (13,05±1,42mm) rates were observed in the same treatment (p<0.05). These results indicate that arginine acts on physiological functions that contribute to the fe-male R. quelen reproductive performance, and such effect is transmitted from mother to the offspring, thus contributing to the first stage of development.

Keywords: Broodstock nutrition, amino acids, nitric oxide

SeSSiOn iii.

GERM CELLS: FROM BASIC SCIENCES TO

APPLIED BIOTECHNOLOGIES

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SPERMATOGONIAL STEM CELLS TRANSPLANTATION AND TRANSGENESIS IN FISH

Luiz Renato de Franca

National Institute of Amazonian Research, Av. Andre Araujo 2936, Bairro Petropolis – Manaus – AM – Brazil. 69080-971; [email protected]

The in vitro genetic manipulation of several organisms has revolutionized our knowl-edge related to the molecular and biological processes.

Therefore, the techniques linked to transgenes have played a major role on the re-cently observed biotechnological progress. Because they are able to pass their genet-ic information to the offspring, the spermatogonial stem cells (SSCs) are considered a unique stem cell on the adult individuals or organisms. In this regard, the genetic manipulation of these cells results on the lifelong production of modified gametes. The SSCs transplantation is an experimental approach that consists on the removal of germ stem cells form the donor testis and their injection to a recipient testis, where they de-velop and continuously produce mature sperm with the donor genetic characteristics.

This valuable technique has recently provided substantial development on agri-culture, on the preservation of threatened or endangered species or zootechnically valuable animals and, particularly, on the development of transgenic animals. From studies beginning about one decade ago in our laboratory, all the necessary proce-dures for SSCs transplantation in adult fish were successfully developed and standar-dized, using the Nile tilapia as the experimental model. These studies were also very important for the recent establishment of specific molecular markers that allowed the isolation of SSCs in the Nile tilapia, as well as the development of a culture system in which we were able to expand in vitro the number of these cells. In this highly favorable scenario, these developed approaches allowed us to investigate important functional and molecular aspects related to the SSCs, particularly those concerning the genetic manipulation of these cells. Besides representing an excellent model for experimental studies, the Nile tilapia is the fish species responsible for the great expansion of fresh water fish production recently observed in our planet. In this regard, we are now using the transplantation of genetically modified tilapia SSCs (using for instance lentivirus as a carrier), as a biotechnological and innovative tool to efficaciously produce transgenic strains of Nile tilapia. We expect that these ongoing approaches will provide a unique opportunity to generate fish strains with superior genetic traits, which will enable the production of fish meat with high quality and even products with high biotechnological and commercial values. Thus, allowing the sustainable fish production and, ultimately, the development and welfare of our society

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Oral presentation, Session III. GERM CELLS: FROM BASIC SCIENCES TO APPLIED BIOTECHNOLOGIES

IMPLEMENTATION OF A GERM STEM CELL TRANSPLANTATION PROCEDuRE FOR THE REGENERATION OF ISOGENIC TROuT LINES

Anne-Sophie Goupil1, Ahmed Maouche1, Alexandra Depincé1, Lionel Goardon, Marjorie Bideau, Nicolas Dechamp2, Edwige Quillet2, Francine Krieg2, Jean-Jacques Lareyre1*, and Florence Le Gac1

1INRA UPR1037, Laboratory of Fish Physiology and Genomics, BIOSIT, Campus de Beaulieu, 35042 Rennes, France.2UMR 1313 GABI, Animal Genetics and Integrative Biology, Domaine de Vilvert, 78352 Jouy-en-Jo-sas, France.3INRA PEIMA, Barrage du Drennec, 29450 Sizun, France *Corresponding author: [email protected]

Isogenic fish lines are valuable animal models for aquaculture research because they allow reliable and reproducible experiments. Isogenic homozygous trout lines were generated at INRA and they are currently used to investigate the genetic and environ-mental determinism of complex traits such as disease resistance, welfare, sexual differ-entiation or feed efficiency. These lines are all-female populations because they were produced using gynogenesis. The present study was aimed to set up a standard and practical experimental procedure that could be easily implemented in fish farms to con-serve and regenerate these important genetic resources. Highly purified (>90%) and partially purified spermatogonial stem cell fractions were obtained from testicular cells of sex-reversed isogenic females (neomales) using centrifuge elutriation or a cushion of percoll, respectively. Cell fractions were injected in the abdominal cavity of conven-tional hatching triploid embryos (320 degree-days) using a cell suspension at 500000 cells/µl. Golden trout embryos, homozygous for a dominant yellow color coat variant, were used as recipient in order to discriminate potential progenies derived from the donor transplanted cells or from the recipient. The presence of donor transplanted cells was investigated from testicular genomic DNA using standard PCR and a panel of diag-nostic microsatellite markers. We showed that the success rate of the gonadal coloni-zation determined at 5 months of age was similar between the highly (74% of positive recipient fry) and partially (70%) purified spermatogonial cell fractions. The percentage of successfully transplanted spermiating males detected between 10 and 13 months of age was also similar between the highly (46%) and partially purified (50%) cell frac-tions respectively. Sperm samples were used to fertilize eggs collected from another isogenic trout line showing a wild type coat. All progenies showed a wild type coat and harbored alleles derived from the donor at all the controlled genetic markers. In addi-tion, sperm DNA genotyping showed that no allele of the golden recipients was pres-ent in the sperm. All together, our data demonstrate that triploid recipients produce functional spermatozoa derived from the donor transplanted cells only.

In conclusion, triploid male recipients show a high efficiency of germ stem cell trans-plantation and produce functional spermatozoa from the donor only. Moreover, we show that lighter procedures for cell preparation can be proposed with low impact on the transplantation success rate. The success rate of the transplantation procedure will be investigated in female trout next winter.

Keywords: Trout, spermatogonial stem cells, transplantation, triploid recipient

Acknowledgements: This study was supported by the Ue Aquaexcel2020 project.

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INTERSPECIFIC TRANSPLANTATION OF BROWN TROuT AND GRAyLING GERM CELLS INTO RAINBOW TROuT: CONSERVATION OF VALuABLE BALKAN TROuT GENETIC RESOuRCES

Jelena Lujić1*, Zoran Marinović1, Simona Sušnik Bajec2, Ida Djurdjevič2, Aleš Snoj2, Béla Urbányi1, Ákos Horváth1

1Szent István University, Department of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary; [email protected] of Ljubljana, Department of Animal Science, Biotechnical Faculty, Groblje 3, Sl-1230 Domžale, Slovenia

High endemism of species and lineages of the fish family Salmonidae require distinct population management strategies for their sustainability and preservation from mixing with introduced lineages. The aim of this study was to develop the germ cell transplan-tation technique for the endangered and endemic Balkan salmonid populations by in-ter-specific transplantation into rainbow trout recipients. Additionally, two experiments were designed to test the appropriateness of different cryoprotectants and their con-centrations for slow-rate freezing of brown trout juvenile gonadal tissue. Firstly, the dis-sociation protocol for the gonadal tissue dissociation was optimized by testing different enzymes. The use of 2 mg/ml of collagenase and 10 µl/ml DNase I was the most optimal. Additionally, labelling of germ cells by the membrane linker dye PKH-26 was optimized. Labelled spermatogonia (SGA) and oogonia (OOG) of brown trout Salmo trutta and gray-ling Thymallus thymallus were then intraperitoneally transplanted into the rainbow trout recipients (3-5 dph larvae). The average survival of recipients 60 days post-transplantation was 85.5±15.3% of control. After dissection of the recipient fry, fluorescently labelled cells were detected within the recipient gonads indicating that both SGA and OOG from both donor species could migrate within the abdominal cavity of the rainbow trout recipients and colonize their gonads. Incorporation rates varied between 23%–30%. Control individ-uals displayed no fluorescence after dissection. In addition to transplantation, we tested the possibility of cryopreservation of the gonad tissue of brown trout as a model for the Balkan salmonids. We tested the effects of 1.3 M concentration of four different cryopro-tectants (methanol - MeOH, glycerol – Gly, ethylene glycol – EG, and dimethyl sulfoxide – Me

2SO) on cell survival, while in the second experiment we tested the effects of three

different concentrations (1.0, 1.3 and 1.6 M) of the most efficient cryoprotectant. Me2SO

yielded significantly higher SGA and OOG viability comparing to the other cryoprotectants (Tukey’s HSD), while the concentration of 1.6 M of Me

2SO was the most suitable concen-

tration for both germ cell types. Overall, the transplantation and cryopreservation meth-ods demonstrated in this study could be used for the conservation and revitalization of genetic resources of endangered and endemic salmonid species or populations and thus give rise to new or improved management strategies for such species.

Keywords: Salmo trutta, Thymallus thymallus, spermatogonia, oogonia, cryopreservation

Acknowledgements: This study was supported by projects nKFiH Snn 116912 in Hun-gary, n4-0045 and P4-0220 in Slovenia, and the Stipendium Hungaricum Scholarship Programme (grant 106360 to ZM). Special thanks goes to the manager of the Bled hatchery (Slovenia) for providing juvenile fish for all the experiments and manager of the Vodomec hatchery (Slovenia) for rearing the rainbow trout fry.

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EPIGENETIC REPROGRAMMING AFTER NuCLEAR TRANSFER IN FISH

Alexandra Depincé1*, Pierre-Yves Le Bail1, Catherine Labbé1

1INRA, UR 1037 LPGP, Campus de Beaulieu, Rennes, France; [email protected]

Somatic cells are a highly convenient diploid support for biodiversity conservation: they carry the genome of both parents, and they can be collected whatever the age or sex of the donor. In fish, cryobanking of somatic cells is highly valuable because eggs and embryos cannot be cryopreserved. With those cells, the regeneration of the donor genotype will however rely on the nuclear transfer technology in which the donor cell nucleus is injected into a recipient oocyte. Although set up in the 1960ies, nuclear transfer in fish still yields severe mortalities and abnormal gene expression (Luo 2009, BOR), preventing the safely and reliably regenerating of valuable genetic resources (<1% fertile adults). It is well described in fish that the embryonic genome is activated around the 10th mitosis (Kane and Kimmel, 1993). At this stage, gamete DNA meth-ylation profile has been fully reprogrammed in a finely tuned sequence to allow early embryonic gene expression (Wu, 2011, Jiang, 2013, Potok, 2013). Nuclear transfer of a non-germinal nucleus (from somatic cell) raises the question of the oocyte ability to reprogram the injected chromatin into an embryo-like pattern.

The purpose of this study was to understand the extent of epigenetic reprogramming during early development in clones. We established the DNA methylation profile of marker genes selected according to their differentially methylated pattern between sperm and somatic cells (donor fin cell).

We studied 29 individual clones at 24h post activation in goldfish. Clone morphology was recorded prior to DNA extraction. After bisulfite conversion, we amplified the pro-motor region of 3 genes involved in early development (nanog, notail and pou2). Then, we measured the CpG methylation of these promoters by pyrosequencing.

We showed that our marker genes had hypomethylated promoter regions in sperm and in fertilized early embryos, while in clones, these regions displayed various patterns of hypermethylation which were close to the donor cell pattern. We also observed that the surviving clones at 24h had numerous morphological defects, and that the quality of the morphology is correlated to the extent of the DNA methylation reprogramming. This strongly suggests that although the injected nucleus faced 10 rounds of mitosis prior to embryonic genome activation, chromatin reprogramming was not thorough enough to set up the proper DNA methylation pattern. This demonstrates the need to improve the donor cell reprogramming in order to improve the nuclear transfer out-come.

Keywords: Somatic cell nuclear transfer, epigenetic reprogramming, DnA methylation, goldfish.

Acknowledgements: Financial support of “investissements d’Avenir“ AnR-11-inBS-0003 (CRB-Anim 2013–2019) and COST AQUAGAMeTe FA 1205.

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STuRGEON PHENOMENON: FIRST EVIDENCE OF PROGENy FROM THREE PARENTS

Viktoriia Iegorova1, Martin Psenicka1, Taiju Saito1, 2

1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Zátiší 728/II, 389 25, Vodnany, Czech Republic; [email protected] (V. Iegorova)2Nishiura Station, South Ehime Fisheries Research Center, Ehime University, Uchidomari, Ainan, Ehime 798-4206, Japan

Polyspermy is known as a penetration of the oocyte cytoplasm by several spermato-zoa. For Acipenseridae family is characteristically monospermic fertilization, but highly concentrated sperm suspensions and quite big amount of micropylles cause polysper-my. It was believed that it is fatal if more than one sperm fuses with the oocyte: poly-spermic embryos develop abnormally and perish before hatching or can develop to abnormal larvae which die. This study clearly refute such statements and demonstrate that sturgeon polysmermic organisms have mostly the same shape as a monospermic and survive well (similar to a control). Moreover, ploidy of such fish is mosaic. Our founding show that sturgeon egg can accept several spermatozoa even from different species and to be a progeny from free parents. Three species were used for fertiliza-tion: Acipenser ruthenus (4n), A. gueldenstaedtii (8n), A. baerii (8n). Two groups were performed: 1). A. baerii eggs were fertilized with mixture of sperm from A. gueldens-taedtii and A. ruthenus, and 2). A. gueldenstaedtii eggs were fertilized with mixture of sperm from A. baerii and A. ruthenus. Fertilized embryos with abnormal cleavage (3, 5, 6, 7, 9, 10 cells) were collected and kept separately until feeding stage. Ploidy was evaluated by flow cytometry (Paa Partec CCA I; Partec GmbH, Münster, Germany) using 4’, 6-diamidino-2-phenylindole (DAPI). Normal state for hybrid is a triploid (3n), produced hybrids from free parents had ploidy 4/6n which clearly demonstrate that two spermatozoa from different species were penetrated into the egg. This aston-ishing phenomenon is emphasized by the fact that it can be generated without any significant artificial intervention: viable individuals composed of three species, namely two species give rise to hybrid zygote and the third species contributes supplementary sperm-derived cells.

Key words: Sturgeon, polyspermy, mosaicism, hybridization

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TRIPLOID OR HyBRID FISH: WHICH IS THE BEST HOST FOR STEM GERM CELLS?

Diógenes Henrique de Siqueira-Silva1,2,3*, Lucas Henrique Piva1,4,Caio Augusto Gomes Goes5, Takafumi Fujimoto6, Taiju Saito7, Letícia Veroni Dragone2, José Augusto Senhori-ni2, Fabio Porto-Foresti5, José Bento Sterman Ferraz3, George Shigueki Yasui1,3

1Centro nacional de Pesquisa e Conservação da Biota Aquática Continental (CePTA-iCMBiO), Pirassununga, São Paulo, Brazil; [email protected] – Universidade Federal do Sul e Sudeste do Pará, instituto de estudo em Saúde e Biológicas (ieSB) Marabá, Pará, Brazil3USP – University of São Paulo, Faculdade de Zootecnia e engenharia de Alimentos Departamento de Medicina Veterinária, Pirassununga, São Paulo, Brazil4UneSP – University estadual Paulista, Campus de Botucatu, Programa de Pós-Graduação em Ciências Biológicas (Zoologia) Botucatu, São Paulo, Brazil5UneSP – Univ. estadual Paulista, Campus de Bauru, Faculdade de Ciências Bauru, São Paulo, Brazil6 Hokkaido University, Faculty of Fisheries Sciences, 3-1-1 Minato-cho, 041-8611 Hakodate, Japan7ehime University, nishiura Station, South ehime Fisheries Research Center, Uchidomari, Ainan, Japan

We aimed to develop an effective procedure to obtain sterile ideal host fish in mass scale, presenting no endogenous germ cells in the germinal epithelium, owning per-manent stem-cell niches able to be colonized by transplanted germ cells. Thus, trip-loids, diploid hybrids and triploid hybrids were produced. To obtain hybrid offspring, oocytes from a single Astyanax altiparanae female were inseminated by sperm from five males (A. altiparanae, A. fasciatus, A. schubarti, Hyphessobrycon anisitsi and Oligosarcus pintoi). Triploidization was conducted by inhibition of the second polar body release using heat shock treatment at 40 °C for two min. At 9-months-age, the offspring from each crossing was histologically evaluated to access the gonadal status of the fish. Variable morphological characteristics of the gonads were found in the different hybrids offspring: normal gametogenesis, gametogenesis without production of gametes, sterile specimens holding germ cells and sterile specimens without germ cells, which were considered “ideal hosts”. However, only in the hybrid derived from crossing between A. altiparanae and A. fasciatus, 100% of the individuals were com-pletely sterile. Among them 83.3% of the male did not presented germ cells inside germinal epithelium, presenting only somatic cells in the gonad. The other 16.7% be-sides sterile presented spermatogonia inside the niches. Such a methodology allows the production of sterile host in mass scale, opening new insights for application of surrogate technologies.

Keywords: Germ cell transplantation; fish chimera; ideal fish; sterile fish; tetra

Acknowledgements: We would like to thanks Centro nacional de Pesquisa e Conser-vação de Peixes Continentais (CePTA – iCMBio).

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EMBRyONIC DEVELOPMENT OF NuCLEO-CyTOPLASMIC HyBRIDS INDuCED By INTERSPECIFIC ANDROGENESIS AMONG SPECIES IN CyPRINIFORMES

Mitsuru Endoh1*, Takafumi Fujimoto1, Etsuro Yamaha2, Katsutoshi Arai1

1Hokkaido University, Faculty and Graduate School of Fisheries Sciences, 3-1-1, Minato-cho, Ha-kodate, Hokkaido, Japan; [email protected] University, Nanae Fresh-Water Station, 2-9-1 Sakura, Nanae, Kameda, Hokkaido, Japan

Nucleo-cyotoplasmic hybrids (Cybrids) comprising nucleus from one species and cy-toplasm from another species, is suitable to study interaction between nucleus and cytoplasm during embryogenesis. Cybrids can be induced by interspecific androgene-sis. Here, we induced cybrids with haploid and diploid genome from different species in Cypriniformes. Haploid cybrids were induced by fertilization of UV-irradiated zebrafish (Danio rerio: DR) eggs with haploid sperm from pearl danio (Danio albolineatus: DA), goldfish (Carassius auratus auratus: CAA) and loach (Misgurnus anguillicaudatus: MA). Interspecific DA-DR (nucleus-cytoplasm) survived until hatching and exhibited severe abnormalities like haploid syndrome. Intergeneric CAA-DR was arrested at late blastula. Interfamilial MA-DR was arrested at embryonic shield stage. Then, diploid cybrids were also induced to compare developmental ability with the haploid cybrids. The diploid DA-DR, which were induced by first cleavage inhibition, survived until hatching but exhibited abnormalities. Diploid cybrids of Carassius and Misgurnus were induced by using diploid sperm from tetraploid silver crucian carp (Carassius auratus langsdorfii: CAL) and sex-reversed clone loach (MAC). Development of CAL-DR was arrested at late blastula, as observed in CAA-DR. Development of MAC-DR was arrested at embryon-ic shield stage, as observed in MA-DR. Finally, expression patterns of zygotic gene, pan-mesoderm marker no tail (ntl), in the three types of haploid cybrids were analyzed by whole mount in situ hybridization. The expression patterns of ntl at sphere and 30–50% epiboly stages in those cybrids were not different from those of diploid zebraf-ish, though the gene expression pattern at 30–50% epiboly stage in CAA-DR could not be analyzed because of the developmental arrest at late blastula stage. In conclusion, developmental ability of cybrids was affected by combinations between nucleus and cytoplasm rather than ploidy level. The expression of zygotic genes was presumably regulated by maternally derived cytoplasm of zebrafish.

Keywords: Chromosome manipulation, cybrid, diploid sperm, embryo, mid-blastula transition

Acknowledgements: This work was financially supported in part by the Japan Society for the Promotion of Science (JSPS), KAKenHi Grant numbers 15H02457 and 17J02645.

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DEVELOPMENT OF PRIOMORDIAL GERM CELLS AND PIGMENT CELLS IN EASTERN LITTLE TuNA, EuTHYnnuS AffiniS, EMBRyOS

Taiju Saito, Rie Goto, Takahiro Matsubara

Ehime University, South Ehime Fisheries Research Center (SEFRC), 25-1 Uchidomari, Ainan, Min-amiuwa-gun, Ehime, 798-4206 Japan; [email protected]

Kawakawa (Eastern Little Tuna), euthynnus affinis, is an animal that carry out the em-bryonic development in the most severe condition. Kawakawa is a species of tuna that mainly lives in the tropical and subtropical waters of the Indo-West Pacific. They spawn eggs in the open ocean from spring to autumn depending on the spawning ground. The eggs have positive buoyancy, and they develop at the water-surface layer. Namely, the embryos need to develop in the extremely intensive ultra violet irradiation under the (sub)tropical sun. Furthermore, the egg has a large oil droplet that is thought to act as a sphere lens. Here, we pose a question: how do kawakawa embryos, especially primordial germ cells, avoid “burning” caused by intense sunlight during their develop-ment? To answer the question, we studied the spawning pattern, the development of primordial germ cells (PGCs) and pigment cells in embryos.

First, we observed spawning behavior of kawakawa in the net cages located at Misho-bay, Ainan, Ehime prefecture, Japan (32°57’57.6”N, 132°30’12.6”E), by using digital vid-eo cameras placed on the bottom and on the cages. The water temperature was logged at 3 hour intervals. We collected eggs by nets from the cages just after spawning, and incubated them at 24 °C to observe the embryonic development. GFP-Bucky ball (buc) mRNA, RFP-zebrafish-nos3 3’UTR mRNA, platinum TALEN RNAs, morpholino oligonucle-otides (MO) were injected into the blastodisc during 1 to 2 cells stage.

Kawakawa started spawning from June when water temperature at the surface reached around more than 23 °C. They exhibited broadcast spawning, which is char-acterized by chasing of females by males and rising above the water surface, before sundown. Aggregations of GFP-positive germ plasm were observed for the first time at the marginal region of the blastodisc at the 2-cell stage. The cells contain GFP-positive germplasm could be isolated by a cell sorter (PERFLOW® Sort, SHIMAZU). PGCs were observed in the germ ring around the yolk plug at the late gastrula period. They aggre-gated into two clusters at the both side of the embryonic body as the embryo develops and finally positioned at the gonadal ridge. Two types of pigment cells appeared in the embryos after 15 hpf. One type was confirmed as melanocytes: they disappeared after injection of antisense MO or platinum TALEN RNAs targeted tyrosinase gene. Another type was unidentified pigment cells, although they seem to be the neural crest origin. They showed brown color in the bright field and emitted green fluorescence when im-aged with GFP filter set. They located underneath the oil droplet and surrounded the PGCs during the process of early embryonic development and disappeared after 3 days post fertilization. These results provoke the assumption that kawakawa spawn eggs before sundown to let the transparent early embryos develop in the absence of intense sunlight. Then the next day, the pigment cells develop as a “sunshade” to prevent the PGCs from intense light during the daytime.

Keywords: Primordial germ cells, pigment cells, genome editing, microinjection, tuna

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EXPRESSION PROFILING OF NEW GERMLINE MARKERS SuGGESTS SuCCESSIVE WAVES OF DIFFERENT SPERMATOGONIAL STEM CELL SuBPOPuLATIONS DuRING RAINBOW TROuT ONTOGENESIS

Ahmed Maouche1, Edouard Curran1, Aude Gautier2, Anne-Sophie Goupil1, Elisabeth Sambroni1, Elodie Dupin de Breyssat1, Yann Guiguen1, Taiju Saito3, Florence Le Gac1, Jean-Jacques Lareyre1*

1INRA UR1037, Laboratory of Fish Physiology and Genomics, Campus de Beaulieu, 35042 Rennes cedex, France; [email protected] BOREA, Biology of Aquatic Organisms and Ecosystems, UNICAEN, MNHN, UPMC, CNRS-7208, IRD-207; Université de Caen Normandie, CS14032, 14032 CAEN, Cedex 5, France3 South Ehime fisheries research center, Minamiuwa, Japan

In adult males, the seasonal or continuous production of spermatozoa in the testes is dependent on spermatogonial stem cells (SSC) that are capable to self-renew or to differentiate into spermatozoa. It is well admitted that adult SSC originate from em-bryonic primordial germ cells (PGCs). However, the molecular mechanisms underlying the production of the SSC stock prior to spermatogenesis onset and the regulation of germ stem cells homeostasis remain poorly understood particularly in fish. Our study was aimed to identify SSC markers suitable to investigate their emergence during on-togenesis and to unravel candidate paracrine regulatory pathways involved in their decision balance to self-renew or to differentiate into committed spermatogonia.

We first generated transgenic zebrafish lines that express the GFP reporter gene in adult SSC or in Sertoli cells. The fluorescent cells were isolated using the FACS technique and their distinct transcriptomes were determined using a next generation sequencing technique (RNAseq, Illumina). Additional samples were included in the transcriptomic analysis (germ cell less testes and mature adult testes). The differential analysis of the transcriptomes between the different testicular cells and tissues allowed us to identify a cluster of genes preferentially expressed in undifferentiated spermatogonia. Some of the clustered genes encode for SSC markers previously proposed in mammalian or fish species. Other genes encode for factors previously involved in the transition between PGCs and spermatogonia in mammalian species. We investigated further additional genes encoding for membrane bound receptors to determine whether they could be involved in the formation of the SSC stock in trout prior to the initiation of the sea-sonal reproductive cycle. A phylogenetic analysis showed that all candidate markers were conserved in teleostean fish species including trout. More, we demonstrated that some of the candidate SSC markers were also preferentially expressed in highly purified trout SSC fractions. Interestingly, the candidate SSC markers showed different temporal expression patterns during trout ontogenesis (from 35 to 125 dpf) sugges-ting the occurence of successive waves of distinct spermatogonial populations prior to spermatogenesis onset. In addition, our data highlight a candidate paracrine regulatory pathway that may play an important role in the formation of SSC stocks before the initiation of the seasonal reproductive cycles.

Keywords: Stem cell biology, Spermatogonial stem cells, transcriptome, spermatogen-esis, fish

Acknowledgements: the study was supported by the AQUAGAMeTe COST.

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MEIOSIS RESuMPTION AND EARLy MITOSIS PATTERN AFTER SOMATIC CELL NuCLEAR TRANSFER IN GOLDFISH

Charlène Rouillon*1, Alexandra Depincé1, Pierre-Yves Le-Bail1 and Catherine Labbé1

1INRA LPGP-Fish Physiology and Genomics, Rennes, France; [email protected], *presenting author

The cryopreservation of genetic resources is of major interest for the security of bio-diversity and for the sustainability of agronomic industry. Cryopreservation of somatic cells, which carry the genome of both parents, is one mean to regenerate functional breeders, but require the mastering of nuclear transfer. This technique involves donor cell injection into a recipient oocyte. The clones obtained possess the same genetic heritage as the fish donor. However, in goldfish, a peak of mortality of clone is observed after the 1000 cells stage, when the mitotic checkpoints are established.

The oocyte receives a differentiated cell whose nuclear dynamics is very different from a spermatozoon. We hypothesized that incomplete reprogramming of the somatic cell by ovarian factors induces a perturbation of the cellular events essential for embryonic development. The aim of this work was to understand the nature of the defects induced after the nuclear transfer, at the time of the meiosis resumption and early mitosis. We had first to set up an in vivo procedure to characterize the nuclear events af-ter meiosis resumption in control fertilized embryos, before analyzing the clones. By a DNA labelling, we have shown that before and after oocyte activation, polar body and maternal chromatin can be characterized but, depending of the eggs, a high heterogeneity in genetic material distribution was observed, making clones defects difficult to assess. As an additional strategy, a comparative study of genetic material distribution and spindle constitution of fertilized embryo and clone was developed. By immunochemistry and DNA labelling, the conformity of embryonic blastomeres di-visions could be analysed. These results allow analyzing the importance of the mitotic dynamics of the donor cell during early embryonic development. The expected out-comes of the project are to identify means to improve the regeneration of individuals by nuclear transfer.

Keywords: nuclear transfer, reprogramming, early mitosis, somatic cell, conservation

Acknowledgements: CR is recipient of an inRA-PHASe and Région Bretagne fellowship. This work is funded by the French CRB Anim project «investissements d’avenir», AnR-11-inBS-0003.

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INDuCED POLySPERMy IN POND LOACH, MiSguRnuS AnguilliCAu-DATuS: CyTOLOGICAL BEHEVIOR OF SPERM NuCLEI INTRuDED INTO DECHORIONATED EGGS

Kota Yokoyama1, Takafumi Fujimoto1, Katsutoshi Arai1*

1Hokkaido University, Faculty and Graduate School of Fisheries Sciences, Minato 3-1-1, Hakodate, Hokkaido, 041-8611, Japan ([email protected])

Micropyle and chorion of teleost eggs prevent polyspermy. Thus, polyspermy can be easily induced by insemination of dechorionated eggs. Previously, we found high fre-quencies of androgenetic embryos with haploid cells, when intra-cytoplasmic injection of multiple numbers of spermatozoa was conducted in pond loach. Here, we observed behaviors of sperm nuclei intruded into water-activated and then dechorionated eggs after compulsory polyspermy.

Wild-type female and orange-mutant male pond loach were used. Ovulation and spermiation were induced by hCG injection. Two min after activation with ambient water, egg chorion was removed by gentle shaking in dechorionation solution (0.1% trypsin, 0.4% urea in physiological saline for freshwater fish). Dechorionated eggs were then rinsed and about 200 eggs were inseminated with sperm solution nine to 12.5 min after the activation. Eggs were individually incubated in 96 well plate. Normally fertilized eggs were used as control group. Survival rates were calculated at the stage of cleavage, blastula, gastrula, somitogenesis, and hatching. Ploidy was assessed by flow cytometry and parentage was genetically analyzed by microsatellite genotyping. Embryos were fixed and prepared for histological sections and microscopy.

Although most embryos from polyspermy exhibited irregular cleavage and abnormal appearance, about 0.2–1.1% of polyspermic embryos survived at hatching. Among 34 embryos examined, 27 were haploids and 4 were mosaic comprising haploid cells. Most haploids were androgenotes, because they had only paternally-derived microsatellite markers. Decondensed sperm nuclei became male pronuclei, but androgenetically de-veloped without fusion with female pronucleus. Tripolar spindle was frequently ob-served and might cause aberrant cleavage to form anuclear blastomeres.

Key words: Androgenesis, dechorionation, fertilization, haploidy, pronucleus

Acknowledgments: This study was supported in part by JSPS-KAKenHi, grant number 25660161 and 15H02457.

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SINGLE AND MuLTIPLE SOMATIC CELLS NuCLEAR TRANSFER IN CRITICALLy ENDANGERED SPECIES, STuRGEON

Effrosyni Fatira1*, Catherine Labbe2, Alexandra Depince2, Victoriia Iegorova1, Kseniia Pocherniaieva1, Hilal Güralp1, Miloš Havelka3, Martin Pšenička1, Taiju Saito1,4

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Laboratory of Germ Cells, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected] 2French National Institute for Agricultural Research, Fish Physiology and Genomics Institute, F-35000 Rennes, France 3Hokkaido University, Faculty of Fisheries Sciences, 041-8611 Hakodate, Japan4Ehime University, South Ehime Fisheries Research Center, 25-1, Ainan, Japan

Reconstruction techniques like somatic cell nuclear transfer (SCNT) could help in stur-geon’s reproduction as it is highly endangered species mainly due to its precious roe. Future protection of the wild sturgeon populations can be achieved by using cloned females for caviar consumption.

The aim of the current project is to examine if SCNT can be applied in real endangered species. Successfully have been established several steps: donor’s fin-tissue dissocia-tion (95±5% cell viability and 275 000±10 000 cells/ml PBS cell density), suitable oo-cytes’ extender solution that keeps the oocytes inactivated and the donor-cells alive, and depth-injection into the oocyte’s animal pole without harming the host. Inter-species single SCNT was performed using as a fin-donor the Russian sturgeon, and as egg-recipient the sterlet. One feeding transplant (28 days post activation) has been resulted (0.5%) exhibiting two ploidy levels (2N and 4N) and sharing the DNA between donor and recipient, as non-enucleated and non-activated oocytes have been used. Concerning the multiple-SCNT, many fin-cells were injected in sterlet recipients (~389 cells/egg). Donor cells were originated from albino sterlet (Intraspecies SCNT) or Rus-sian sturgeon or beluga (Interspecies SCNT). Only 3 embryos from beluga x sterlet group could successfully reach the 2/3 of epiboly (2.3%) and the use of species-spe-cific primers revealed chimerism in 2 embryos. The rest groups albino x sterlet and Russian x sterlet could successfully formed the blastopore in gastrula stage (11.1% and 7%, respectively) and thereafter stopped their development.

Both single and multiple fin-cells transplants exhibited normal development accord-ing to the control group (sterlet embryo). In addition, it seems that in sturgeon-SCNT the critical stage for transplants’ survival is gastrula and not the blastula like is in Tele-ostean-SCNT. Overall, the current project proves to be the first evidence of application of cloning technique to real endangered species.

Keywords: interspecies SCnT, intraspecies SCnT, chimera, single and multi fins micro-injection, sturgeon

Acknowledgements: The study was supported from Grant Agency of the University of South Bohemia in České Budějovice; GAJU Fatira 068/2017/Z 2017.

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FIRST EVIDENCE IN FISH OF ALTERNATIVE DEAD EnD gEnE SPLICING

Ana Carina Nogueira Vasconcelos1,2*, Anna Octavera1, Danilo Pedro Streit Jr. 2, Goro Yoshizaki1

1Tokyo University of Marine Science and Technology, Department of Marine Biosciences, 108-0075 Tokyo, Konan, 4 Chome-5-7, Japan; [email protected] Univerity of Rio Grande do Sul, Department of Animal Science, Bento Gonçalves Avenue 7712, Porto Alegre, Rio Grande do Sul, Brazil

The Amazon species Colossoma macropomum is one of the highly exploited species in Brazil due the appreciated flavor and rapid growth. However, declining of its natural resources due to overexploitation and the climate change is concerned. Although germ cell cryopreservation and transplantation can be a silver bullet to preserve their genetic resources semi-permanently, their germ cell behavior has not been analyzed to date. Therefore, in this study, we isolated tambaqui dnd homolog as a molecular marker for primordial germ cells (PGCs) and undifferentiated mitotic germ cells, in order to ob-tain basic information essential for germ cell transplantation. First, the total RNA was extracted from the gonads of immature and mature tambaqui. RT-PCR was performed with degenerate primers that were designed using the highly conserved regions of dead end gene (dnd) homologs from various fish species. Amplified cDNA fragments were sequenced and 5’ and 3’ rapid amplification of cDNA ends (RACE) was performed. The whole gonads of tambaqui were fixed using Bouin’s solution, cut into 4 µm-thick sections and used for in situ hybridization with dnd probe. The cloned cDNA had an open reading frame of 1194 bp that begins with the start codon, ATG, at the position 79 and stop codon, TAA, at the position 1272. The amino acid sequence was inferred to encode 398 amino acid residues, which showed 84% identity to Pygocentrus nattereri dnd gene. Phylogenetic analysis revealed that the sequence obtained in this study belongs to the dnd family. RT-PCR products showed that dnd was detectable only in testes and ovaries of immature and mature fishes, and three different fragments of dnd were found in tambaqui as a result of the alternative splicing. In situ hybridization showed that the cells had strong positive signal in oocytes and weak signal in oogonia. Thus, we successfully isolated tambaqui homolog of dnd and confirmed that it can be used as a potential germ cell marker. Further study to determine the function of differ-ent transcripts of dnd is currently ongoing.

Keywords: Molecular biology, Tambaqui, sequencing, primordial germ cells, gene ex-pression

Acknowledgements: The study was supported by Grant-in-Aid for Scientific Research (KAKenHi – Japan) on innovative Areas, ‘Mechanisms regulating gamete formation in animals’ (#25114005) and Conselho nacional de Desenvolvimento Científico e Tecno-lógico (CnPq – Brazil).

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SPERMATOGONIAL STEM CELLS TRANSPLANTATION IN A LITTLE SHARK, THE DOGFISH SCYliORHinuS CAniCulA L.

Laura Gribouval1,2, Aude Gautier1, Pierrïck Auvray2, Pascal Sourdaine1*

1Université de Caen Normandie, UMR BOREA, Biology of Aquatic Organisms and Ecosystems, UNICAEN, MNHN, UPMC, CNRS-7208, IRD-207; CS14032, 14032 CAEN, Cedex 5, France; [email protected] 2KELIA, Parc Technopolitain Atalante Saint Malo, Saint-Malo, France

The continuous production of spermatozoa during adult life is sustained by the sper-matogonial stem cells (SSCs). Our aim is to develop their study on the shark S. canic-ula. This cartilaginous fish is at a key phylogenetic position for evolutionary studies concerning spermatogenesis. Ten years ago, we started the SSC characterization of the dogfish and developed cell-ultrastructure, proteomic, molecular and cell-culture approaches. That way allowed us identification of markers of differentiating sper-matogonia such as hmgb3 (High-mobility group box 3) or mcm6 (Minichromosome maintenance complex component 6) and markers of undifferentiated spermatogonia such as gfra1 (glial cell line-derived neurotrophic factor family receptor alpha 1), pou2 (an homolog of the pluripotency factor POU5F1) and SSEA4 (stage-specific embryonic antigen 4). Those markers allowed to identify signaling involved in the control of SSCs. A long-term in vitro maintenance of spermatogonia has been successfully obtained by addition of GDNF (glial cell line-derived neurotrophic factor), the ligand of the GFRa1 receptor, with the formation of clones of spermatogonia expressing stem cell char-acteristics. Amplification of SSCs by culture, gave us the opportunity to consider cell transplantation experiments in order to demonstrate their stemness by their ability to colonize host gonads. Due to the presence of a complex male genital tract, with clasp-ers and epididymis, cell transplantation in embryos was envisaged rather than through-out the urogenital papilla of adults as commonly done for teleosts. Based on previous reports on developmental stages and our own histological observations, the stage of the host embryo corresponding to the timing of primordial germ cell colonization was chosen for our preliminary transplantation assays with PKH26 labelled SSCs. For this, host embryos were kept in their eggshell wall to prevent rupture of the particularly vulnerable vitelline vesicle. Presently, embryos were maintained alive during one week only after transplantation but this allowed us to detect fluorescent cells in the recipi-ent. This is still underway and its success would be pioneering for sharks and could be, in the future, an approach for chondrichthyes preservation.

Key words: Spermatogonia, stem cells, culture, transplantation, evolution, sharks

Acknowledgements: This study was supported by CRBn / FeDeR. Laura Gribouval ben-eficiated of a PhD grant CiFRe AnRT KeLiA.

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ESTABLISHMENT OF IN VITRO CuLTuRE CONDITIONS OF STuRGEON GERM CELLS

Xuan Xie1,2,3#, Ping Li1,2,3#, Martin Pšenička2,3, Huan Ye1,3, Kseniia Pocherniaieva 2,3, Jie Ma1,3, Lingbing Zeng1,3, Chuangju Li1,3 and Qiwei Wei1,3,*

1Chinese Academy of Fishery Sciences, Key Laboratory of Freshwater Biodiversity Conservati-on, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Wuhan, China; [email protected] of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic3Sino-Czech Joint Laboratory for Fish Conservation and Biotechnology, Yangtze River Fisheries Institute, Chinese Academy of Fishery Sciences and Faculty of Fisheries and Protection of Waters, South Bohemian University, Wuhan, China and Vodňany, Czech Republic

To expand germ cells populations and sustain the supply for long periods for transplantation, we established basal culture conditions for germ cells in endangered species, sturgeons, and improved their mitotic activity by eliminating gonad soma-tic cells, supplementing with growth factors, and replacing fetal bovine serum (FBS). Germ cells cultured in Leibovitz’s L-15 medium (pH 8.0) supplemented with 5% FBS at 21°C had a higher rate of survival and mitotic activity. Proliferation of germ cells was promoted and maintained for long periods by elimination of gonad somatic cells and culture under feeder-free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor. Replacing FBS with a new serum-free culture me-dium improved germ cells proliferation. Mitotic activity increased nearly 11-fold at 10 d of culture compared with initial conditions and was maintained for >34 d. Morphology remained similar to that of fresh germ cells for at least 38 d. Finally, more than 40 days cultured germ cells showed colony and transplantability after transplantation. This stu-dy provides useful information for culturing germ cells in sturgeons. The development of germ cell culture could be useful in germ-cell xenotransplantation and bioenginee-ring.

Keywords: Sturgeon, germ cell culture, glial-cell-derived neurotrophic factor, leukemia inhibitory factor, serum-free, transplantation

Acknowledgements: This study has been financially supported by the Special Scientific Research Fund for Central non-profit institutes, Chinese Academy of Fishery Sciences (grant number 2015B02YQ01), the Special Fund for Agro-scientific Research in the Public interest of the Ministry of Agriculture of China (grant number 201203086), the national natural Science Foundation of China (grant number 31402301), the Minis-try of education, Youth and Sports of the Czech Republic-projects “CenAKVA” (grant number CZ.1.05/2.1.00/01.0024), “CenAKVA ii” (grant number LO1205 under the nPU i program), and the Czech Science Foundation (grant number 16-02407Y).

SeSSiOn iV. GAMETE “OMICS”

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SMALL REGuLATORy TRANSCRIPTOME IN FISH GONADS, GAMETES AND EARLy EMBRyOS

Igor Babiak1*, Teshome Tilahun Bizuayehu1,2, Christopher Presslauer1, Asan Meera Sa-hib Haja Mohideen1

1Nord University, Faculty of Biosciences and Aquaculture, PO Box 1490, 8049 Bodø, Norway; [email protected] 2Present address: University of Bergen, Department of Informatics and Sars International Centre for Marine Molecular Biology, 5008 Bergen, Norway

Small non protein-coding RNAs (ncRNAs) are involved in various regulatory processes of an organism, yet their prevalence and functions during the initial stages of deve-lopment remain largely unknown. To have an insight into the landscape and functions of ncRNAs during germline and embryonic development, we have constructed a hybrid See-Thru-Gonad zebrafish line, which enables in vivo gonad imaging and functional studies. We analysed the dynamics of microRNA (miRNA) in gonads and embryos, and performed functional characterization of chosen miRNAs. Although miRNAs are gene-rally highly conserved in Metazoans, their regulatory features can be highly varying. We performed comparative analyses of gametic and embryonic miRNAomes of several phylogenetically distant fish species, including Atlantic salmon, Atlantic halibut, Atlan-tic cod, and zebrafish. Also, we elucidated the exact origin of “sperm miRNA“ and “egg miRNA“, which can actually be composed of both germline and somatic supporting cell originating miRNAs. We demonstrated the role of miRNA in egg developmental potential. Piwi protein-interacting RNAs (piRNAs) have known functions in silencing of transposable elements in the germline; tRNA-derived fragments (tRFs) are another abundant class of ncRNAs with implicated functions in cell proliferation and stress re-sponse. We characterized the dynamic expression of both classes throughout the go-nadal and embryonic development of zebrafish. In conclusion, our work shows massive occurence, dynamic changes in abundance, and functional relevance of small ncRNAs during critical stages of germline and embryonic development in fish.

Keywords: embryo, germline, miRnA, piRnA, tRF

Acknowledgements: We thank FishmiR team (J. Postlethwait, T. Desvignes, P. Batzel, J. Snydes – Oregon University; P. Aleström, L. Martin – norwegian University of Life Sciences; H. Aanes – Oslo University), colleagues from nord University (M. Kopp, K. Razmi, T.e. Jørgensen, J.M.O. Fernandes, S. Johansen), and M. Mommens (Aquagen SA), for their contribution to the studies reviewed in the current presentation. The study was supported by nord University, Research Council of norway (# 213825), and MABiT program (#AF0057).

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Oral presentation, Session IV. GAMETE “OMICS”

THE INVALIDATION OF THE FSHR GENE IN ZEBRAFISH LEADS TO A GAIN OF BODy WEIGHT, A BIASED SEX-RATIO AND A DECREASE IN SPERM quALITy

Elisabeth Sambroni*, Amélie Patinote, Florence Le Gac, Jean-Jacques Lareyre

LPGP, INRA, 35000 Rennes, France; [email protected]

In fish, Fsh is an important hormone regulating both gonadal functions, gametogene-sis and steroidogenesis. The biological actions of Fsh depend on its binding to a mem-brane receptor (Fshr) that belongs to the superfamily of the G protein-coupled recep-tor (GPCR). This study aimed at better understanding the physiological implications of Fshr signalling in fish. Using the CRISPR/Cas9 method we disrupted the fshr gene in zebrafish. We selected three CRISPR sites in the 10th exon of the gene to ensure a de-letion easily detectable by standard PCR. Two mutant lines were generated from inde-pendent F0 founders. An expected large deletion within the 10th exon (>1200 bp) was confirmed by DNA sequencing in both lines. This deletion led to a predicted truncated protein in which the whole transmembrane and intracellular domains are lacking. Het-erozygous (fshr+/-) male and female F1 siblings were mated to generate homozygous (fshr-/-) F2 mutants. The expression levels of normal and truncated fshr transcripts were consistent with the number of gene copies in the 3 genotypes. As previously reported (Zhang et al, 2015), homozygous mutants were all fertile males suggesting that Fshr is required for female sex differentiation and/or oogenesis. Besides, our study also demonstrated additional phenotypes. First, heterozygous zebrafish population (fshr+/-) showed a female biased sex ratio. Moreover, the body weight of heterozygous females was about 30% higher than that of non-mutated females. Likewise, the body weight and the length of the homozygous males were significantly increased compared to the non-mutated and heterozygous males. We also observed a low sperm count and quality in the homozygous mutant. Such phenotypes resemble to those described in mice KO for FSHR. To gain insights into the underlying mechanisms we measured the expression levels of genes involved in gametogenesis or steroidogenesis. In mature adult males the expression levels of transcripts encoding for different steroidogenic enzymes were decreased in heterozygous and much more in homozygous fish. Inter-estingly, they were also decreased in heterozygous females. Our data suggest that Lhr cannot compensate for the loss of the steroidogenic activity of the Fsh receptor signal-ling. Furthermore, we provide evidence for the first time in fish that the loss of a single functional copy of fshr affects both reproductive and growth traits.

Keywords: Zebrafish, Fsh receptor, Crispr/Cas9, steroidogenesis, gametogenesis

Acknowledgements: This work was supported by the BiOSiT research infrastructure.

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CRISPR/Cas9 DISTuRBANCE OF THE MuLTIPLE VITELLOGENIN (VTG) SySTEM: ESSENTIAL FuNCTIONS OF DIFFERENT VTG TyPES DuRING ZEBRAFISH EGG, EMBRyO AND LARVAL DEVELOPMENT

Ozlem yilmaz1,3*, Amelie Patinote1, Thaovi Nguyen1, Emmanuelle Com2, Charles Pine-au2, Amaury Herpin1, Julien Bobe1

1INRA, UR1037, Laboratory of Fish Physiology and Genomics, Campus de Beaulieu, 35042 Rennes Cedex, France.2Protim, Inserm U1085, Irset, Campus de Beaulieu, 35042 Rennes Cedex, France3Present address: University of Bergen, Institute for Biology, 5034, Bergen, Norway, [email protected]

Involvement of the multiple vitellogenin (Vtg) system in vertebrate yolk formation and oocyte growth has been extensively studied, but little is known about specific contributions of the different types of Vtg to the developmental competence of eggs or resulting embryos and larvae. The objective of this study was to investigate these contributions using CRISPR/Cas9 technology in the zebrafish (Danio rerio), an estab-lished biomedical model for vertebrate development. The complex zebrafish vtg gene repertoire includes five type 1 (vtgs1, 4, 5, 6, & 7), one type 2 (vtg2), and one type 3 (vtg3) genes encoding three different types of proteins, Vtg1, Vtg2 and Vtg3, respec-tively. Our study targeted type 1 vtgs for knock out (KO) collectively using common target sites and targeted vtg2 and vtg3 employing gene-specific targets. Pure lines of zebrafish carrying single type mutations, proven by conventional PCR genotyping and sequencing, for vtg1 and vtg3 have been established at generation F4; development of pure vtg2 mutant lines is still in progress. Homozygous vtg1 and vtg3 KO F4 zebraf-ish larvae exhibited clear morphological phenotypes involving abdominal edema and spinal cord defects while heterozygous vtg2 KO F3 zebrafish eggs exhibited vitelline membrane defects resulting in embryo mortality. Although no differences in fecundity, fertility, egg diameter or larval size were noted between KO and wild type (Wt) females for any line, embryonic and larval survival rates and egg hatching rates were altered by vtg gene KO. The KO of genes encoding type 1 Vtgs resulted in no larval survival after 20 days post fertilization (dpf), while vtg3 KO resulted in high levels of late embryo mortality, delayed hatching, and reduced hatching rates, with only 3–15% progeny sur-vival after 20 dpf. The results of this study have, for the first time, definitely shown that type 1 Vtgs and Vtg3 are essential for embryonic and larval development and survival while Vtg2 may have important roles at earlier stages, during egg and early embryo development.

Key words: CRiSPR/Cas9, vitellogenins, zebrafish, egg, embryonic and larval develop-ment, abdominal edema, vitellin membrane

Acknowledgements: This study was supported by the Region Bretagne in France (SAD-2013)-FiSHeGG (8210); Project #13009218), the eC-Marie Skłodowska-Curie Actions within the frame of the ieF program (FP7-PeOPLe-2013-ieF; FiSHeGG: Project # 626272) and Maternal Legacy (AnR-13-BSV7-0015).

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DuPLICATION OF NANOS1 GENE AND EXPRESSION IN DOGFISH GONADS

Laura Gribouval1, Pascal Sourdaine1, Jean-Jacques Lareyre2, Johanna Bellaiche2, Florence Le Gac2, Sylvie Mazan3, Aude Gautier1*

1UMR BOREA, Biology of Aquatic Organisms and Ecosystems, UNICAEN, MNHN, UPMC, CNRS-7208, IRD-207; Université de Caen Normandie, CS14032, 14032 CAEN, Cedex 5, France; [email protected] UPR1037, Laboratory of Fish Physiology and Genomics, BIOSIT, Ouest-Genopole, Campus de Beaulieu, 35042 Rennes, France3CNRS-UPMC-Sorbonne Universités, UMR 7232, Oceanological observatory, 66650 Banyuls sur mer, France

Nanos are highly conserved RNA-binding proteins involved in the translational con-trol. In Vertebrates, three nanos genes have been identified except in Teleosts which displayed an additional copy of nanos1. Nanos proteins are well known for their impli-cation in the germline cell development and the maintenance of germline stem cells. In mammals and fish, Nanos1 was predominantly detected in brain but also described in gonads, Nanos2 generally presented an expression limited to germline stem cells and Nanos3 an expression in primordial germ cells. Nanos3 was also found in oocytes in fish and in various stages of spermatogonia in mice. Chondrichthyes, which includes chimera, sharks and rays, have a key phylogenetic position at the root of Vertebrates. In an evolutionary purpose, this study aimed to identify nanos genes and their expression profiles in a chondrichthyan species.

in silico analysis revealed the presence of four nanos genes in Chondrichthyes. Phyloge-netic and syntenic analyses suggested that nanos1 gene was duplicated prior the diver-gence between Chondrichthyes and Osteichthyes. Shark and ray displayed both copies, similarly to spotted gar and coelacanth. In contrast, Teleost fish would have lost a gene copy and duplicated the second one (re-named t-nanos1 (a/b)); Mammals would have kept the other copy only (re-named m-nanos1). nanos2 and nanos3 were also found in whale shark and chimerae genomes but not in dogfish transcriptomic databases. Due to its abun-dance on our coast, the small-spotted dogfish Scyliorhinus canicula was used as a model to study the expression profiles of m-nanos1 and t-nanos1. By real-time PCR, m-nanos1 was mainly detected in brain whereas t-nanos1 showed a more ubiquitous expression pat-tern in male and female. In the polarized testis, m-nanos1 had a large distribution, like Nanos1 in human and rodents, whereas t-nanos1 expression progressively decreased from the niche to meiotic areas. Both transcripts were localized by in situ hybridization in testic-ular germ cells, oocytes in primordial follicles and follicular cells in vitellogenic follicles. This was confirmed by immunohistochemistry with a particular localization of tNanos1 protein, associated to chromosomes in spermatogonia and primary spermatocytes in division. To conclude, this study brought a new insight in nanos gene evolution in Vertebrates with a duplication of nanos1 prior the emergence of Gnathostomes and the maintenance of only m-nanos1 in mammals and t-nanos1 in Teleosts. As both transcripts were found in shark gonads, their respective functions in reproduction would be to explore.

Keywords: nanos, germ cells, follicular cells, Chondrichthyes, gene evolution

Acknowledgements: This study was supported by CRBn / FeDeR. Laura Gribouval ben-eficiated of a Ph.D. grant CiFRe AnRT KeLiA.

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NOVEL MATERNAL-EFFECT GENES SHED NEW LIGHT ON THE MOLECuLAR MECHANISMS CONTROLLING FISH EGG quALITy

Caroline T. Cheung1, Thaovi Nguyen1, Aurélie Le Cam1, Jérôme Montfort1, Julien Bobe1

1INRA, UR1037 Fish Physiology and Genomics, Campus Beaulieu, Rennes, [email protected]

In fish, as in other animals, maternal-effect genes (i.e. genes expressed by the moth-er and are critical for embryonic success) play important roles in the molecular mech-anisms that control the egg’s ability to be fertilized and subsequently develop into a normal embryo. Over the last few years, we have initiated several genomic screens to identify novel maternal effect genes that would broaden our knowledge of the mo-lecular mechanisms behind fish egg quality. These studies were conducted with spe-cial interest for evolutionary conserved genes/mechanisms that would be shared by evolutionary distant species. This work was carried out using zebrafish (Danio rerio), a species with short generation time that is therefore especially suitable for genome editing using the Crispr/CAS9 technology.

in silico analysis has revealed two nucleoplasmin 2 (npm2) genes, designated as npm2a and npm2b, the latter of which has been previously described as a maternal-ef-fect gene in several vertebrates including zebrafish, Xenopus, and mouse. We showed that the two npm2 genes are locally duplicated from an ancestral npm2 gene and that both npm2a and npm2b are predominately expressed in the ovary. Functional analy-ses using the CRISPR/Cas9 system showed that both npm2 genes are maternal-effect genes that play essential yet distinct roles during early development. npm2a functions very early during embryogenesis, at or immediately after fertilization, while npm2b is involved in processes leading up to or during zygotic genome activation.

In order to expand our knowledge on the repertoire of maternal-effect genes, we performed microarray analysis on zebrafish fertilized eggs at the one-cell stage to ob-tain a global portrait of the egg transcriptome to determine if it has any association with developmental competence and to identify new candidate maternal-effect genes. Among the differentially expressed genes, fam105ba and slc29a1a were found to be increased in good quality eggs and predominantly localized in the ovaries. Thus, knock-out models of these two genes using the CRISPR/Cas9 system were created, and we observed remarkable subfertility in both transgenic animals whereby the embryos from these mutants failed to develop and were arrested at the earliest stage.

These novel findings will broaden our knowledge on the evolutionary diversity of maternal-effect genes and underlying mechanisms that contribute to vertebrate repro-ductive success.

Keywords: zebrafish, egg, transcriptome, mutant, npm2

Acknowledgements: This study is funded by AnR (Agence nationale de la Recherche/French national Research Agency) Maternal Legacy (AnR-13-BSV7-0015).

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INVOLVEMENT OF THE TRANSCRIPTIONAL COACTIVATOR NCOA7 DuRING INITIAL STAGES OF GAMETOGENESIS IN EuROPEAN SEA BASS (DiCEnTRARCHuS lAbRAx)

Cinta Zapater1*, Berta Crespo1, Iciar Muñoz1, Patricia Pinto2, Silvia Zanuy1, Ana Gómez1

1Institute of Aquaculture Torre la Sal, CSIC, Castellon, Spain; [email protected]

2Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal

Recent studies in European sea bass using a hemigonadectomy approach and tran-scriptomic analyses have revealed several genes potentially involved in the onset of puberty in female gonads. One of these genes is ncoa7 that encodes a nuclear recep-tor co-activator that, in mammals, increases the transcriptional activity of the estrogen receptor (Esr). This study is aimed at the molecular and functional characterization of the ncoa7 gene and its role during gametogenesis. We have cloned the complete cDNA of sea bass ncoa7 and studied its expression in different male and female tissues showing high expression in brain and gonads. Functional analyses using HEK293 cells co-transfected with Ncoa7 together with the estrogen, androgen and progesterone receptors, and exposed to E2, Testosterone or 17,20β-P, showed that Ncoa7 is able to increase the transcriptional activity of all the different receptors, suggesting that can control different processes in early gametogenesis. Quantification of the expression of ncoa7, esr1, esr2a and esr2b in the ovary and testis of adult sea bass during a whole reproductive cycle showed different expression patterns among stages. In females, an increase of ncoa7 expression is observed during vitellogenesis coinciding with an increase of esr2b expression and E2 levels in the ovary, reinforcing the hypothesis that Ncoa7 is involved in the onset of puberty in female sea bass. In males, the highest expression of ncoa7 was observed in immature testes overlapping with the highest ex-pression of esr1 and high levels of E2 in plasma. In addition, Ncoa7 and Esr1 colocalized in type A spermatogonia of immature testis, and further co-immunoprecipitation stud-ies demonstrated that Ncoa7 was bound to Esr1 in these gonads. Finally, treatment of immature testis with E2 increased proliferation of type A spermatogonia as found by phospho-Histone H3 (Ser10) immunocytochemistry, what together with the previous results suggest that both factors may be involved in spermatogonia proliferation.

Keywords: ncoa7, estrogen receptor, gametogenesis, transcriptional activation, sper-matogonia proliferation

Acknowledgements: Funded by AGL2015-67477-C2-1-R, Aquagenomics (CSD2007-00002), PROMeTeOii-214/051.

SeSSiOn V. GAMETE STORAGE

AND PRESERVATION

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GAMETE AND GERM CELL PRESERVATION AND STORAGE: PERSPECTIVES AND THE REALITy

Ákos Horváth1*

1Szent István University, Faculty of Agricultural and Environmental Sciences, Institute of Aqua-culture and Environmental Safety, Department of Aquaculture, H-2100 Gödöllő, Páter Károly u. 1., [email protected]

Preservation of gametes and more recently germ cells keeps attracting scientists and remains a popular scientific topic. This includes cryopreservation of fish sperm, short-term storage of sperm exposed to various extenders, studies on the chilling sensitivity of fish embryos, as well as cryopreservation or vitrification of testicular or ovarian tis-sues or cell suspensions. Scientists keep developing methodologies and protocols and involve new species in these investigations.

In spite of all these efforts, application of preservation and storage technologies to aquaculture practice is very limited. The primary area of application is the establishment of frozen gene banks in order to conserve the genetic resources of valuable taxa or varieties. These frozen gene banks are typically established and maintained at the pre-mises of research institutions or universities. They operate on a non-profit basis, thus, require continuous funding and use of taxpayers’ money, in other words, they are not a commercially viable. Gamete preservation is applied in isolated cases to actual spe-cies conservation, however, this is done on an individual basis and again, as all conser-vation actions, involves public or private funding without commercial profit.

While cryopreservation of sperm developed into a multi-million dollar business in dairy cattle breeding, very few examples of commercial application of fish gamete preservation and storage are known. Individual selection has been applied very sporadically in aquacul-ture, male or female fish do not have an individual value like they do in case of dairy bulls.

Significant efforts have been made by the scientific community to overcome this particular problem. These included investigations of the dietary regimes of breeders to improve sperm quality, experimentation on alternative preservation methods (such as vitrification), development of high-throughput preservation technologies and their standardization with detailed description of each function and finally, continuous im-provement of protocols in cultured species. The last three-four years also saw a rapid development in the preservation of early-stage germ cells such as primordial germ cells (PGC-s), spermatogonia and oogonia for the purpose of their transplantation into suitable recipients.

What can we thus expect from the future? Gamete preservation and storage will remain an interesting scientific topic that will attract scientists. It will also be applied in indivi-dual cases (as it is today), however, profit-oriented commercial application will probably have to wait until individual selection becomes integral part of breeding in aquaculture.

Keywords: Gamete, preservation, storage, challenges

Acknowledgements: The work was supported by the european Regional and Development Fund and the Government of Hungary within the project GINOP-2.3.2-15-2016-00025.

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Oral presentation, Session V. GAMETE STORAGE AND PRESERVATION

OPTIMIZATION AND STANDARDIZATION OF PROTOCOLS FOR EuROPEAN EEL SPERM SHORT-TERM STORAGE AND CRyOPRESERVATION OF LARGE SPERM VOLuMES

J. Germán Herranz-Jusdado1, Victor Gallego1, David S. Peñaranda1, Christoffer Rozen-feld1, Luz Pérez1, Juan F. Asturiano*1

1Universidad Politécnica de Valencia, Instituto de Ciencia y Tecnología Animal, Grupo de Acuicul-tura y Biodiversidad, Camino de Vera s⁄n 46022 Valencia, Spain; [email protected]

Maturation in captivity of European eel (Anguilla anguilla) requires costly hormonal treatments that normally lead to asynchronic maturation between sexes. Therefore, optimization of sperm storage conditions and cryopreservation protocols are of great importance for successful artificial fertilization. With this motivation, we conducted a series of factorial experiments to determinate the optimal conditions for short-term storage of sperm, and to improve the available protocols for European eel sperm cryo-preservation, applying them to larger sperm volumes.

In a short-term storage experiment, sperm was stored at different dilution ratios (1/10 and 1/50) and temperatures (4 and 20 °C) and storage in constant agitation or still was tested as well. In addition, for the sperm cryopreservation of large volumes (cryovials of 2 and 5 ml), different cooling rates (1 and 3 cm above liquid nitrogen during 15 and 20 minutes), and different extender compositions (methanol control, methanol and FBS, methanol and BSA or methanol and egg yolk) were tested. Sperm kinetic parameters were analyzed by a CASA system both in fresh and short- or long-term stored samples.

In relation to short-term storage trial, sperm quality did not show significant differ-ences in the first 24 hours after sperm collection between the different storage condi-tions tested. For longer time, 4 °C and 1/50 dilution showed significantly better results than the other conditions. Cryopreservation showed good motility results with sam-ples frozen in cryovials of 2 and 5 ml, and the best conditions were cooling 1 cm above liquid nitrogen independently of the cooling time tested (15 and 20 minutes). Further-more, the combined use of methanol (10%) and egg yolk (5%) as cryoprotectants, showed significant higher motility values (close to 50%) than the control (only 10% methanol), whereas the addition of FBS (20%) and BSA (5%) showed a significant reduction of the sperm motility. The establishment of these storage and cryopreserva-tion protocols will be a great improvement for European eel artificial reproduction and for genetic cryobanking.

Keywords: european eel, cryopreservation, egg yolk, sperm, CASA

Acknowledgements: This study was funded by the european Union’s Horizon 2020 re-search and innovation program under the Marie Skłodowska-Curie grant agreement no 642893 (iMPReSS) and the COST Office (COST Action FA1205: AQUAGAMeTe). VG has a postdoc grant from the UPV (PAiD-10-16).

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PROTECTIVE ROLE OF ANTIFREEZE PROTEINS DuRING CRyOPRESERVATION OF STERLET (ACiPEnSER RuTHEnuS) SPERMATOZOA

Miaomiao Xin1*, Anna Shaliutina-Kolešová1, Mohammad Abdul Momin Siddique1, Jan Štěr-ba2,3, Borys Dzyuba1, Sergii Boryshpolets1, Vitaliy Kholodnyy1, Otomar Linhart1, Li Ping1

1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic; [email protected] of South Bohemia in České Budějovice, Faculty of Science, Department of Chemistry and Biochemistry, Branišovská 1760, 37005 České Budějovice, Czech Republic.3Institute of Parasitology, Biology Centre of Academy of Sciences of the Czech Republic, Branišov-ská 31, 37005 České Budějovice, Czech Republic.

Cryoinjuries of fish spermatozoa during cryopreservation can affect spermatozoa mo-tility traits, DNA integrity and membrane structure as well as fertilizing ability and larval survivability. Antifreeze proteins could preserve sperm quality and stabilize spermatozoa membrane structure during cryopreservation. In the present study, two antifreeze pro-teins (AFPI and AFPIII) were used to understand their protective role on sperm quality and membrane integrity during cryopreservation of sterlet Acipenser ruthenus spermato-zoa. In both antifreeze proteins was used a concentration of 0, 0.1, 1, 10, and 100 ug/ml with an extender (30 mM sucrose, 1 mM KCl, 25 mM Tris-HCl, 10% methanol, and pH 8.5). Spermatozoa motility parameters were investigated by computer-assisted semen analy-sis (CASA). Membrane integrity of spermatozoa were estimated by flow cytometry using SYBR green staining for the membrane of living cells and propidium iodide staining for the membrane of degenerate cells. After dilution in activation medium, fresh spermato-zoa showed 85±3% of motility and 160±14 µm.s-1 of curvilinear velocity at 15 s post-ac-tivation. The highest curvilinear velocity (146±15 µm.s-1) in cryopreserved spermatozoa was observed in the samples frozen with 10 ug/ml of AFPI at 15 s post-activation. The highest post-thawed motility (58±6%) was obtained in cryopreserved spermatozoa with 1 ug/ml of AFPIII at 15 s post-activation. A decrease of motility (44±2%) and curvilinear velocity (132±14 µm.s-1) was obtained in cryopreserved spermatozoa without antifree-ze protein at 15 s post-activation. Live/dead proportions indicated that the spermato-zoa cryopreserved with 10 ug/ml of AFPI had lowest amount of damage (16±7%) of plasma membrane, and the cryopreserved spermatozoa without antifreeze proteins had 46±11% damage of plasma membrane. Our results showed that addition of antifreeze proteins have a positive effect on the quality of spermatozoa during cryopreservation but highly dose dependent. Results from this study will provide an insight into the pro-tective mechanism of antifreeze protein on fish sperm that could help to standardize cryopreservation protocol for sturgeon and other fishes.

Keywords: Cryopreservation, AFPi, AFPiii, membrane integrity, motility trait

Acknowledgements: This study was supported by the Ministry of education, Youth and Sports of the Czech Republic – projects “CenAKVA” (no. CZ.1.05/2.1.00/01.0024) and “CenAKVA ii” (LO1205 under the nPU i program), and COST (no. LD14119), by the Grant agency of the University of South Bohemia in Ceske Budejovice (no. 125/2016/Z), and by the Czech Science Foundation (no. 16-02407Y).

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DEVELOPMENT OF SPERM VITRIFICATION PROTOCOLS FOR ENDANGERED SALMONIDS: ADRIATIC GRAyLING, THYMAlluS THYMAlluS and MARBLE TROuT, SAlMO MARMORATuS

Eszter Kása1*, Gergely Bernáth1, Zoltán Bokor1, Béla Urbányi1, Kinga Katalin Lefler1, Dušan Jesenšek2, Ákos Horváth1

1Szent István University, Department of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary, [email protected]ška družina Tolmin, Trg 1. maja 7, SI-5220 Tolmin, Slovenia

Vitrification is the solidification of a liquid into an amorphous or glassy state which can be attained at very fast cooling rates (106–1010 °C/s). Recently, several studies have been published on the vitrification of fish sperm, but no information is available regarding the vitrification of sperm of the salmonid species investigated in this study.

The Adriatic grayling is a genetically isolated population of the grayling (Thymallus thymallus), while the marble trout (Salmo marmoratus) is a native fish species of the peri-Adriatic drainage area including the Soča river basin in Slovenia. They are affected by intensive stockings of non-native fish species that leads to the problems with hy-bridization, competition and predation. Thus, the role of cryopreservation for preserv-ing the genetic material of these two salmonids is very important.

Vitrification was successfully applied to the sperm of the Adriatic grayling and marble trout. Experimental individuals of both species were mainteined at the facilities of the Angling Association of Tolmin, Slovenia. Sperm was collected, diluted in species-spe-cific non-activating media containing cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). A fertilization trial was carried out to test the effectiveness of vitrification in case of grayling. Additionally, the effect of trehalose supplementation in the cooling media was tested in case of Adriatic grayling sperm vitrification, while numerous different proto-cols and two cooling devices (inoculating loop and Cryotop) were compared in case of the ultra-rapid cooling of marble trout sperm.

The vitrification method for Adriatic grayling sperm resulting the highest post-thaw motility (7.5±6.5%, fresh control: 92.4±3%) was as follows: 1:1 dilution ratio (v/v), grayling extender, 30% cryoprotectant (15% methanol + 15% propylene-glycol), cool-ing device: Cryotop, 2 µl droplets. In case of marble trout sperm the post- thaw motility was 8.6±0.7% (fresh control: 78.4±23.3%), with the following protocol: 1:1 dilution ratio (v/v), with 40% cryoprotectant (20% methanol and 20% propylene-glycol), cool-ing device: Cryotop, with 2 µl of sperm suspension. Viable embryos were produced by fertilization with vitrified grayling sperm (hatching: 13.1±11.7%, control: 73.9±10.4%).

Keywords: Spermatozoa vitrification, ultra-rapid cooling, fish sperm cryopreservation, adriatic grayling, marble trout

Acknowledgements: The work was funded by the nKFi grant number K-109847.

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GuIDELINES FOR ZEBRAFISH SPERM CRyOPRESERVATION AND STORAGE

Patricia Diogo1,2*, Gil Martins2, Ana Eufrásio1, Rita Nogueira1, Isa Quinzico1, Tomé Silva3, Elsa Cabrita2, Paulo Gavaia2,4

1University of Algarve, 8005-139 Faro, Portugal; [email protected] 2University of Algarve, Centre of Marine Sciences, , 8005-139 Faro, Portugal3SPAROS Lda., Área Empresarial de Marim, Lote C, 8700-221 Olhão, Portugal.4University of Algarve, Department of Biomedical Sciences and Medicine, 8005-139 Faro, Portugal

Zebrafish (Danio rerio) sperm cryopreservation was first achieved more than 30 years ago, however until today there is still a lack of standardization which is translated into high variability in post-thaw sperm quality and in vitro fertilization success. Success-ful cryopreservation depends on high quality sperm, which can only be ensured by having high quality breeders. Consequently, broodstock selection and management is a priority to improve cryopreservation, and therefore, this study aimed to characterize optimal age and sperm stripping frequency in zebrafish. Furthermore, one of the most important bottlenecks in germ cell cryopreservation is the liquid nitrogen requirement for storage. Thus, we propose an alterative storage in ultrafreezer (-150 °C), since germ cells need to be stored below -130 °C. To study optimal donor selection, fish age and sperm stripping frequency was assessed in wild type males (AB) and in a transgenic strain (tg(runx2:egfp)). Males were sampled at 6, 8, 12 and 14 months of age. For each age, males were sampled at day 0 and at 2, 7 and 14 days after the first stripping. Males were anesthetized and sperm collected by abdominal massage and immediately diluted with 10 µl of Hank´s Balanced salt solution (HBSS) at 300 mOsm.kg-1. Sperm quality was assessed using motility parameters with the CASA system (Proisier, Spain), while viability was determined by flow cytometry using PI/Syber green. Results showed that tg(runx2:EGFP) strain displayed significantly higher sperm motility than AB. Youn-ger males (6 and 8 months) show improved sperm motility in both strains. Although stripping frequency had no significant effect on sperm motility, it induced an effect in terms of membrane viability. While AB fish recovered membrane viability 14 days after stripping, tg(runx2:EGFP) did not. The male donor selection guidelines obtained from this trial were then used for a trial in a ultrafreezer, where males with 6 months of age were sampled as previously described, pooled and their sperm cryopreserved with 10% DMF in HBSS and either: 1) inserted directly in a ultrafreezer, 2) freezed at –20 °C/min in a programmed biofreezer and stored in liquid nitrogen, or 3) freezed using the same system but stored in a ultrafreezer (-150 °C). Sperm quality was assessed in terms of sperm motility, viability and apoptosis (Annexin V). Afterwards, conditions 2) and 3) were followed along storage time (1, 4 and 12 weeks) and motility, viability and fertili-zation ability were assessed. The knowledge obtained by these experiments supports the establishment of guidelines for male zebrafish selection to optimize cryopreserva-tion and storage methods.

Keywords: Sperm cryopreservation, zebrafish, breeder selection, ultrafreezer

Acknowledgements: The study was supported by FCT grant project number SFRH/BD/97466/2013. We would like to the students Filipa Beça and Ana Domingos.

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PTAT PRECONDITIONING OF ZEBRAFISH EMBRyOS IN ORDER TO INCREASE THEIR CHILLING RESISTANCE

Ákos Horváth1*, Tímea Kollár1, Gergely Bernáth1, Béla Urbányi1, Zsolt Csenki-Bakos1, Bernadett Faragó2, Katalin Szabó2, Csilla Budai2, Eszter Losonczi2, Judit Cserepes2

1Szent István University, Faculty of Agricultural and Environmental Sciences, Institute of Aqua-culture and Environmental Safety, Department of Aquaculture, 2100 Gödöllő, Páter Károly u. 1., Hungary; [email protected] 2Applied Cell Technology Ltd., Budapest, Hungary

Pressure triggered activation of tolerance (PTAT) was applied to zebrafish embryos in order to increase their resistance to chilling and to improve their survival during an arrest in their development Chilling sensitivity is one of the main obstacles in the development of fish embryo cryopreservation protocols. At the same time, arrest of embryonic development by lowering the temperature of incubation would represent a significant advantage for micromanipulation studies by allowing synchronization of embryos.

Zebrafish embryos (26-somites and Prim-5 stage) were subjected to hydrostatic pressure treatment (PTAT) and then chilled for 24 hours at 0 °C. PTAT treatment sig-nificantly improved the survival of embryos and larvae. Hatch (PTAT: 38±3%, control: 23±4%), 10-day survival (PTAT: 17±3%, control: 4±2%) as well as 30-day survival (PTAT: 2±1%, control: 0%) results were all significantly higher for the PTAT treated group. No chilled but untreated embryos survived beyond day 19 post fertilization, whereas PTAT treated and chilled embryos developed to sexually mature individuals and spawned.

One-cell zebrafish embryos were subjected to PTAT-treatment and then chilled at 4 °C for one hour. It was noted that chilling reversibly arrested the development of zebrafish embryos. PTAT treatment improved the survival of embryos assessed at 24 hours (16±6%) and 48 hours (15±5%) compared to the control (5±4% at 24 hours and 4±4% at 48 hours). However, the timing of chilling had a significant impact on the sur-vival with the highest percentage of surviving embryos recorded in the control chilled at 10 minutes post fertilization (40±9% at 24 hours and 29±8% at 48 hours).

We found that PTAT preconditioning improves the chilling survival of advanced zebraf-ish embryos and hatch rates alone are not suitable indicators of post-chilling survival. Although PTAT preconditioning helps to improve the chilling survival of early zebrafish embryos, this improvement depends on the timing of chilling.

Keywords: Zebrafish, embryos, chilling, PTAT, survival

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SPERM FROM THE ENDANGERED AMAZONIAN FISH HYPAnCiSTRuS zEbRA CAN BE SuCCESSFuLLy COLLECTED AND CRyOPRESERVED

Leandro Godoy1*, Henrique Dias2, Jôsie Caldas2, Nayara Cruz2

1Federal University of Rio Grande do Sul, Department of Animal Science, Av. Bento Gonçalves 7712, Porto Alegre, Brazil; [email protected] 2Nilton Lins University, Aquaculture Graduate Program, Av. Prof. Nilton Lins 3259, Manaus, Brazil

Hypancistrus zebra is an endemic ornamental fish from the Brazilian Amazon and is critically endangered by the construction of a hydroelectric plant on its habitat and illegal fishing. In an attempt to establish a germplasm bank for the species, this work aimed to develop a protocol for sperm collection from this tiny fish (4.54±0.71 g) using “CPE” carp pituitary extract (1, 3 and 5 mg/kg) and Ovopel™ (1.25, 2.00 and 2.75 mg/kg). Efficiency of the hormonal treatment was evaluated by sperm motility rate, spermatozoa lifespan and membrane integrity, using eosin-nigrosine staining. The hor-monal treatments were efficient in increasing the seminal fluid, allowing the collection and manipulation of the sperm. Among the 48 fish used (8 per treatment), all males responded to treatments 1.25 and 2.75 mg/kg Ovopel™ and 3 mg/kg CPE. At doses 1 and 5 mg/kg CPE only 1 male (12.5%) did not respond, and three animals (37.5%) did not respond to induction at 2 mg/kg Ovopel™. The volume of sperm collected ranged from 6 to 15 μl. The evaluated parameters did not present a significant difference between the hormones and concentrations tested, with an average motility rate of 87.31±9.37%, a long lifespan of 14.70±1.67 min and membrane integrity of 80.52±10, 60%. Cryobiology results indicate that H. zebra spermatozoa are very sensitive to low temperature exposure and dimethylsulfoxide was more efficient than methanol in pro-tecting them from cryoinjuries. In preliminary freezing tests, we got 18.0±16.4% of survival and our studies are underway to optimize the freezing protocol and to create a germplasm bank for this rare and endangered Amazonian species.

Keywords: Zebra Pleco, Loricariidae sperm stripping, spermatozoa lifespan, ichthyo-fauna conservation, gamete banking

Acknowledgements: The graduate students have been scholarship supported by FA-PeAM and CAPeS Foundation. We would like to thank iBAMA and Dr. Luciano Jensen for the logistics support and fish donation.

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THE EFFECTS OF DIFFERENT PRESERVATION METHODS ON IDE (lEuCiSCuS iDuS) SPERM AND THE LONGEVITy OF SPERM MOVEMENT

Gergely Bernáth1, Zsolt Csenki-Bakos1, Zoltán Bokor1, Levente Várkonyi1, József Mol-nár1, Alexandra Kajtár1, Tamás Szabó1, Ádám Staszny1, Árpád Ferincz1, Krisztián Szabó2, Béla Urbányi1, Balázs Csorbai1

1Szent István University, Department of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary2Dinnyési Halgazdaság Kft., 7-es u., H-2485 Dinnyés, Hungary

The ide (Leuciscus idus) is an important gamefish in numerous European countries. The interest for the artificial propagation is increasing. However, the information re-garding to different sperm storage and preservation methods are very limited in this species. In our study, the effect of 48 hours chilled storage and a formerly developed cryopreservation method were investigated on ide sperm motility and fertilizing capac-ity (hatching and larval deformity). The longevity of sperm movement (at 10, 20, 30, 60, 90, 120 seconds following activation) in fresh and thawed sperm was also tested. A broodstock of wild caught ide individuals (16 males and 9 females) was maintained at the recirculating system of the Department of Aquaculture in Szent István Univer-sity, Gödöllő (Hungary). Males and females were hormonally induced only before the cryopreservation and fertilization experiment using carp pituitary in a dose 3.5 (males) and 5 (females) mg body weight kg-1. Fish were stripped 48 hours following injection. Sperm motility was investigated using a CASA (computer-assisted sperm analysis) sys-tem and larval deformity was evaulated with a Stereo microscope (20X magnification). Cryopreservation of sperm was carried out using a sugar based extender (200 mM glu-cose, 40 mM KCl, 30 mM Tris, pH 8, Horváth et al. 2012) and a dilution ratio 1:9 com-bined with 10% of methanol as cryoprotectant. Samples were freezed 3 cm above the liquid nitrogen for 3 minutes. For fertilization, fresh (25µl/group) and thawed sperm (250µl/group) of 5 males were selected. Eggs from 3 males was separated for the fertilization trial (0.5g: 2–300 eggs/group). For motility assessment, as well as for fer-tilization an activating solution for cyprinids (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8, Saad et al. 1988) was used. Sperm motility showed significant reduction both after and 24 and 48 hours of chilled storage in comparison with fresh group. No significant differ-ences were observed among groups in sperm longevity both in fresh and thawed ide sperm at 10-120 seconds. Freshly stripped sperm showed moderate motility whereas motility following thawing did not decreased significantly (fresh control-49±19%, cryo-preserved-22±22%). A similar result was observed during fertilization (hatching rate: fresh control – 22±15%, cryopreserved – 33±18%). A low ratio of deformed larvae was measured both in fresh control (3±2%) and the thawed group (4±3%) after hatching whereas significant difference between the two groups was not observed.

Keywords: ide, sperm motility, chilled storage, longevity of movement, cryopreserva-tion

Aknowledgements: The work was supported by the european Fisheries Fund Fisheries Operative Programme iii. axis, european Fisheries Fund for Renewable Fisheries pro-vided by the eU and Hungary. This study was supported by the GinOP 2.3.2–15–2016–00004 project: “establishing the sustainable angling-aimed management of Lake Ba-laton.”

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FREEZING SENSITIVITy OF SuRuBIM-DO-PARAÍBA, STEinDACHnERiDiOn PARAHYbAE OOCyTES

Taís Silva Lopes1*, Eduardo Antonio Sanches2, Danilo Caneppele3, Elizabeth Romagosa1

1Fishery Institute of São Paulo, APTA, SAA, Av. Francisco Matarazzo, 455, São Paulo, SP, Brazil. [email protected]ão Paulo State University – UNESP, Registro, SP, Brazil. 3Hydrobiology and Aquaculture Station of São Paulo Energy Company – CESP, Paraibuna, SP, Brazil.

Studies to better understand the mechanisms of loss of viability of oocytes, as well as to increase longevity, and even conserving oocytes over a long period – cryopre-servation are necessary in order to conserve endangered fish species, such as the su-rubim-do-Paraíba. For this it is necessary to use different cryoprotectants solutions (CPAs) to increase the resistance of oocytes to freezing protocol. In the present study, the aim was investigated the freezer sensitivity in differents oocytes stages of suru-bim-do-Paraíba. Immature (diameter <1.7mm) and mature oocytes were obtained from a broodstock at the Hydrobiology and Aquaculture Station of the CESP, Paraibuna, SP, Brazil. Oocytes were incubated in Hank’s or 90% L15 solutions containing different CPAs: (1) 0.1–0.4M sucrose + 1–2M methanol, (2) 1–4M methanol X 1–4M propylene glycol X 1–4M DMSO, for mature oocytes; (3) 0.5M sucrose or fructose + 2M methanol or PG or DMSO, (4) 0.25–1M fructose + 1–4M DMSO, for immature oocytes. All the treatment was kept for 120 min at -5.9±2.8 °C. Control treatment, only Hank or 90% L15 solutions were carried out. Three replicates for each treatment and control were used. Evaluations were made by viability tests: membrane integrity staining in 0.2% Trypan blue (TB) for immature oocytes and fertilization (%F) rate for mature ones. Results showed that mature oocytes being the most sensitive to lower temperatures, because there were not % F. Immature oocytes may be used all cryoprotectants tested in different concentrations, but the statistically superior was the CPA with fructose and DMSO and, there was no statistical difference between the concentrations of this CPA. This may indicate that for this specie, the immature stages have shown a lower freezer sensitivity than the mature one. 1M Fructose + 4M DMSO appears to be the best CPA to protect surubim-do-Paraíba immature oocytes from cold (about -8 °C).

Keywords: Crypreservation, cooling, gametes, oocytes, freshwater fish

Acknowledgements: The study was supported from FAPeSP project number 2014/21215-8.

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EFFECT OF EXTENDER SuPPLEMENTED WITH DIFFERENT ANTIOXIDANTS ON MOTILITy, DNA DAMAGE AND FERTILIZING ABILITy OF WILD AFRICAN CATFISH (ClARiAS gARiEPinuS) SPERM

İlker Yavaş1, yusuf Bozkurt2*, Zafer Cantekin3, Tuğba Korkmaz Yavaş4

1Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Reproduction and Arti-ficial Insemination, 31200, Antakya, Hatay, Turkey2Iskenderun Technical University, Faculty of Marine Sciences and Technology, Department of Aqu-aculture, 31200, Iskenderun, Hatay, Turkey; [email protected] 3Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Microbiology, 31200, Antakya, Hatay, Turkey4 Republİc of Turkey, Ministry of Food – Agrıculture and Livestock, Hatay Control Laboratory Dire-ctorates, 31200, Antakya, Hatay, Turkey

African catfish (Clarias gariepinus) has been considered one of the most suitable species for aquaculture. Antioxidants play an important role in sperm motility, integrity, metabolism, and function, by protecting the cells against oxidative damage. Damage to sperm function has been successfully minimized in several mammalian species by the addition of antioxidants to the extender media prior to cryopreservation. From this point of view, the present work was designed to assess the influence of different antioxidants such as catalase (100, 250, 500 U/l), uric acide (0.25, 0.5, 1 mmol/l) and peroxidase (100, 250, 500 U/l) incorporated with two different ionic extenders on post-thaw motility, viability, DNA damage and fertilizing ability of cryopreserved African catfish sperm. Semen samples diluted at the ratio of 1:10 by the extender were subjec-ted to cryopreservation process. Following, the semen was loaded into 0.25 mL straws and the straws were placed on a rack 4 cm above of the liquid nitrogen in a styrofoam box. Sperm freezing was performed in liquid nitrogen vapour (-140°C)during10minandafterthattimethestrawswere immersed into liquid nitrogen (-196°C).Thesemensampleswerestored ina liquid nitrogen container untill analyses. The cryopreserved sperms were thawed in a water bath (35 °C) for 5 s and evaluated for fertilization. Thawed sperm was activated using 3‰ NaCl and observed under microscope for de-termination of motility, motility durations and viability (three replicates). Fertilization was conducted at ratio of 3x106 spermatozoa/egg. DNA integrity was determined by membrane-permeant DNA stain, SYBR-14, in combination with propidium iodide (PI) using fluorescent microscope. Supplementation of 250 U/l peroxidase showed better cryoprotective effect for sperm motility, duration of motility and viability (p<0.05) when compared to catalase and uric acide. In addition, higher fertilization rates were determined with phosphate buffer saline (PBS) solution containing peroxidase and uric acide (p<0.05). Peroxidase at dose of 250 U/l gave the best score (34.2±4.2%) when compared to the other antioxidants reducing DNA damage (P<0.05).

Keywords: African catfish, cryopreservation, extender, antioxidant, DnA damage

Acknowledgements: This study was supported by Mustafa Kemal University Research Fund (MKU-SRF-15103).

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CRyOPRESERVATION EFFECT ON STERLET SPERM VIABILITy AND PROTEIN CONTENT AFTER LIVE/DEAD CELLS SEPARATION By PERCOLL DENSITy GRADIENT

yevhen Horokhovatskyi1*, Mariola A. Dietrich2, Ievgen Lebeda1, Sergii Boryshpolets1, Pavlo Fedorov1, Marek Rodina1, Borys Dzyuba1

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected] 2Polish Academy of Sciences, Institute of Animal Reproduction and Food Research, Department of Gametes and Embryo Biology, Tuwima 10, 10-748 Olsztyn, Poland

In many fish species, sperm cryopreservation procedures lead to deleterious effects and significant decrease of spermatozoa viability. After freeze-thawing process, sperm suspension contains viable, non-viable and cryodamaged spermatozoa. Usually, such a frozen-thawed sperm suspension is the object of routine cryobiological studies, aim-ing to evaluate the damage of cryopreservation on sperm proteins, lipids, etc. However, the presence of a varying percentage of cryodamaged spermatozoa in the sperm sus-pension probably obscures the real cryopreservation effects on the fraction of sperma-tozoa that survived the freeze-thawing process and saved suitable motility parameters. This fraction is the only one potentially involved into the fertilization process. There-fore, a more precise estimation of the cryopreservation effects on spermatozoa that survived freezing-thawing governed the design of our experiments by including a sepa-ration step of cryodamaged spermatozoa and specific analysis of the viable sperm frac-tion. For this purpose, a Percoll density gradient centrifugation (“Percoll method”) was selected as a suitable technique for separation of fish sperm. Fresh sperm of sterlet males, as well as cryopreserved one, were divided into 2 groups, either with or without application of the “Percoll method”. At each step of the experiment, sperm quality was evaluated by video microscopy with integrated computer-assisted sperm analysis soft-ware (CASA) and flow cytometry with live-dead sperm viability analysis. Sperm motility and percentage of live cells in the cryopreserved bulk fell to 40% and 63% respectively. However, the percentages of motile and live cells following separation were higher than 90 % in both cases. Using two-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with MALDI-TOF/TOF mass spectrometry, significant changes in 20 proteins were identified when comparing fresh and cryopreserved sperm, while the content of only one protein was significantly changed when comparing fresh and cryopreserved sperm resulting from the “Percoll method” separation. Thus, the results of this study show that the spermatozoa surviving the freezing-thawing process are much less af-fected by cryodamages than those in the unselected suspension. However, for a clear-er understanding of cryopreservation damaging nature, further researches should be more deeply performed.

Keywords: Cryopreservation effect; sperm motility, Percoll separation; proteomic anal-ysis.

Acknowledgements: This study was financially supported by the COST Action FA1205 AQUAGAMeTe, COST (no. LD14119), by the Grant agency of the University of South Bo-hemia (no. 125/2016/Z) and by the Czech Science Foundation (no. P502/15-12034S).

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CRyOPRESERVATION OF ZEBRAFISH SPERMATOGONIA: SLOW-RATE FREEZING VS VITRIFICATION

Zoran Marinović1*, Qian Li2, Jelena Lujić1, Eszter Kása1, Yoshiko Iwasaki2, Béla Urbányi1, Ákos Horváth1, Goro Yoshizaki2

1Szent István University, Department of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary; [email protected] University of Marine Science and Technology, Department of Marine Biosciences, Tokyo 108-8477, Japan

With the acceptance of zebrafish as a model organism, and with the development of several hundreds of transgenic lines, currently there is a need for keeping these lines beyond common practices of keeping breeding colonies. Even though sperm cryopres-ervation has been developed, lack of egg and embryo cryopreservation protocols hin-der cryogenic storage of whole genetic resources of a line. In order to overcome this, we have developed protocols for the cryopreservation of zebrafish spermatogonia by optimizing both slow-rate freezing and vitrification methodology. By testing different cryoprotectants (dimethyl sulfoxide – Me

2SO, ethylene glycol – EG, propylene glycol –

PG and glycerol – Gly) and concentrations (1 M, 1.3 M and 1.6 M), we have determined that the usage of 1.3 M Me

2SO yielded the highest survival after slow-rate freezing (~

65%). Additionally, we have tested the effects of different sugars (glucose, trehalose, fructose, sucrose) in different concentrations (0.1 M and 0.3 M) and protein (1.5% BSA, 1.5% FBS, 10% skim milk powder and 1.5% egg yolk), however these variables did not have a significant effect on the survival of spermatogonia after thawing. As for vitrifica-tion, we have tested three equilibration and three vitrification media with each of them containing different combinations and concentrations of methanol (MeOH), PG and Me

2SO. By utilizing the needle immersed vitrification method (NIV), testes were pinned

on an acupuncture needle, passaged through equilibration and vitrification media and plunged into liquid nitrogen. Equilibration media containing 1.5 M MeOH + 1.5 M PG and vitrification media containing 3 M PG + 3 M Me

2SO yielded the highest survival rates

after warming (~ 50%). Additionally, the repeatability of the optimal vitrification pro-tocol was demonstrated by obtaining spermatogonia survival rates of 50–70% in five different zebrafish lines (wild AB type, casper, leopard, vasa [vasa::egfp] transgenic line and Wilms tumor [wt1::egfp] transgenic line). In the present study we demonstrate for the first time an efficient method for the cryogenic storage of zebrafish spermatogo-nia. Cryopreserved spermatogonia could be then transplanted into suitable recipients (optimally sterilized zebrafish larvae) in which they would colonize the recipient gonad, proliferate and give rise to functional gametes of both sexes.

Keywords: Spermatogonia, Danio rerio, transplantation

Acknowledgements: This study was supported by projects nKFiH Snn 116912 in Hun-gary and the Stipendium Hungaricum Scholarship Programme (grant 106360 to ZM).

SeSSiOn Vi. ANTHROPOGENIC

CONTAMINANTS AND FISH GAMETES

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ENDOCRINE DISRuPTING CHEMICALS IN AquATIC ENVIRONMENTS: EFFECTS ON FISH GAMETES

Oliana Carnevali*, Stefania Santangeli, Isabel Forner Piquer, Francesca Maradonna

Università Politecnica delle Marche, Dipartimento di Scienze della Vita e dell’Ambiente, Via Brecce Bianche, 60131 Ancona, Italy; [email protected]

Endocrine Disrupting Compounds (EDCs) are ubiquitous chemical compounds used in numerous consumer products as plasticizers or detergent that have been shown to have unforeseen impacts on the ecosystem and human health interfering with the endocrine systems.Large efforts have recently been devoted to discover the mechanisms of action of these chemicals. EDCs interacting with the endocrine system may cause alterations at different levels of biological organization, from the molecular to the individual and the population level. The potential for their deleterious effects must be considered rela-tive to the regulation of hormone synthesis, secretion, metabolism and actions and the variability in regulation of these events across the life cycle. The developmental stage at which EDC exposure occurs, is also a critical consideration in understanding their effects. Over the past 25 years, extensive research in vertebrate species has identified a wide variety of genomic actions that are altered by exposure to anthropogenic chemicals with hormone-like activity through their interactions with nuclear receptors. In addi-tion, many pollutants have been shown to interfere with non-genomic (non-classical) actions but this mechanism of endocrine disruption is still poorly understood. Their ac-tion at DNA level is also starting to be known, producing DNA mutations and changes in epigenetic pathways inducing specific mechanisms of toxicity and cellular responses.In the last years, the number of publications describing the effect of EDCs on fish repro-duction, focusing on the deregulation of the hypothalamus- pituitary-gonadal axis as well as on gamete quality, significantly increased. Depending on their ability of mimick-ing endogenous hormones, the EDCs may differently affect male or female reproductive physiology. Inhibition of gametogenesis, development of intersex gonads, alteration of the gonadosomatic index have been largely documented. In males, alterations of sperm density, motility and fertility have been observed in several wild species living in polluted areas as well as in those exposed in laboratory to different chemicals. In female, oocyte growth and maturation, occurrence of apoptotic/authophagic processes and epigenetic changes have been recently described. Gamete viability is considered as one of the major indicator of reproductive endocrine disruption, of greatest concern is also the fact that adverse effects in the gonads have the potential to affect subsequent generation(s) through the germline. Therefore, when EDCs introduce epigenetic changes during early development, they permanently alter the epigenome in the germ line, and these changes can be transmitted to subsequent generations. Evidence of transgenerational transmis-sion has important implications for the reproductive health and fertility of animals and humans, significantly increasing the potential biohazards of pollutants.

Keywords: endocrine disruptors, teleost, biomarkers, epigenetic, gamete quality

Acknowledgements: Supported by the Ministry of Health – RiCeRCA FinALiZZATA2009 “Food and environmental safety: the problem of the endocrine disruptors” to OC; 2012–2015 COST european Cooperation in the field of Scientific and Technical Re-search “AQUAGAMeTe” to OC and by PRin 2010–2011 prot 2010W87LBJ to OC.

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Oral presentation, Session VI. ANTHROPOGENIC CONTAMINANTS AND FISH GAMETES

HISTOPATHOLOGICAL ASSESSMENT OF GONADAL TISSuE OF FISH EXPOSED TO ANTHROPOGENIC CONTAMINANTS, SOuTH AFRICA

Ina Wagenaar1, Irene Barnhoorn2

1University of Johannesburg, Department of Zoology, P.O. Box 524, Auckland Park 2006, Johannesburg, South Africa; [email protected] 2University of Venda, Department of Zoology, Private Bag X5050, Thohoyandou, Limpopo Province, South Africa, 0950

Five impoundments in the North-West Province, South Africa were selected to assess the reproductive health. These impoundments are known for its poor water quality and are characterized by eutrophication and algal blooms due to the high levels of phosphates and nitrates. The primary pollution sources are debatably Sewage Treatment Works, mining activities as well as effluent from surrounding agricultural areas. The freshwater indicator fish species, Clarias gariepinus, was used to assess and compare the reproductive health status of fish from the selected. The Marico Bosveld Dam which receives water from the Marico River and classified as being in an unmodified natural ecological state, was chosen as a reference site. The reproductive health status was done by determining the gonadosomatic indices (GSI), staging of the gonadal development and the gonadal histopathology. The histopathological changes were quantified following a semi-quantitative histological protocol to determine the testes index (I

T).

Adult C. gariepinus were sampled using gill nets (n=20). Blood was drawn and a necropsy performed. For the histology a qualitative and quantitative histological assessment were performed on the testes. Focus was placed on reproductive health aspects by staging the gonads according to their reproductive development. Water samples were analyzed for selected physical parameters, selected metals (ICP-MS) and endocrine disrupting chemicals (EDCs). The OCs (o’p- and p,p’ of DDT, DDD and DDE), PCBs (Dieldrin, Aldrin, Endrin, α-endosulfan, αendosulfan Heptachlor, Heptachlor epoxide, Lindane and Arochlor) and alkylphenol, nonylphenol (NP) and organochlorine (OC) were determined (detection limit = 0.50µg/L).

The results showed distinct macroscopic differences in the testes comparing with the reference site. Macroscopic morphology of the testes of fish from the polluted impoundments showed abnormal growths on the surface of the testes, cholesterol granuloma and necrotic testes tissue. The water quality results of these impoundments and the effect on the reproductive health will be elucidated. In conclusion, the increase in anthropogenic contaminants had a greater effect on the reproductive health status of C. gariepinus when compared to the reference site. According to the selected parameters assessed, it seemed like the anthropogenic contaminants had increasing detriment upon fish health.

Keywords: Reproduction, pesticides, histopathology, staging, eDCs

Acknowledgements: This study was financially supported by the national Research Foundation of South Africa (nRF Grant no. 86056) and the University of Venda for assistance during the field sampling.

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SIMuLTANEOuS EXPOSuRE TO ENVIRONMENTALLy RELEVANT CONCENTRATIONS OF DROSPIRENONE AND GESTODENE CAuSED INTERSEX IN COMMON CARP, CYPRinuS CARPiO L.

Hana Kocour Kroupová1, Pavel Šauer1, Jitka Tumová1, Christoph Steinbach1, Oksana Go-lovko1, Jana Máchová1, Hans Komen2, Vít Profant1, Roman Grabic1*

1University of South Bohemia in České Budějovice, South Bohemian Research Center of Aquacul-ture and Biodiversity of Hydrocenoses, Faculty of Fisheries and Protection of Waters, Zátiší 728/II, Vodňany, Czech Republic; [email protected] Wageningen University, Animal Breeding and Genomics Centre, PO Box 338, 6700 AH Wagenin-gen, the Netherlands

Synthetic progestins are mostly used as active ingredients in women’s oral contra-ceptives or other hormonal preparations. As a consequence, they are consumed in relatively high amounts and their concentrations may reach up to tens of ng/L in the aquatic environment. Recently, it has been shown that even such low concentrations can negatively affect reproduction in model fish species. The aim of this study was to investigate the effects of synthetic progestins, gestodene and drospirenone, on sex differentiation in common carp (Cyprinus carpio L.). Common carp were constantly exposed to concentration levels of 2 ng/L of single progestins (gestodene or drospire-none) and to their mixture (2+2 ng/L). Moreover, two control groups were included (clean water control and solvent control with 0.0005% dimethyl sulfoxide). The ex-posure started 24 hours after fertilisation of eggs and was concluded when the fish reached 160 dph (days post-hatch). During this time sexual differentiation is normally completed. The test was run in four replicates. Baths were changed every 24 hours and water temperature and oxygen saturation was kept at 21 °C and > 80%, respectively. During the exposure and at the conclusion of the test, samples for histology and gene expression were taken. Chemical analysis proved that real concentrations of tested progestins did not differ significantly from the nominal ones. Simultaneous exposure of common carp to drospirenone and gestodene caused increased incidence of intersex (32%) when compared to both control groups (3%). On the other hand, exposure to single progestins did not lead to any statistically significant changes in sex ratio and occurrence of intersex individuals. Given that fish are most probably exposed to a mix-ture of progestins (or other steroids) in natural aquatic environment, we may expect serious consequences of such conditions for gonadal development in exposed fish.

Keywords: Fish, gonad development, histology, pharmaceuticals, synthetic progestins

Acknowledgement: Supported by the Ministry of education, Youth and Sports of the Czech Republic – projects ‘CenAKVA’ (no. CZ.1.05/2.1.00/01.0024), ‘CenAKVA ii’ (no. LO1205 under the nPU i program), project of the Czech Science Foundation (no. 16-09709Y) and GAJU (no. 012/2016/Z).

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Oral presentation, Session VI. ANTHROPOGENIC CONTAMINANTS AND FISH GAMETES

in ViTRO TOXICOLOGy TEST SySTEM BASED ON COMMON CARP (CYPRinuS CARPiO) SPERM ANALySIS

Tímea Kollár*, Eszter Kása, Árpád Ferincz, Balázs Csorbai, Béla Urbányi, Zsolt Csenki-Ba-kos, Ákos Horváth

Szent István University, Faculty of Agricultural and Environmental Sciences, Institute of Aquacul-ture and Environmental Safety, Depatment of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary; [email protected]

The effect of seven heavy metals on the motility parameters of common carp (Cy-prinus carpio) sperm was investigated in order to develop an in vitro toxicology test system. More than 100 million vertebrates are used for laboratory experiments every year worldwide; 10-11 million of them in the EU. The aim of the EU is to reduce the number of animals used for scientific purposes and to develop alternative methods for these fields (e.g. in toxicology). Because of this in vitro test systems are recently widely used in ecotoxicology. Fish sperm could be a suitable in vitro toxicological test system. Despite all of this, only a few studies have been published in which fish sperm was used as a toxicological model.

Common carp sperm was exposed in vitro to different heavy metals (Cr, Zn, Cd, Hg, As, Cu, Ni) in various concentrations. The tested concentrations were defined based on preliminary range finding tests. Progressive motility (the percentage of forward moving cells, PMOT), curvilinear velocity (VCL, µm/s) and linearity of the moving (LIN, %) were measured with Computer-assisted Sperm Analysis (CASA) system at 30-minute inter-vals of the 4-hour exposure in order to find the most feasible and sensitive parameter for toxicological monitoring.

We found that PMOT is the most adequate one of the three investigated parameters: dose-response curves were generated in case of each heavy metal, moreover, the toxic effect increased paralel with the increase of exposure time. VCL was less sensitive: al-though dose-response curves were generated in all cases, less changes were observed in this variable upon exposure of heavy metals. LIN proved the least affected parame-ter: decreasing dose-response curves were generated only in case of Hg and As. Cu triggered and increase of LIN, furthermore, there was no effect on LIN in case of Ni. The order of toxicity expressed as PMOT was as follows: Hg > As > Cd > Zn > Cu > Cr > Ni.

Consequently, the progressive motility of carp sperm proved to be a suitable model for in vitro toxicological monitoring. It can be an accurate, fast bioindicator of aquatic pollutions. Its application cannot replace the in vivo tests entirely, but it can be amena-ble for preliminary range finding tests to estimate the possible toxic effect of chemicals in vitro. In addition, the use of fish sperm in toxicology tests can reduce the release of hazardous wastes; thereby its application should be considered with respect to envi-ronmental protection and animal protection.

Keywords: Carp, sperm, in vitro, CASA, progressive motility, curvilinear velocity, linearity

SeSSiOn i. SPERMATOGENESIS

AND SPERM quALITy

POSTER PRESENTATIONS

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INVESTIGATION OF MALE MATuRATION OuT-OF- AND DuRING THE SPAWNING SEASON IN SICHEL (PElECuS CulTRATuS)

Zoltán Bokor1, József Molnár1, Árpád Ferincz1, Levente Várkonyi1, Zsolt Szári2, Ferenc Fodor2, Tibor Tulipán2, Gábor Nagy2, Béla Urbányi1, Gergely Bernáth1

1Szent istván University, Department of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary2Balaton Fish Management non-Profit Ltd, Horgony u. 1., H-8600 Siófok, Hungary

The sichel (Pelecus cultratus) is an endemic cyprinid fish of the Hungarian river sys-tem and also the Lake Balaton. The improvement of propagation process can support the reintroduction of sichel and to protect the native populations. However the data available regarding to the males maturation and to the sperm quality is limited. In our experiments preliminary data (standard length-Sl, body weight-Bw, testis weight-Tw, sex ratio, sperm motility) was collected in out-of- (March, 2017) and during the spawning season (May, 2017) caught sichel (Pelecus cultratus). The maturation status was evaluated according to the sperm motilty assessment. Out-of-season individuals were collected using a flood gate trap from the Lake Balaton. During the spawning season fish were collected by angling. The ichthyological and motility parameters (out-of-season) were measured at the seat of the Balaton Fish Management Non-Profit Ltd., Siófok (Hungary). The motility parameters were recorded during the spawning season at the Department of Aquaculture in Szent István University, Gödöllő (Hungary). The testis was dissected and measured. Prior to the CASA analysis, testis was cut in small pieces and squeezed using a mesh (200µm). During the spawning season a part of sperm was released from the testis during the dissection into the Petri dish before squeezing. In the mentioned samples, motility was compared in the squeezed and the collected sperm. Motility parameters (progressive motility, curvilinear velocity-VCL and straightness-STR) were measured using a CASA (computer-assisted sperm analy-sis) system. For motility assessment, an activating solution for cyprinids (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8, Saad et al. 1988) was used. Out-of-season 8 males (18%, Sl: 28±2 cm, Bw: 185±57g, Tw: 1>g) and 36 females (82%, Sl: 29±2 cm, Bw: 213±32g) were collected. Following squeezing, motility was not observed in the sperm samples. During the spawning season, 4 males were caught (Sl: 25±0.5 cm, Bw: 126±13g, Tw: 1±0.3g). A significantly higher progressive motility was recorded in the group collect-ed from the Petri dish (84±8%) in comparison with the squeezed group (24±9%). No significant difference was found in VCL (collected-74±25µm/s, squeezed-60±11µm/s) and STR (collected-85±4%, squeezed-89±2%). Out-of-season only the 18% of the in-vestigated individuals was male which can make difficulties in the establishment of a broodstock from wild caught fish. Prior to the spawning season, males were imma-ture according to the results of the motility assessment. During the spawning season, motility was observed in all males. However, the negative effect of squeezing of testis before motility measurement was also observed.

Keywords: Sichel, ichtyological parameters, maturation, sex ratio, motility

Acknowledgements: This study was supported by the GinOP-2.3.2-15-2016-00004 pro-ject: „establishing the sustainable angling-aimed management of Lake Balaton.“

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Poster presentation, Session I. SpermatogeneSiS and Sperm quality

SPERMATOGENIC CELL T-TyPE CA2+ CuRRENTS ARE POTENTIALLy REGuLATED By ARACHIDONIC ACID (AA)

O. Bondarenko1,2,*, I. López-González1, A. Darszon1

1Universidad Nacional Autónoma de México, Departamento de Genética del Desarrollo y Fisi-ología Molecular, Instituto de Biotecnología, Avenida Universidad 2001, Chamilpa, 62210 Cuer-navaca, Morelos, México2University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; *[email protected]; [email protected]; [email protected]

The concentration of intracellular Ca2+ ([Ca2+]i) regulates many fundamental physi-

ological processes in different cell types including sperm. Specifically, it plays a key role in signaling, regulating motility, capacitation and the acrosome reaction. [Ca2+]

i is

significantly influenced by plasma membrane Ca2+ permeable channels. Among other ion transporters, voltage dependent Ca2+ channels play significant roles in [Ca2+]

i reg-

ulation. The role of T-type voltage dependent Ca2+ channels (CaV3) in mature sperm

physiology remains unclear, since their currents have not been recorded in mature sperm, even though previous studies proved their presence in mammalian spermato-zoa. Contrary to sperm, spermatogenic cells express T-type Ca2+ currents (I

CaT) encoded

by CaV3.1 and Ca

V3.2 genes which could play a relevant role in the spontaneous Ca2+

oscillations occurring during spermatogenesis. In the present study ICaT

were recorded in the whole cell configuration in mouse spermatocytes or round spermatids.

How these ICaT

are regulated during spermatogenesis is still unclear. A previous report documented the presence of the α/β hydrolase domain-containing protein 2 in the male human germ line, which can be activated by different hormones and produces arachidonic acid (AA) and glycerol from 2-arachinoylglicerol (Miller et al, 2016, Science 352 (6285), 555–559.). In this study we investigated the potential regulation of sper-matogenic cell T-type Ca2+ currents by AA. Whole cell I

CaT electrophysiological record-

ings in spermatogenic cells were obtained in the absence or presence of different AA concentrations. AA inhibits I

CaT in a dose-dependent manner with an IC

50=186 nM, with-

out shifting the I-V curve. ICaT

lacks run down in control conditions and AA-induced ICaT

inhibition was reached 5 min after addition and was stable through time. Short-term incubation (2 min) inhibition of I

CaT by AA can be completely recovered by perfusion

with external media containing 1% BSA. However, longer incubation periods (20 min) and/or higher AA concentrations reduce the BSA potency to revert the AA-induced ICaT

inhibition. These results suggest that AA incorporates into the spermatogenic cell plasma membrane and the level of its incorporation depends on AA concentration and incubation time. Finally, preliminary results show AA does not modify the time to peak or the inactivation kinetics of the I

CaT suggesting a reduction of the available Ca2+ chan-

nel fraction as the inhibitory mechanism of ICaT

.

Acknowledgements: OB is a fellow from DGAPA-UnAM (Mexico). This work was sup-ported by COnACyT Fronteras 71 to AD; PAPiiT/UnAM: in205516 to AD, and in204914 to iLG; niH RO1 HD038082-13 to AD; „CenAKVA“ (no. CZ.1.05/2.1.00/01.0024), “Ce-nAKVA ii“(no. LO1205 under the nPU i program) and COST (no. LD14119) and by the Grant agency of the University of South Bohemia (no. 125/2016/Z).

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CHANGES OF SPERM MORPHOLOGy, VOLuME, DENSITy AND MOTILITy PARAMETERS IN NORTHERN PIKE DuRING THE SPAWNING PERIOD

Volodymyr Bondarenko*, Miroslav Blecha, Tomas Policar

University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]

Sexually mature males (BW = 1600±150 g and TL = 235±30 mm) in Northern pike (esox lucius L.) were randomly selected from a production pond and tagged to record changes in their sperm quality parameters (spermatozoa morphology, sperm volume, density, and motility parameters) during the spawning season. Stripping was perfor-med at the beginning of February, March and April with one month interval as begin-ning, middle and end of spawning period. The morphological and motility parameters changed significantly during the reproductive season with following trends. Only, head width wasn‘t changed during the spawning season. Head length was the longest at the middle and shortest at the beginning and end of spawning period. The longest spermatozoa and its flagellar length were found at the middle (TL = 38.24±0.37 µm and 35.14±0.26 µm) and shortest at the beginning of spawning period (TL = 34.81±0.29 µm and 32.53±0.18 µm). Other morphological characters (such as midpiece length, anterior width and posterior length) were always the lowest at the beginning of spa-wning period. Sperm volume was changed from 0.33±0.3 ml in February, 0.43±0.2 ml in March to 0.24±0.1 ml in April, and density from 16.2±0.2×109 spermatozoa ml-1 in February, 19.4±0.2 ×109 spermatozoa ml-1 in March to 4.8±0.2×109 spermatozoa ml-1 in April. Same sperm velocity was observed in all spawning terms at 10 and 20 seconds after activation. Higher velocity was found at 30 and 40 seconds after activation in sperm collected at the middle and the end of spawning period. The percentage of mo-tile spermatozoa at 10 seconds after activation did not show a significant difference between the term of the spawning. Significantly, higher percentage of motile sperm was observed at 20, 30 and 40 seconds after activation in sperm sampled at the end of spawning period. This study supports the hypothesis that longer spermatozoa swim faster.

Keywords: Gamete, esox lucius L., male, reproduction, quality

Acknowledgements: Our study was supported by following projects: “CenAKVA” (no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii” (no. LO1205 under the nPU i program), nAZV projects QJ1510117 and QK1710310.

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OVARIAN FLuID IMPACTS FLAGELLA BEATING AND BIOMECHANICAL METRICS OF SPERM BETWEEN ALTERNATIVE REPRODuCTIVE TACTICS

Ian Anthony Ernest Butts1*, Galina Prokopchuk2, Vojtěch Kašpar3, Jacky Cosson3, Trevor Edgar Pitcher4,5

1Auburn University, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn, Alabama, USA; [email protected] 2Czech Academy of Sciences, Biology Centre, Institute of Parasitology, České Budějovice, 370 05, Czech Republic3University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Centre of Aquaculture & Biodiversity of Hydrocenoses, Research Insti-tute of Fish Culture & Hydrobiology, Vodnany 389 25, Czech Republic4University of Windsor, Department of Biological Sciences, Windsor, Ontario, Canada, N9B 3P45University of Windsor, Great Lakes Institute for Environmental Research, Windsor, Ontario, Can-ada, N9B 3P4

Alternative reproductive tactics (ARTs) are prevalent in nature, where smaller para-sitic males typically have better sperm quality than larger territorial guard males. At present, it is unclear what is causing this phenomenon. Our objective was to gain insights into sperm form and function by examining flagella beating patterns (beat frequency, wave amplitude, bend length, bend angle, wave velocity) and biomechanical sperm metrics (velocity, hydrodynamic power output, propulsive efficiency) between wild spawning Chinook salmon ARTs. Ovarian fluid (OF) and milt were collected to form a series of eight experimental blocks, each composed of OF from a unique female and sperm from a unique pair of parasitic jack and guard hooknose males. Sperm from each ART were activated in river water and OF. Flagella parameters were evaluated from recordings using high-speed video microscopy and biomechanical metrics quantified. We show that ART had an impact on flagella beating, where jacks had a higher bend length and bend angle than hooknoses. Activation media also impacted the pattern of flagella parameters, such that beat frequency, wave velocity, and bend angle declined, while wave amplitude of flagella increased when OF was incorporated into activation media. Furthermore, we found that sperm from jacks swam faster than hooknoses and required less hydrodynamic power output to propel themselves in river water and OF. Jack sperm were also more efficient at swimming than hooknoses and propulsive efficiency increased when cells were activated in OF. Results demonstrate that sperm biomechanics may be driving divergence in competitive reproductive success between ARTs.

Keywords: Oncorhynchus tshawytscha, Spawning, Reproductive strategy, Sperm com-petition, Cryptic female choice

Acknowledgements: This research was funded by nSeRC; Ministry of education, Youth and Sports of the Czech Republic – projects “CenAKVA” (South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses; no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii” (no. LO1205 under the program “national Program of Sustainability i” (nPU i); COST Office (Food and Agriculture COST Action FA1205: AQUAGAMeTe); Ala-bama Agricultural experiment Station; and Hatch program of the niFA-USDA.

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OPTIMIZATION OF SODIuM AND POTASSIuM CONTENT AND PH IN COMMON CARP CYPRinuS CARPiO L. ARTIFICIAL SEMINAL PLASMA

Beata I. Cejko1*, Ákos Horváth 2, Radosław K. Kowalski1

1Polish Academy of Science, Department of Gamete and Embryo Biology, Institute of Animal Re-production and Food Research, Olsztyn, Poland; [email protected] István University, Department of Aquaculture, Institute of Aquaculture and Environmental Safety, Gödöllő, Hungary

The effect of sodium and potassium concentrations as well as various pH values on the motility of common carp sperm during a short-term storage trial was investigated. Sperm was collected from individual common carp Cyprinus carpio L. males during the reproductive period. After collection, each sample of sperm was diluted in immobilizing solutions characterized by various proportions of sodium and potassium. Solutions contained 2 mM CaCl

2, 1 mM Mg

2SO

4, 20 mM Tris at pH 8.0 and was supplemented by

following sodium and potassium concentrations: 0 mM NaCl, 150 mM KCl (variant A), 20 mM NaCl, 130 mM KCl (variant B), 40 mM NaCl, 110 mM KCl (variant C), 75 mM NaCl, 75 mM KCl (variant D), 110 mM NaCl, 40 mM KCl (variant E), 130 mM NaCl, 20 mM KCl (variant F) and 150 mM NaCl, 0 mM KCl (variant G). Sperm was diluted tenfold in the various solution and stored at 4°C. Sperm motility was measured using a CASA system during 72h of storage. To sperm acivation 10 mM Tris buffer containing 100 mM NaCl at pH 9.0 and osmolality of 200 mOsm kg-1 was used. Immediately after dilution, sperm motility was high (90%) in each variants and in the control. After 72h of sperm storage, the highest motility of sperm was noted in variant E (80%) in comparison to variant A (10%), variant B (20%), variant C (25%), variant D (60%), variant F (70%), variant G (50%) and the control (25%). Next, sperm from common carp males was collected and diluted in the most effective solution that contained 110 mM NaCl + 40 mM KCl (variant E) at different pH values i.e. 7.0; 7.5; 8.0; 8.5 and 9.0. Sperm was diluted tenfold and stored at 4 °C. Sperm motility was measured using a CASA system during 72h. Immedi-ately after dilution sperm motility was remained at the high level (90%) in the control group and in solution at pH of 8.5 and 9.0 (85%). After 72h of sperm preservation, the highest motility of sperm was noted in variant E at pH 7.5 (75%) in comparison to buffer at pH 7.0 (65%), pH 8.0 (60%), pH 8.5 (65%), pH 9.0 (65%) and in control group (50%).

To conclude, the most promising option for common carp short term sperm preser-vation is solution containing 2 mM CaCl

2 + 1 mM Mg

2SO

4 + 20 mM Tris + 110 mM NaCl

+ 40 mM KC at pH 7.5. Sperm may be preserved in these conditions for 72h without significant change in motility.

Keywords: Common carp, Sperm, Sodium, Potassium, CASA

Acknowledgements: The presented study was supported by the grant of KnOW Consor-tium “Healthy Animal – Safe Food”, MS&He Decision no. 05-1/KnOW2/2015.

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CHANGES OF SPERMATOZOA LIPID COMPOSITION IN RELATION TO THERMO-ACTIVATION PROCESS IN BuRBOT, lOTA lOTA

Hadiseh Dadras*, Sabine Sample, Tomas Policar, Miroslav Blecha, Borys Dzyuba

University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Wa-ters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, Vodnany 389 25, Czech RepublicE-mail: [email protected]

Fish spermatozoa are normally immotile in semen and become vigorously motile af-ter dilution of seminal plasma by aqueous solutions. It is reported that sperm of bur-bot, Lota lota became spontaneously motile at temperatures >5 °C in seminal plasma, indicating controlled temperature conditions in a range of 3–5 °C are an important factor for handling of semen. In turn, no spontaneous activation of North American burbot, Lota lota maculosa sperm in seminal plasma was observed when temperature was raised from 2 to 10 °C. These results show contradiction impact of temperature on burbot sperm function which could propose species-specific thermo-activation phe-nomenon which is not observed in other fish species. In addition, sperm membrane lipid composition has been proposed to play a prominent role in specific function of sperm cell, because it promotes the creation of micro-domains with different fluidity and permeability traits, required for fertilizing ability. To date, the physiological pro-cesses which induce spontaneous sperm motility at high temperatures in burbot has not been investigated. Therefore, this study was designed to explore thermo-activation phenomenon in burbot sperm in relation to changes of lipid composition. For this study sperm of five individual males were incubated at two media including : seminal fluid and activation medium (10 mM Tris-HCl buffer, pH 8.5, 125 mmol L-1 NaCl+ 2 mmol L-1 KCl) under two temperatures (4 and 30 °C). And then, analysis on lipid composi-tion after incubation of semen samples were done. According to our investigations, spermatozoa were immotile in the seminal plasma at temperatures between 4 and 20 °C. At higher temperatures spontaneous motility was observed. While, at 30 °C most of the spermatozoa were motile. So, in vitro fluctuation of temperature could not be considered as an effective factor for handling of semen. In the present study there was no alter in lipid composition including fatty acid composition, total lipids (cholester-ol and phospholipid) and phospholipid classes at different conditions. These results show contradiction impact of temperature on burbot sperm function which could pro-pose species-specific thermo-activation phenomenon. Moreover, lipid composition has shown no contribution in this phenomenon. Further studies are required to identify how a unique thermo-activation mechanism may be affected by other factors involved.

Keywords: Lipid, Lota lota, motility, spermatozoa, thermo-activation

Acknowledgments: The study was financially supported by the Ministry of edu-cation, Youth and Sports of the Czech Republic through projects: CenAKVA (no. CZ.1.05/2.1.00/01.0024), CenAKVA ii (no. LO1205 under the nPU i program), COST (no. LD14119), by the Grant Agency of the University of South Bohemia in Ceske Bude-jovice (no. 125/2016/Z) and nAZV project (no. QK1710310).

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SPERMATOZOON STRuCTuRE, LIPID COMPOSITION AND MOTILITy IN RELATION TO INTERNAL FERTILIZATION IN FRESHWATER STINGRAy POTAMOTRYgOn MOTORO

Borys Dzyuba1*, Sabine Sampels1,3, Viktoriya Dzyuba1, Alexandre Ninhaus Silveira2, Mar-tin Kahanec1, Rosicleire Veríssimo Silveira2, Vitaliy Kholodnyy1, Hadiseh Dadras Asyabar1, Marek Rodina1, Sergii Boryshpolets1

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]ão Paulo State University, Ilha Solteira, Faculty of Engineering, Department of Biology and Zoo-techny, Monção street, 226, 15385-000, Ilha Solteira, SP, Brazil3Swedish University of Agricultural Sciences, Department of Molecular Sciences, PO Box 7015, 75007 Uppsala

All extant groups of Elasmobranches have internal fertilization but the structure of male reproductive organs is very uncommon: sperm passes from internal organs via the cloaca, but the male copulating organ (clasper) is not close to the cloaca. This suggests that sperm contacts the water medium before fertilization. Because of this involvement of environ-ment, external signalling in sperm motility activation cannot be excluded even though fer-tilization is internal. Thus, spermatozoa of Elasmobranches should possess specific struc-ture and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes that occur during fertilization. Additionally, sperm motility in this taxa is not well understood. The current study examined spermato-zoon structure, lipid composition and motility in different environmental conditions for Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from the cloaca of mature males (n=7) during the natural spawning period. Sperm motility was examined in seminal fluid, uterine fluid, fresh water, and artificial media of different osmolality and ionic composition. Spermatozoon structure was investigated by routine scanning and transmission electron microscopy. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Sperm motility was evaluated by high speed (2000 frame/s) video recordings. Spermatozoa of P. motoro are relatively large cells: total length 146±4 µm, tail length 75±3 µm, flagellar structure comprises 9+2 axoneme and two longitudinal columns, cork-screw like head is of 71±8 µm. Spermatozoa FAs consisted of 33±1% saturated, 28±1% mono-unsaturated, and 41±1% poly-unsaturated FAs, and a high content of n-6 FAs (32±2%) was noticed. Ratio between different FAs suggests low tolerance of membrane to hypotonicity. Helical flagellar motion was observed in uterine or seminal fluids or media of 300–380 mOsm/kg and resulted in spermatozoon progression associated with rotation of the head. After dilu-tion with fresh water, spermatozoa were immotile and had compromised structure. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without sperm contact with hypotonic environment to preserve fertility, associated with specific spermatozoa structure, lipid composition and motility mode.

Keywords: elasmobranches, sperm motility, membrane, lipids, internal fertilization

Acknowledgements: The study was supported by the Czech Science Foundation (Project no. 16-03754S), by the Ministry of education, Youth and Sports of the CR (“CenAKVA” no. CZ.1.05/2.1.00/01.0024, “CenAKVA ii” no. LO1205 under the nPU i program) and COST (no. LD14119) and by the Grant agency of the University of South Bohemia (no. 125/2016/Z).

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EVIDENCE OF SPERM MATuRATION IN FRESHWATER STINGRAyS POTAMOTRyGON MOTORO

Viktoriya Dzyuba1*, Alexandre Ninhaus Silveira2, Martin Kahanec1, Rosicleire Veríssimo Silveira2, Jan Sterba3, Marek Rodina1, Borys Dzyuba1

1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected] São Paulo State University, Ilha Solteira, Faculty of Engineering, Department of Biology and Zootechny, Monção street, 226, 15385-000, Ilha Solteira, SP, Brazil3 University of South Bohemia in České Budějovice, Faculty of Science, Institute of Chemistry and Biochemistry, Branišovská 1760, 370 05 České Budějovice, Czech Republic

Sperm maturation as a process through which morphologically complete spermatozoa acquire the ability for motility activation and fertilization is well known for many groups of animals. In contrast to most fish with external fertilization, a large number of animals have internal fertilization where it is the epididymis of male reproductive tract which is necessary to complete spermatozoa maturation. Sperm maturation in the epididymis of cartilaginous fishes has been proposed but this still must be proven. We used nine ma-ture freshwater stingrays Potamotrygon motoro collected during the natural spawning period to verify epididymal sperm maturation and to describe the changes in sperma-tozoon motility parameters during this process. Sperm samples were collected from: 1) cloaca, 2) testis, 3) epididymis, and 4) seminal vesicle. Uterine fluid (UF) and seminal fluids (SF) were obtained by centrifugation (2000 g for 30 min) of liquids from uterus and sperm samples respectively. Sperm was diluted with two activating media (300 mM sucrose and 150 mM NaCl), SF or UF, and spermatozoon motility was analysed by video microscopy to estimate percent of motile cells (motility rate) and spermatozoon curvilin-ear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity of the curvilinear path (LIN), straightness of the average path (STR) using integrated system for semen analysis (ISAS, Proiser, Spain). Sperm concentration in samples collected directly from seminal vesicle (2.7±0.8 x 109 cells/ml) was higher than those collected from clo-aca (1.7±0.7 x 109 cells/ml), indicating that during ejaculation sperm can be diluted by urine. There was no significant difference in SF osmolality between sperm collected from seminal vesicle (362±7 mOsm/kg) and from cloaca (380±15 mOsm/kg). Spermatozoa collected from seminal vesicles and cloaca were motile in each activating media tested. The highest motility parameters were recorded in in SF (VCL 28±4 µm/s, VSL 22±4 µm/s, VAP 23±4 µm/s, LIN 78±12 %, STR 97±2%). Spermatozoa from testes and epididymis did not initiate motility in the solutions. Our results confirmed the existence of a sperm mat-uration process in P. motoro which quite probably occurs after sperm pass through epidid-ymis. Cellular mechanisms underlying this process should be further studied.

Keywords: cartilaginous fishes, internal fertilization, sperm maturation, sperm motili-ty, epididymis

Acknowledgements: The study was supported by the Czech Science Foundation (Proj-ect no. 16-03754S), by the Ministry of education, Youth and Sports of the CR (“CenAK-VA” no. CZ.1.05/2.1.00/01.0024, “CenAKVA ii” no. LO1205 under the nPU i program) and COST (no. LD14119) and by the Grant agency of the University of South Bohemia (no. 125/2016/Z).

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THE EFFECTS OF GiNGER ON ZEBRAFiSH SPERM MOTiLiTy

İlker Keskin1, Sude Atmaca1, Aygül Ekici 2*

1Istanbul University, Institute of Graduate Studies in Science and Engineering. Department of Aquaculture2Istanbul University Faculty of Fisheries, Department of Aquaculture, Ordu Street No:200 34130 Fatih/ İstanbul TURKEY; [email protected]

Ginger is a species belonging to the Zingiberaceae family called Zingiber officinale. This species is used as a spice from roots or rhizomes. Ginger is a plant that origin was South East Asia that is widely cultured in İndia, China, Nigeria, Sierra Leone, Indone-sia, Bangladesh, Australia, Fiji, Jamaica and Nepal. The primarily intended purpose is that fresh ginger is used as a sweetener and as a secondary use, dry ginger is used as powder ginger, ginger oil and resin. Studies have shown that ginger has the effect of reducing DNA breaks in human. On the other hand, in rats, the increase in testicular volume, sperm quality and quantity as well as the increase in serum testosteron level and histopathologically detected lesions improved. The zebrafish is used model to stu-dies in many areas. In this study, it is preferred because the generation time is short. Male fish have been kept in same aquarium with female fish for sperm release and gi-ven feed to be effective soon. In the study, powder ginger which is sold in tablet form was given once a day as dosage of 240 mg for 30 days. When evaluated the behavioral point of view, it was observed that ginger-fed groups had a higher intake of feeds than the control groups and faster in aquarium. Despite not being statistically significant, a decrease in body weight was detected. Osmolality of sperm; 295 mOsm/kg in the ginger-fed group and 320 mOsm/kg in the control group. It was detected that values of sperm motility duration is average 75±2sn in both groups. A significant difference was found when spermatological characteristics of the two groups were compared (p<0.05). VAP,VSL and VCL values were higher in ginger-fed group than in the control group.

Keywords: Ginger, zebrafish, Danio rerio, sperm motility

Acknowledgements: We would like to thank to Prof.Devrim MeMİŞ providing us Com-puter-assisted sperm analysis system.

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uLTRASTRuCTuRAL DISTRIBuTION OF CALCIuM DuRING SPERMATOGENESIS OF ZEBRAFISH, DAniO RERiO

Amin Golpour, Martin Psenička, Hamid Niksirat

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]

Calcium plays a variety of vital regulatory functions in many physiological and bioche-mical events in the cell. The aim of this study was to describe the ultrastructural distri-bution of calcium during different developmental stages of spermatogenesis in a mo-del organism, the zebrafish (Danio rerio), using a combined oxalate–pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and sperma-tozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly de-tectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The no-table changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.

Keywords: Oxalate-pyroantimonate, quantification, testis, ultrastructural localization

Acknowledgements: The study was financially supported by the Ministry of education, Youth and Sports of the Czech Republic, projects CenAKVA (no CZ.1.05/2.1.00/01.0024) and CenAKVA ii (no. LO1205 under the nPU i program), by the Grant Agency of the Uni-versity of South Bohemia in CeskeBud ejovice (125/2016/Z) and by the Czech Science Foundation (no. P502/13/26952S).

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FERTILIZING ABILITy OF GAMETES AT DIFFERENT POST-ACTIVATION TIMES AND THE SPERM-EGG-RATIO IN THE ARTIFICIAL REPRODuCTION OF PIKEPERCH SAnDER luCiOPERCA

Jiri Kristan1*, Daniel Zarski2,3, Miroslav Blecha1, Tomas Policar1, Oleksandr Malinovskyi1, Azin Mohagheghi Samarin1, Katarzyna Palinska-Zarska2, Joanna Nowosad2, Slawomir Krejszeff2 & Dariusz Kucharczyk2

1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research In-stitute of Fish Culture and Hydrobiology, Zatisi 728/II 389 25 Vodnany, Czech Republic; [email protected] and Mazury University in Olsztyn, Department of Lake and River Fisheries, ul. Oczapows-kiego 5, PL 10-719 Olsztyn-Kortowo, Poland3Szent István University, Faculty of Agricultural and Environmental Sciences, Institute of Aquacul-ture and Environmental Safety, Department of Aquaculture, 2100 Gödöllő, Páter K. u. 1, Hungary

The time period during which egg and spermatozoa retain their fertilizing ability after contacting with water, was evaluated in pikeperch (Sander lucioperca). In addition, suc-cess of in vitro fertilization was examined regarding to the sperm-to-egg ratio (SER). In the first trial, eggs were placed in petri dishes containing 5 mL of the hatchery water, to which freshly collected and pooled sperm were added to each sample at 0, 15, 30, 60, 90, 120, 150 and 180 s post-egg activation. The eggs retained their fertility for at least 30 s after contacting with water. The second trial tested the maximum time period during that spermatozoa retained fertilizability after contacting with water. Milt (50 µL) was collected from each male and added to 5 mL of the water in petri dishes. Thereafter eggs were added at 0, 5, 15, 30, 60 and 75 s post-sperm activation. Delays exceeding 15 s affected negatively on the fertilization success. The third trial examined the optimum SER; in which was found that 100 x 103 spermatozoa per egg was the minimum ratio to ensure fertilization rates above 70%. Overall, the data clarified some biological interactions of gametes in the artificial propagation of pikeperch.

Keywords: Fertilization success, gamete activation, Sander lucioperca, sperm-egg-ra-tio, reproduction

Acknowledgements: The study was financially supported by projects of the Min-istry of education, Youth and Sports of the Czech Republic: „CenAKVA“(no. CZ.1.05/2.1.00/01.0024) and “CenAKVA ii“(no. LO1205 under the nPU i program), nAZV projects QK1710310, QJ1510117 and GAJU project 060/2016/Z. The research was partially funded by the University of Warmia and Mazury, Olsztyn, Poland grant (no. 0808-0801).

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THE EFFECTS OF CHILLED STORAGE AND THE pH OF THE ACTIVATING SOLuTION ON DIFFERENT MOTILITy PARAMETERS IN BuRBOT (lOTA lOTA) SPERM

Levente Várkonyi1, Zoltán Bokor1, Balázs Csorbai1, Zsolt Szári2, István Ittzés3, Zoltán Szabó3, Daniel Żarski4, Béla Urbányi1, Gergely Bernáth1

1Szent istván University, Department of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary2Balaton Fish Management non-Profit Ltd, Horgony u. 1., H-8600 Siófok, Hungary3Self-entrepreneur, H-2425 nagykarácsony, Hungary4University of Warmia and Mazury, Department of Lake and River Fisheries, Olsztyn, Poland

Burbot (Lota lota) is an autochthonous species in Europe. Development of burbot pro-pagation and rearing is a need in Hungary for two reasons: natural populations (mainly from lake Balaton) should be strengthened and rising economic importance of the spe-cies creates a demand. The expansion of our knowledge regarding to the burbot sperm movement can enhance the imrovement of the propagation process. In our experiments a broodstock of burbot males (n=17) was maintained at the Department of Aquacul-ture, in Szent István University, Gödöllő (Hungary). Fish was kept at a temperature 2–3 °C. Spermiation was hormonally induced using 1 Ovopel (D-Ala6, Pro9NEt)-mGnRH+me-toclopramide)) bodyweight kg-1. Sperm was collected 3 days following injection. In our first experiment, motility parameters were investigated using a CASA (computer-assis-ted sperm analysis) system during 96 hours chilled storage (4 °C) in 24 hours interval. Sperm (n=6) was activated using a basic ionic solution (50mM NaCl, 30 mM Tris, pH 8.5, Lahnsteiner 2011.). In our second experiment, the motility parameters of 7 males were compared using the above mentioned ionic solution, prepared with two different pH (4.4 and 8.5). Chilled storage caused a significant reduction in progressive motility already after 24 hours (0h: 49±24%, 24h: 12±7%). Significant decreasing tendency was record-ed only after 72 hours in DAP (distance average path, 0h: 26±4µm/s, 72h: 19±9µm/s), DSL (distance straight line, 0h: 21±5µm/s, 72h: 17±8µm/s), VAP (average path veloci-ty, 0h: 59±9µm/s, 72h: 43±21µm/s), and BCF (beat cross frequency, 0h: 28±2Hz, 72h: 18±10Hz) parameters. No significant reduction was observed in DCL (distance curved line), VCL (curvilinear velocity), VSL (straight line velocity), STR (straightness), LIN (lin-earity), WOB (wobbling), and ALH (amplitude lateral head displacement) parameters up for up to 96 hours storage. A significantly higher progressive motility (49±22%), DAP (25±5µm/s), DCL (31±4µm/s), DSL (20±5µm/s), VAP (57±10µm/s), VCL (70±9µm/s), VSL (47±11µm/s), and BCF (27±2Hz) was measured with the activating solution buffered on pH 8.5 in comparison with pH 4.4 (progressive motility-14±19%, DAP-16±5µm/s, DCL-19±7µm/s, DSL-13±4µm/s, VAP-36±13µm/s, VCL-44±17µm/s, VSL-29±9µm/s, and BCF-18±9Hz). No significant differences were measured in STR, LIN, WOB, and ALH parame-ters using the two solutions. Chilled storage and the pH of the two activating solution had different effects on the various motility parameters of burbot sperm.

Keywords: Chilled storage, pH, burbot, motility, CASA parameters

Acknowledgements: The work was supported by the european Fisheries Fund Fishe-ries Operative Programme iii. axis, european Fisheries Fund for Renewable Fisheries provided by the eU and Hungary as well as the Department for Angling and Fisheries, Ministry of Agriculture.

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SPERM quALITy ANALySIS OF NORMAL SEASON AND OuT-SEASON By PHOTOPERIOD MANIPuLATION OF MALE RAINBOW TROuT BROODSTOCK (OnCORHYnCHuS MYKiSS)

Momin Momin, Devrim Memiş*

Istanbul University, Fisheries Faculty, Aquaculture Department, Ordu Street, No: 200, Laleli, Is-tanbul, Turkey; [email protected]

Sperm quality parameters of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) were investigated during normal spawning season (November-January) and out-season (July-August) spawning by photoperiod manipulation. Normal spawning males (N=5) were kept in open concrete pond under natural condition. Photoperiod manipulated males (N=5) were treated with artificial LED light (50 lumens/m2) in closed concrete pond, where application started with summer photoperiod at 8D:16L from January to April, 2016, then winter photoperiod at 16D:8L to the end of the July 2016. Commercial trout feed (10 mm diameter pellet at a rate of 2% of their total body weight) was given twice a day in both groups during the study period. The mean weight and body length of males were 1193.3±183.15 g and 45.05±2.25 cm, respectively. Sperm was collected through abdominal massage at the end of the July, 2016 from the photoperiod ma-nipulated group (PMG) (water temperature 15.4±1.2 °C) and at the middle of the De-cember, 2016 from the normal spawning group (NSG) (water temperature 8.8±0.2 °C) without using any anesthetic drug. Motility parameters of spermatozoa were assessed by CASA. Volume of sperm, osmolality of seminal plasma, density of sperm, percentage of motile spermatozoa (Mot), curvilinear velocity (VCL) and duration of motility were measured for each male. Tukey (HSD) test was done and no significant differences were found in volume of sperm, pH of sperm and osmolality of seminal plasma be-tween PMG and NSG. Nevertheless, density of sperm, duration of motility, percentage of motile spermatozoa (Mot) and curvilinear velocity (VCL) are significantly different (p <0.05). Though some differences were found but these two groups also showed simi-larities in some parameters. The results of the study showed that good quality sperm can be collected in summer by using only artificial light where the quality parameters are almost similar to the sperm of the normal season of rainbow trout broodstock.

Key Words: Rainbow trout, photoperiod, CASA, out-season, sperm quality

Acknowledgment: This work was supported by Scientific Research Projects Coordina-tion Unit of istanbul University. Project number 24374. We would like to thank Gökhan Tünçelli and Dr. Güneş Yamaner for their cordial help throughout the study.

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GONADOTROPIN SIALyLATION IN EuROPEAN SEA BASS (DiCEnTRARCHuS lAbRAx), ANOTHER WAy OF ACTIVITy REGuLATION?

Gregorio Molés*, Cinta Zapater, Silvia Zanuy, Ana Goméz

Instituto de Acuicultura de Torre la Sal (IATS-CSIC). Fish Physiology and Biotechnology De-partment. Ribera de Cabanes s/n, 12595, Castellon, Spain; [email protected]

The pituitary gland synthesizes and secrets differently glycosylated forms of gona-dotropins that differ in their oligosaccharide structure. In the case of FSH, several enzy-mes are involved in its post-translational glycosylation processing; particularly the sia-lyltransferases (STs) that transfer sialic acid residues into the FSH carbohydrate chain. Previous studies with recombinant sea bass Fsh showed that, as occurs in mammals, less sialylated isoforms exhibit higher in-vitro biological activity but shorter plasma half-life than their more sialylated counterparts. These facts suggest that gonadotropin heterogeneity represents an alternative mechanism of the pituitary gland to regulate the intensity and duration of gonadotropin stimuli in the gonads. In addition, the com-position of the FSH carbohydrate moiety is likely regulated by the endocrine milieu, and the GnRH and gonadal sex steroids have been proposed as the main regulators of FSH microheterogeneity.

Our aim in this study was to know the expression profile of several sea bass ST ge-nes in male pituitary during a whole reproductive cycle and to elucidate whether tre-atments with gonadal steroids (Estradiol, Testosterone, Dihydroxyprogesterone) and LHRHa can affect the expression of these genes in a pituitary primary cell culture, and therefore to the potential sialylation of sea bass Fsh.

Keywords: Follicle Stimulating Hormone, sialyltransferase, gonadotropin activity regu-lation

Acknowledgements: Founded by MineCO (AGL 2015-67477-C2-1-R), CSiC (201640e073) and GV (PROMeTeO ii-2014/051).

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IMPACT OF GONADAL STEROIDS ON PITuITARy GONADOTROPINS IN MALE EuROPEAN SEA BASS (DiCEnTRARCHuS lAbRAx)

Gregorio Molés1*, Cinta Zapater1, Patricia Pinto2, Soledad Ibañez1, Ana Goméz1

1Instituto Acuicultura de Torre la Sal (IATS-CSIC). Fish Physiology and Biotechnology Department. Ribera de Cabanes s/n, 12595, Castellon, Spain; [email protected] 2University of Algarve. Centre of Marine Sciences (CCMAR). Faro. Portugal

The follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central en-docrine regulators of gametogenesis in vertebrates. Gonadotropin-releasing hormones (Gnrh) have been postulated as the main regulators of the synthesis and secretion of Fsh and Lh since long. In addition, sex steroids produced by the gonads have a feed-back effect modulating the availability of gonadotropins. All these effects at the level of pituitary have a direct impact in gametogenesis progression.

Previous in vivo studies in sea bass, during the sexual resting period (August), showed that Gnrh injections stimulated Lh synthesis and release, but had no effect on the expression of the Fsh beta-subunit gene (fshb). At the same time, different steroid implants almost suppressed basal fshb expression, while activated the expression of lhb in the pituitary.

To elucidate how this system is organized in the pituitary of sea bass, we analysed in a first step the annual profile of male pituitary expression of the estrogen, androgen and progesterone receptors, in relation with the different stages of spermatogenesis, the pituitary gonadotropin content and the circulating levels of sex steroids. In addi-tion, immunohistochemistry studies have been performed to identify the pituitary cells containing steroid receptors. Next, we have used an in vitro pituitary primary cell cul-ture stimulated with Estradiol, Testosterone Dihydroxyprogesterone and a Luteinizing Hormone-Releasing Hormone analogue (LHRHa) to analyse their impact on gonadotro-pin synthesis and release.

It has been confirmed the strong effect of LHRHa on sea bass Lh release, while this effect is more weak on Fsh release and decreases with the advance of spermatogene-sis. On the other hand, the main evidence of repression for both gonadotropins is due to Estradiol, with major intensity at the beginning of spermatogenesis.

Keywords: Sex steroids, Gnrh, gonadotropin regulation

Acknowledgements: Founded by MineCO (AGL 2015-67477-C2-1-R), CSiC (201640e073) and GV (PROMeTeO ii-2014/051).

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THE INFLuENCE OF SPERM ENZyMES INHIBITION ON THE PERCENTAGE OF FERTILIZED CARP EGGS

Beata Sarosiek1, Katarzyna Dryl1, Joanna Nowosad2, Dariusz Kucharczyk2

1Polish Academy of Sciences, Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Tuwima Str. 10, 10-748 Olsztyn, Poland; [email protected] of Warmia and Mazury in Olsztyn, Department of Lake and River Fisheries, Poland

Our former studies revealed that suppressing of activity of some fish gametes en-zymes (siberian sturgeon, rainbow trout) caused decrease in percentage of fertilized eggs. The aim of this study was to test how addition of: ammonium molybdate (4; 1; 0.25mM), saccharolactone (1; 0.2; 0.04mM), gossypol (1; 0.25; 0.06µM) and acet-amide (0.5; 0.13; 0.03M) – inhibitors of respectively: acid phosphatase, β-glucuroni-dase, lactate dehydrogenase and β-N-acetylglucosaminidase (β-NAGase) affected the percentage of fertilized eggs. Additionally, influence of mannitol (0.5; 0.13; 0.03M) – another β-NAGase inhibitor increasing buffer osmolality – was checked. Doses of inhibitors and mannitol were set during preliminary studies. It was proved that percent-age of fertilized eggs was not decreased by addition of any inhibitors. Only mannitol addition decreased the percentage of fertilized eggs. It need to be stated, that none of parameters of sperm movement were negatively affected by any enzymes’ inhibitors. Mannitol however, apart from the lowest dose, caused severe decrease in all analyzed parameters of sperm movement. Thus, in this case, decrease in percentage of fertilized eggs was connected with negative influence of mannitol to carp sperm movement ap-paratus. Carried out experiment showed that increase of osmolality up to 650 mOsm/kg (by addition of acetamide and mannitol) is not critical parameter during carp’s eggs fertilization. If so, the percentage of fertilized eggs would be similar in both acetamide and mannitol variants. Activity of β-NAGase, acrosomal enzyme, in carp’s seminal plas-ma was very high, compared to activity of other enzymes and amounted about 600U/l. For comparison purposes – rainbow trout plasma was about 500–550 U / l, and in this species, the addition of acetamide resulted in complete inhibition of fertilization. Why inhibition of any variants of analyzed enzymes’ activity did not affect negatively percentage of carp’s fertilized eggs? It was possible that enzymes in carp’s sperm and oocytes were so well protected, that access of inhibitors to enzymes during concep-tion was limited and resulted in inhibitors’ failure. Other possibility was that duration of inhibitors activity was too low (2–3 min.). Further experiments will be needed to clarify these questions.

Keywords: Carp, eggs,enzyme inhibition, acrosomal enzyme, fertilization

Acknowledgements: The study was supported from funds appropriated to institute of Animal Reproduction and Food Research, Polish Academy of Sciences.

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REPRODuCTIVE CyCLE AND SPERMATOGENESIS OF PIRACANJuBA (bRYCOn ORbignYAnuS) IN CAPTIVITy

Danilo Pedro Streit Jr1*, Éverton Luís Zardo¹, Daniel Antônio Rotili¹, Lis Santos Marques¹, Itamar Cossina Gomes¹ , Marcelo Bernardi¹ , Damião Guedes², Júlia Giora³1Universidade Federal do Rio Grande do Sul, Faculdade de Agronomia, Produção e Conservação da Biodiversidade das Espécies Aquáticas (AQUAM), Av. Bento Gonçalves, 7712, Porto Alegre/RS, Brazil; [email protected] Meio Biótico Serviços Ambientais. Rua Sebastião Laurentino da Silva 126, APT 604, Córrego Grande, Florianopolis/SC, Brazil³Universidade Federal do Rio Grande do Sul, Instituto de Biociências, Laboratório de Ictiologia, Av. Bento Gonçalves, 9500, Porto Alegre/RS, Brazil.

Piracanjuba (Brycon orbignyanus) is a neotropical species, occurring naturally in Paraná, Paraguay, Uruguay and Paraná River basins. Their natural populations are declin-ing dramatically in recent years due to overfishing, environmental pollution, reduced supply of natural food and construction of hydroelectric dams. B. orbignyanus is often used in repopulation programs in Brazil, since it is considered an environmental indi-cator and is among listed species threatened with extinction. The knowledge of mor-phological changes throughout the reproductive cycle, as well as the technical ability in reproductive biotechnologies, are essential issues to be successful in reintroduction programs. Thus, the aim of the present study was to describe the spermatogenesis and the reproductive cycle of the piracanjuba under cultivation conditions, in order to elab-orate a maturity scale for the species. Animals were euthanized in immersion of Ben-zocaine solution (50 mg/L) for biometry, dissection and removal of testicles. Samples were collected in two years, in all seasons, with monthly and quarterly collections in the first and second year, respectively. Animals are between 3.9 cm and 32.5 cm in size and weigh between 0.62 g and 416 g. For the histological analysis, following fixation in 10% paraformaldehyde, the samples were dehydrated in graded alcohol up to 95% (70%, 80% and 95% for 24 hours in each stage) and embedded in blycol methacrylate infiltration solution (Leica historesin). Three-micron thick sections were cut on a mi-crotome (RM2245, Leica), mounted on glass slides and stained with Toluidine Blue. To date, three reproductive phases were identified: immature, initial development, and de-veloping. The immature phase is marked by the presence of small, translucent testicles containing only spermatogonia and lumen still imperceptible. The “initial development” phase presents differentiated spermatogonia (A and B) and cysts of primary spermato-cytes. The developing phase presents all stages of cellular development: spermatogo-nia A and B, primary and secondary spermatocytes, spermatids, and even spermatozoa in the lumen of the seminiferous tubules. This description of the maturation scale will help to understand the processes related to sexual differentiation, spermatogenesis and reproductive cycle of males of piracanjuba kept under cultivation conditions.

Keywords: Germ cells, Spermatocytes, Gametogenesis, Maturation scale, Gonadal de-velopment

Acknowledgements: To CnPq and CAPeS for the granting of postgraduate scholarships. To BAeSA / eneRCAn for the funding of the study research project.

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NILE TILAPIA AS A VERSATILE MODEL FOR STuDyING HORMONAL CONTROL OF SPERMATOGENESIS in ViVO AND Ex ViVO

Michelle Thönnes, Marlen Kotte, Katja Steinborn, Alexander Froschauer and Frank Pfen-nig*

Technische Universität Dresden, Institute of Zoology, Chair of Zoology and Developmental Biolo-gy, D-01217 Dresden, Germany; *[email protected]

Dominant and territorial behavior is well known in cichlids. In the Nile tilapia (Oreo-chromis niloticus), males form stable social hierarchies where the social status of an individual can influence its reproductive fitness and success. Fish with high social sta-tus (especially dominant males) have well developed gonads. Such dominant and terri-tory-holding tilapia males are also characterized by high plasma levels of gonadotropic and androgenic hormones. Dominant animals suppress reproduction and fertility of the subordinates by aggressive behavior. In the aquaria at our institute we have ob-served long-term stable social hierarchies that make the Nile tilapia an excellent model fish to elucidate the influence of social rank on the reproductive axis. We analyzed and compared the expression of key genes of gonad development in males with different social status. One goal of our studies is to learn about the role of Amh (Anti-Müllerian hormone) in Nile tilapia. Amh is an important regulator of germ cell development and steroid hormone production in vertebrates. The interrelations of Amh with gonado-tropins and androgens are in the focus of our experiments using an ex vivo cultiva-tion system for testes fragments. This allows the testing of hormones or chemical compounds without animal experiments. Histological analysis using PCNA (marker of S-phase of cell cycle) antibodies revealed that the testis fragments can be cultured without any structural loss for 9 days or longer. We used this testis cultures to study the effect of pituitary extracts and selected hormones on gene expression of amh and other marker genes of testis development. Our data can contribute to unravel the mechanisms of gonadotropic action in Nile tilapia, an economical important species of aquaculture.

Keywords: nile tilapia, social hierarchy, spermatogenesis, testis explants, ex vivo, Amh

Acknowledgements: This work was supported by the Deutsche Forschungsgemein-schaft (DFG FP683/5-1)

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A RESEARCH ON FERTILIZATION SuCCESS AND GAMETE quALTy PARAMETERS OF ENDEMIC WILD TROuT (SAlMO SP.) IN TuRKEy

Devrim Memiş, Gökhan Tunçelli, Güneş Yamaner

Istanbul University, Fisheries Faculty, Aquaculture Department, Laleli, Fatih, Istanbul, Turkey; [email protected]

Endemic fish species are decreasing in their natural habitats. Aquaculture is using for the protection of biodiversity. Especially endangered fish species moved to the rearing systems for the stock enhancement and monitoring program. Wild fish species are cultured and adapted to the hatchery conditions and then release to the their natural environment. The reasons of decreasing of endangered fishes are face on some impor-tant problems such as water pollution, lose of habitat, Hydro Electric Power Stations, sand-mines, sets, dams, over fishing etc. Migratory fishes are more affected by the disrupted river systems.

In this study, minimum weight of 10 g and maximum weight 65 g wild trout fish (Sal-mo sp.) are captured by electroshock device in the Bıçkıdere Stream (40°34‘42.99“K; 29°55‘57.60“D) in Kocaeli Province in january 2014. Captured wild fish were brou-ght to the Sapanca Inland Fish Culture Research and Applied Unit. Experimental fis-hes are kept in circular fiberglass tank, 1.5 m diameter at mean water temperature 11.79±4.55 °C. Broodstock fed with commercial trout feeds till their first spawning.

Two years later, total 5 female (476.32±96.48 g) and 2 male (529±51 g) individuals has spawned at 8.4°C water temperature in November 2016. The sperm quality para-meters were investigted with the Computer Assisted Sperm Analysis (CASA) system. Values of total motility, progressive motility, sperm volume, VCL, VSL, VAP, LIN and STR parameters are; 70,41%, 43,26%, 7,5 ml, 93,79 µm/s, 74,06 µm/s, 84,23 µm/s, 70,35% and 80,64% respectively. The average of eggs diameter was ±3,84 mm and 6.809 fer-tilized eggs placed to vertical incubation system at 12,5°C constant well water in the hatchery. Fertilization rate was calculated as 95%. After 23 days, eyed-eggs stage was observed with 27,92%. The hatched larvae rates was calculated as 6.93% after 5 days . The larvae (1.17% of hatched) was started feeding with starter commercial trout feeds after 29 days from fertilization.

The result of the study shows wild Salmo sp. which are captured from natural habitat can be able to adapt to the hatchery conditions. However, we need more researches to get high survival rate of larvea. Moreover, we should focus on optimal feed formulation for broodstock and larvea to get more quality gamete of this species. Their adaptation to the hatchery and water conditions should be improved.

Keywords: endemic wild trout, Salmo sp., sperm quality, fertilized egg, adaptation

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CHARACTERIZATION AND SHORT-TERM STORAGE OF THE PATAGONIAN BLENNy (ElEginOPS MAClOVinuS) SPERM

Patricio ulloa-Rodríguez1*, Pablo Contreras1, Elías Figueroa1,2, Manuel Lee-Estevez1, Iván Valdebenito2, Jenny Risopatrón1, Jorge Farías1

1Universidad de La Frontera, Faculty of Engineering and Sciences, Chemical Engineering Depart-ment, Francisco Salazar 01145 Temuco, Chile; [email protected] University of Temuco, Research Nucleus on Animal Production, Rudecindo Ortega 02950 Temuco, Chile

Patagonian blenny (eleginops maclovinus; known in Chile as róbalo) resides mainly in the southern coasts of South America and its population have been decreasing due to overexploitation. Little is known about the reproduction behaviour of the Patagonian blenny, and even less about its reproduction biology. Fish sperm of several other teleo-sts are described in literature, but research regarding fish sperm from native species from Chile is only beginning. Understanding the ability for Patagonian blenny sperm to sustain short-term storage is a key step to carry out artificial propagation of this species. In this aspect, sperm function markers (membrane and DNA integrity; reac-tive oxygen species generation), cell respiration/mitochondrial-function markers (mi-tochondrial membrane potential; dynamics of the ATP content; oxygen consumption), and also the pH and osmolarity of the extracellular medium during cold storage could be highly helpful for designing an in-vitro management protocol for Patagonian blenny. Patagonian blenny spermatozoa structure and ultrastructure are consistent with sper-matozoa from modern teleosts with external fertilization, measuring ~40 μM length, a head of ~0.8 μM diameter, and containing 3 to 5 spherical mitochondria. Semen contains ~15×109 spz mL-1, an osmolarity of ~345 mOsm kg-1 and pH of ~7.5. Analyses of sperm function were done during 14-day cold storage, under diluted (1:1 with Cort-land solution) and undiluted conditions. The use of Cortland solution do improve the storage time from 3 to 7 days approximately, allowing better gas exchange, preventing desiccation and keeping membrane integrity better. Factors that affect most the stor-age time are reactive oxygen species (ROS) generation and unwanted motility process activation during storage, produced probably by osmolarity and ion content differen-ces. In further experiments, a Cortland solution with modifications will be designed in order to address these drawbacks and improve storage time, and fertilizing capacity experiment will be done.

Keywords: Aquaculture, fish sperm, sperm function, robalo

Acknowledgements: The study was supported by Conicyt (Chile) doctoral scholarship grant no. 21140852, and Fondecyt project no. 1151315 (JF).

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SPERMATOGENESIS PROCESS AND SuPPORT CAPACITy OF SERTOLI CELLS IN Astyanax Altiparanae (CHARACIFORMES)

Rosicleire Veríssimo-Silveira1*, Maira da Silva Rodrigues1, Patrícia Postingel Quirino1,

Diógenes Henrique de Siqueira-Silva2, Alexandre Ninhaus-Silveira1

1São Paulo State University UNESP, Ilha Solteira School of Engineering, Department of Biology and Zootechnics, Av. Brasil, 56, 15385-000 Ilha Solteira, SP, Brazil; [email protected] University of South And Southeast of Pará UNIFESSPA, Faculty de Health and Biological Sciences. Folha 31, quadra 07, 68507-590 Nova Marabá, PA, Brazil

Aimed the morphological and stereological analysis of the different types of germ cells and the number of Sertoli cells per cyst in Astyanax altiparanae testes during spermatogenesis, being the first contributing to the better understanding this event in Characiformes. Testes of 25 male specimens were sampled for light microscopy. Diam-eters of the cell nuclei were measured. Then the samples were submitted to 24 serial cuts and the amount of Sertoli cells and germ cells were counted, from undifferentiat-ed spermatogonia to final spermatids. Based on the number of spermatogonia B per cyst it was estimated that spermatogonia undergoes at least 9 mitotic divisions before differentiating into primary spermatocytes. There are four spermatogonial types, un-differentiated spermatogonia A* (Aind*), undifferentiated spermatogonia (Aind.), dif-ferentiated spermatogonia (Adif.) and type B spermatogonia. The number of Sertoli cells increased gradually from a single cell associated with isolated undifferentiated spermatogonia (Aind.*) to approximately 10 cells involving the spermatocyte cysts in leptotene/zygote, which should be related to a greater complexity of cellular events occurred during the meiotic stage. The number of germ cells dramatically increase from spermatogonia A to spermatogonia B. However, the quantity of spermatocyte in lep-totene/zygotene stage decreased inside the cysts when compared to spermatogonia B, representing a loss of approximately 36% of the total number of cells. This fact is, probably, due to natural events of programmed cell death, in order to ensure the best development of these cells during the processes of sperm production. The carrying capacity of the Sertoli cells varied according to the type of germ cell and the spermato-genesis yield at the end of spermatogenesis was 43%. Thus, the stereological analysis provides a better understanding of the spermatogenesis of this species and for the other species of fish.

Keywords: Germ cells, spermatogenesis, stereology, testicular morphometry, fish

Acknowledgements: The study was supported by the FAPeSP – Foundation for Research Support of the State of São Paulo (Project no. 2014/23379-8 and 2013/24527-8).

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TRANSMEMBRANE SIGNAL TRANSDuCTION uNDERLyING HERRING SPERM-ACTIVATING PROTEINS (HSAPS) DEPENDENT ACTIVATION OF HERRING SPERM MOTILITy

Kaoru yoshida1, Manabu Yoshida2

1Toin University of Yokohama, Faculty of biomedical Engineering, Yokohama, Kanagawa 225-8503, Japan; [email protected] of Tokyo, Misaki Marine Biological Station, Misaki, Miura, Kanagawa 238-0225, Japan

Sperm of the Pacific herring, Clupea pallasi, are immotile in seawater or diluted seawater upon spawning in the environment. They become motile when two egg-de-rived molecules affect to their membrane. One is herring sperm-activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These factors coopera-te to assist fertilization: Diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequi-site for sperm to enter the micropylar canal and fertilize the egg (G.N. Cherr, et al., Int. J. Dev. Biol. 52: 743–752, 2008). The difference of these motility patterns are thought to correspond to differing levels of [Ca2+]

i increases above the level hound in immotile

sperm: SMIF is two times above the HSAPs-induced increase. Sperm motility initiation by SMIF depended on decreased extracellular sodium (<350 mM) and could be induced in the absence of SMIF in very low sodium seawater. Motility initiation depended on > 1 mM extracellular calcium. Calcium influx caused by SMIF involved both the opening of voltage-gated calcium (Cav) channels and reverse sodium-calcium (Na+/Ca2+) exchange (C.A. Vines, et al., PNAS 99: 2026–2031, 2002). While HSAPs interact with prolyl endo-peptidase (PEP) on sperm tail, suggesting it is a receptor molecule of sperm motility activation. (K. Yoshida, et al., Develop. Growth. Differ. 41: 217–225, 1999). A 19-amino acid peptide from the carboxy terminus of HSAP possesses the majority of motility activating activity and localize in the head of sperm. Differences in these localizations have not yet been solved, moreover, these concerning membrane-molecules, such as Cav channels, sodium-calcium (Na+/Ca2+) exchanger (NCX), and PEP were not revealed. Therefore, we report here on attempting to clone and to identify these molecules in testis of the Pacific herring, Clupea pallasi.

Keywords: Sperm motility, Cav channels, na+/Ca2+ exchanger, prolyl endopeptidase, egg-derived molecule

SeSSiOn ii. OOGENESIS AND EGG quALITy

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SPOTTED WOLFFISH (AnARHiCHAS MinOR) EGG quALITy PARAMETERS AND BIOCHEMICHAL COMPOSITION INFLuENCE IN EGG DEVELOPMENT

José Beirão1*, Einar Egeland1, João Santana1,2, Sylvie Bolla1

1Nord University, Faculty of Biosciences and Aquaculture, NO-8049 Bodø, Norway; [email protected] of Algarve, Faculty of Sciences and Technology, Faro, Portugal

Spotted wolffish (Anarhichas minor) has been considered one of the most promis-ing species for species diversification in cold-water marine aquaculture. Nevertheless, there are some challenges in this potential successful story. The long egg incubation period, ranging from 800 to 1000 day-degrees (dd), lasts for up to 5 months, is very labor-intensive and with unpredictable results. Therefore, spawns quality indices need to be defined that correlate in first place with fertilization rates but also with hatching and larvae survival rates. In this study, we analyzed different basic egg quality param-eters that can be easily measured in the hatcheries from the time of stripping prior to fertilization (oocytes), as well as the biochemical composition of the stripped eggs. This data will be compared with the eggs development, hatching and larvae survival. Unfertilized eggs were collected from different spawns for basic egg quality parame-ters analysis and for biochemical analysis. The following biochemical parameters were measured: fatty acids, phospholipids, amino acids and carotenoids. Each spawn was fertilized with a pool of sperm and transfer to an incubation system. The following measures were taken during the incubation period: fertilization rate measured at 22h (between 2 and 4 cell stage), development rate at 300dd and 600dd, hatching rate, yolk conversion ratio and early survival rate (after two weeks of hatching). The com-parison between the basic quality parameters and the eggs development rate will help build evaluation schemes that can be used to select rearing protocols, breeders and gametes for in vitro fertilization. Simultaneously, the results of the stripped eggs bio-chemical composition will help in the development of broodstock feeds for the spot-ted wolffish.

Keywords: Spotted wolfish, egg quality, fatty acids, amino acids

Acknowledgements: This study was supported by the WOLFeGG (project number 269726) funded by the RFF-nord from norway. AMinOR AS provided the access to spotted wolffish gametes and gave support with egg incubation.

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Poster presentation, Session II. OOGENESIS AND EGG QUALITY

REPRODuCTIVE BIOLOGy OF THE LuMPFISH (CYClOPTERuS luMPuS) AND ITS APPLICATION TO AquACuLTuRE

Mia Berwick1*, Craig Pooley1,Carlos Garcia de Leaniz1

1Swansea University, Centre for Sustainable Aquaculture, Singleton Park, SA2 8PP, Wales; [email protected]

Cleaner fish such as the lumpfish are being used increasingly as a biological control agent against parasitic copepod infection in salmon cages. These sea lice can cause significant damage and/or mortality to farmed salmon at great economic cost. With chemical methods becoming redundant due to the evolution of resistance, there is now more need than ever to find alternative solutions to reducing lice on salmon. This novel industry needs refinement for it to be considered as a sustainable alternative to chemical methods. The industry is currently dependent on wild caught lumpfish as brood stock are susceptible to various health problems and there are limited records of these fish being kept until maturation in aquaculture systems. The overarching ob-jective of this research is to further our understanding of lumpfish reproductive biology and seek to optimize its efficiency; eventually closing the breeding cycle in captivity.

Preliminary investigations have surrounded improving egg incubation and husbandry protocols to increase the efficiency of artificial reproduction in captivity. Degumming the egg mass via the use of a protease enzyme has been investigated in order to re-move the adhesive gum layer of eggs. Egg characteristics, hatch rate and survival to wean of larvae under commercial settings; in a recirculating aquaculture hatchery set-up, were monitored. The use of a novel disinfectant was also explored after fertilisation in the hope of reducing notifiable disease transmission such as VHSv. Furthermore, the possibility of an interaction between the degumming and disinfection treatments was tested.

Preliminary results have showed that the use of a protease enzyme was effective in degumming the eggs which is likely to reduce the challenges associated with husband-ry of marine, adhesive eggs including improved disinfection and incubation. Buffodine treatment at 150 ppm was not lethal to lumpfish eggs and resulted in similar surviv-al to controls. Initial trials highlight the necessity for further in-situ research on this species so as to tailor and improve current husbandry procedures to ensure optimal productivity and welfare conditions. Future research aims to look at the nutritional requirements of captive lumpfish in order to increase gamete production as well as the non-destructive stripping of males.

The present study represents a significant step forward in ensuring a secure and sus-tainable supply of lumpfish for the salmon farming industry.

Keywords: Cleaner fish, Adhesive eggs, Degumming, Disinfection, Artificial reproduc-tion

Acknowledgements: This project is supported by the Welsh government and the euro-pean Regional Development Fund.

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DETERMINATION OF THE OVARIAN MATuRITy IN SIBERIAN STuRGEON (ACIPENSER BAERII) AND STERLET (ACiPEnSER RuTHEnuS) WITH uSE OF uLTRASOuND AND ITS SuSCEPTIBILITy IN HORMONALLy STIMuLATED REPRODuCTION

Michał Blitek1, Mirosław Szczepkowski2, Katarzyna Dryl1*

1Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland; [email protected] Fishery Institute, Department of Sturgeon Fish Breeding, Olsztyn, Poland

Nowadays sturgeons have become one of the most valued species of commercial fish. They are cultivated mostly for their eggs which, when processed, become caviar. Effective assessment of ovarian maturity allows more effective usage of females in spawning process. The most commonly used method of maturity evaluation is trocar insertion due to its speed and simplicity of performance. However this method is inva-sive and potentially infectious which may lead to fish loss. Therefore, attempts are made to develop just as fast, noninvasive methods of gonadal maturity assessment. In the presented study ultrasound imaging (USG) was used to evaluate eggs size as an indica-tor of ovarian maturity stages of two sturgeon species: Acipenser baerii and Acipenser ruthenus in order to determine whether selected specimen are suitable for being sub-jected to effective prespawn hormonal stimulation. Researches were conducted in the Acipenserid Fish Hatchery in Pieczarki, the Inland Fishery Institute in Olsztyn, Poland on January to March 2016 and February to April 2017. 70 females of siberian sturgeon (Acipenser baerii; body weight: 7–16 kg, age: 9–12 years) and 35 females of sterlet (Acipenser ruthenus; body mass: 1–3 kg; age: 4 to 6 years) were examined. Investiga-tions were performed using Honda Electronics HS–2100 portable ultrasonograph ma-chine equipped with a 7,5 MHz linear probe in a paramedial position. Egg size, presence of ovarian fluid and fatness of gonads were analysed. Females recognized as mature and ready to spawn were those whose egg size exceeded 2.3 mm for Acipenser baerii and 1.5 mm for Acipenser ruthenus. It was possible to harvest eggs from 34 hormonally stimulated females (LHRH 0.1 mg/kg BW) out of 36 specimens of A. baerii and 11 out of 13 individuals of A. ruthenus assigned for spawning on the basis of the ultrasound examination. Determination of ovarian maturity using ultrasound method were of 94% and 84% accuracy for A. baerii and A. ruthenus groups respectively. According to ob-tained results, ultrasonography is an effective method for qualification to hormonal stimulation in order to efficient egg collection.

Keywords: ultrasound, sturgeon, gonadal maturity, egg size, caviar

Acknowledgements: The study was supported from Project ”PneUFiSH” (OR-61724-OR1400001/10) and funds appropriated to institute of Animal Reproduction and Food Research, Polish Academy of Sciences.

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EFFECT OF FOOD RESTRICTION ON REPRODuCTIVE PERFORMANCES AND EGG quALITy IN THE TROuT OnCORHYnCHuS MYKiSS

Emilie Cardona1*, Jerome Bugeon1, Violette Thermes1, Violaine Colson1, Sandrine Ski-ba-Cassy2, Julien Bobe1

1INRA, UR1037 Fish Physiology and Genomics, F-35000 Rennes, France; [email protected] .2INRA, UMR1419 Nutrition Metabolism and Aquaculture, F-64310 Saint-Pée-sur-Nivelle, France.

Rainbow trout (Oncorhynchus mykiss) egg production is a key sector for French Aquaculture. This industry relies on a well-controlled reproductive cycle and the exten-sive use of photoperiod control to obtain year-round production. Trout eggs produc-ers are however facing some variability in the female response to the onset of repro-duction by photoperiod (number of weeks needed to reach maturation) and female synchronization within the same tank. Moreover, food management practices are also very diverse between producers and not always designed to optimize egg production. Among these practices, food restriction was used. This practice has the advantage of limiting fish aggressive behavior and reducing egg production cost but it is not being applied in optimum condition.

The aim of this study was to target periods where feeding can be modulated (re-duced) without impacting egg production.

In this study, we compared the effects of three feeding strategies during the last six months before the reproduction on egg production. The following feeding schemes were used: females are fed (1) ad libitum, (2) 80% of ad libitum – restriction (3) 80% of ad libitum (3 months) followed by ad libitum (3 months), on reproductive perfor-mances and eggs quality of Oncorhynchus mykiss trout. Two artificial photoperiods were applied to obtain a reproduction period during spring: a three months long pho-toperiod (20 hours light, 4 hours dark from December to March, 2017) followed by a three months short photoperiod (8 hours light, 12h hours dark from March to June, 2017).

The results of our study show a better growth of broodstock fed ad libitum in com-parison to restricted fish. No difference was observed with the intermediate treatment. Liver lipid metabolism and egg quality biomarkers are currently being investigated us-ing molecular analysis. Egg size and number will be assessed with visilog imaging soft-ware, and developmental success will be monitored.

Key words: Trout, reproductive performances, egg quality, lipid metabolism, imaging software

Acknowledgements: This study is funded by FeAMP (nutriegg n° 4320164674).

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uSING MuLTIPLE REGRESSION TO PREDICT THE EFFECTS OF STIMuLATED HORMONAL REPRODuCTION IN 10 BREEDING LINES OF THE COMMON CARP, CYPRinuS CARPiO

Beata Irena Cejko1,ElżbietaBrzuska2

1Polish Academy of Sciences, Institute of Animal Reproduction and Food Research, Department of Gamete and Embryos Biology, Bydgoska 7, 10-243 Olsztyn, Poland; [email protected] 2Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture, Gołysz, 43-520 Chybie, Poland

The aim of the research was to investigate whether predictions for effectiveness of reproduction expressed in percentage of living embryos after 36-h spawn incubation can be sufficiently precise by knowing the origin, age and body weight of females, the agents used for stimulating ovulation, the weight of the eggs obtained, the fertiliza-tion percentage and percentage of living embryos after 24-h incubation. The material used for calculations included results from 15 reproductive seasons of the common carp from 10 breeding lines (three Polish: 2,3,6; three Hungarian: 0,W,7; Lithuanian B; French F; Israeli D; and Yugoslavian J). In 455 fish used for reproduction, CPH was used to stimulate ovulation in 265 females (0.3+2.7 mgkg-1), while 190 females were treated with Ovopel [(D-Ala6, Pro9NEt-mGnRH-a) + metoclopramide] (1/5+1 pellet/kg-

1). Multiple regression equations were derived for both agents within every line, using the percentage of living embryos (36h) as the dependent variable. Calculations were performed in three variants. In the first one, the independent variables in the equation were age, body weight of the females and the weight of the eggs obtained. The second variant included an additional variable, fertilization rate (12h), while the third one also included the percentage of living embryos (24h). From the total of 51 equations deri-ved, we selected those which were most precise and adequate for the data analysed. Three independent variables included in the analysis, i.e. age, body weight of females and the weight of eggs (g), generally did not result in satisfactory precision of predic-tion. The precision of estimation and accuracy of prediction were significantly increased by including four independent variables: age and body weight of females, egg weight (g) and the fertilisation rate (12h). In most variants analysed, the coefficient of deter-mination (R2) calculated for the group of fish treated with CPH was higher than for fish stimulated with Ovopel.

Keywords: Artificial reproduction, living embryos, multiple regression predictions, carp

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IMPACT OF MATERNAL EXPOSuRE TO HIGH TEMPERATuRE ON EGG quALITy AND SuBSEquENT OFFSPRING BEHAVIOR

Danielle Zanerato Damasceno1,2*, Morgane Cousture1, Claudiane Valotaire1, Aurélie Le Cam1, Thao Vi Nguyen1, Julien Bobe1, Violaine Colson1

1INRA LPGP, UR1037 Fish Physiology and Genomics, F-35000 Rennes, France; [email protected], Aquaculture Center of São Paulo State University, CEP-14884-900, Jaboticabal- SP, Brazil

In the current context of global climate warming, aquaculture fish are exposed to varying environmental factors including suboptimal temperatures at specific periods of their lifecycle. Fish are highly sensitive to extreme or abnormal (i.e. outside of the nor-mal physiological range) temperatures throughout their lifecycle. This is especially true for key periods such as critical steps of gamete formation. The reproductive period, during which the female gamete undergoes final oocyte maturation, is very sensitive to suboptimal temperature exposure, even for short periods of time. The direct impact on gamete – especially the female gamete – quality has been thoroughly investigated in many temperate species. Despite this well documented negative impact on egg quality and subsequent embryonic development, the long-term effects have been poorly inves-tigated and data on subsequent fish performance and adaptive capacities are scarce. The aim of the study was to investigate the intergenerational consequences of mother exposure to abnormal temperature on offspring behavioral plasticity in rainbow trout (Oncorhynchus mykiss). Sixty females at end of oogenesis were kept in normal (12 °C) and high (17 °C) water temperature. Fish were checked for ovulation every 2–3 days. Eggs (approximately 500 per female) were fertilized with a pool of sperms and incubat-ed at 10 °C for 138 days after fertilization. Developmental success was monitored at 3 stages: eyed stage (18 dpf), hatching (33 dpf) and yolk-sac resorption (70 dpf). In each phase one sample of embryos was frozen in -80 °C for molecular analysis. Between 75 and 138 dpf behavioral phenotyping was performed using tests designed to evaluate fear and learning performance. For fear we used an open-field test, a sudden event test and an emergence test. For learning we used a spatial learning task with a T-maze. Developmental success was significantly lower in embryos originating from females exposed to 17 °C, in comparison to embryos originating from females held at 12 °C. An overall developmental success of 86% was monitored in the control (females held at 12°C) group while it was only 47% in the eggs originating from females exposed to high temperature (17 °C). The behavior performance showed that progeny of 17 °C fish presented a phenotype towards more proactive profile but with a slower learning than the control group. This shows that the thermal stress during late oogenesis triggers an increase in embryonic mortality and differences in behavior in the progeny. Molecular analyses are currently in progress to understand the molecular mechanisms mediating the intergenerational impact of maternal exposure to high temperature on egg quality and offspring adaptive capacities.

Key words: Fish reproduction, rainbow trout, global climate warming, oocyte, fish lar-vae

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BIOCHEMICAL CHARACTERISTICS OF OVARIAN FLuID OF SALMONIDAE FISH AND STERLET

Joanna Nynca, Mariola A. Dietrich, Mariola Słowińska, Halina Karol, Ewa Liszewska, Bea-ta I. Cejko*, Beata Sarosiek, Sylwia Judycka, Katarzyna Dryl*, Radosław K. Kowalski

Polish Academy of Sciences, Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Olsztyn, PolandPolish Academy of Sciences, Institute of Animal Reproduction and Food Research, Poland; [email protected]

The biochemical composition and pH of ovarian fluid (OF) can reflect the quality of the eggs and has influence on sperm motility parameters. Biochemical characteristics of the OF surrounding the oocyte may play a critical role in determining oocyte quality and the subsequent potential to achieve fertilization and embryo development. The aim of this study was the comparative biochemical characteristics of OF of four Sal-monidae species: rainbow trout (Oncorhynchus mykiss, n=10), brook trout (Salvelinus fontinalis, n=11), sea trout (Salmo trutta, n=8), whitefish (Coregonus lavaretus, 12) and one sturgeon species: sterlet sturgeon (Acipenser ruthenus, n=12). The correla-tions between OF parameters were also evaluated. Ovarian fluid parameters such as protein concentration (ranged from 1.4 to 5.4 mg/ml) and LDH (ranged from 54 to 70 U/L) were similar for examined fish species. The value of alkaline (ALP) and acid phos-phatase (AcP) were also similar, except for brook trout and whitefish, for which the highest value of ALP and AcP were noticed, respectively. Ovarian fluid of brook trout, sea trout and whitefish was characterized by the same level of total antioxidant capa-city (TAC; 0.09-0.12 mM Trolox/L) in comparison to the lower values of rainbow trout and sterlet (0.05 and 0.009 mM Trolox/L, respectively). Sterlet OF was distinguished by the highest superoxide dismutase (SOD; 10 U/L) )and the lowest osmolality (226 mOsmol/kg) in compare with Salmonidae OF (2 U/L and 295 mOsmol/kg for SOD and osmolality, respectively). A few correlations between OF biochemical parameters were found to be common for part of fish species, e.g. a correlation between protein con-centration and LDH as well as between LDH and AcP. Some of correlations were only found in particular species. Biochemical analysis revealed that composition of the ova-rian fluid of the investigated species quantitatively differs. Species-specific differences existed in the occurrence of AcP, ALP, TAC and SOD. The latest was not detectable in OF of sea trout. Species-specific fluctuations in components of the ovarian fluid partially could result from variation in post-ovulatory maturation within the coelomic cavity, in the physiological status of the female and in egg quality. The results of the present study provide basic characteristics regarding the biochemical composition of the OF in four salmoniformes species and one sturgeon species.

Keywords: Salmonidae, Sterlet, Ovarian fluid, Biochemical composition

Acknowledgements: The study was supported from Project ”PneUFiSH” (OR-61724-OR1400001/10) and funds appropriated to institute of Animal Reproduction and Food

Research, Polish Academy of Sciences.

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REPRODuCTIVE PERFORMANCE IN RAINBOW TROuT, OnCORHYnCHuS MYKiSS: EXCLuSIVE STuDy OF PROBIOTIC EFFECT ON FEMALE BROODSTOCK

Erfan Akbari Nargesi, Bahram Falahatkar*, Mir Masoud Sajjadi

University of Guilan, Faculty of Natural Resources, Fisheries Department, Sowmeh Sara, Guilan, Iran *[email protected]

In recent years, the benefits of probiotics in aquaculture are well proven. However, studies in this base are limited in term of reproduction. This study aimed to investi-gate the effect of probiotics on reproductive performance of female rainbow trout, Oncorhynchus mykiss. Bio-Aqua probiotic complex was used for this purpose. To per-form the test, 60 females with an average initial weight of 2247.92±34.12 g were fed for an 8-week with diets containing 0 (control), 0.5, 1 and 2 g Bio-Aqua probiotic kg-1 diet. At the end of the experiment, matured females anesthetized by clove powder extract and then stripped manually. Semen from two males was used to fertilize each female’s eggs. Fertilized eggs were transferred to incubators and kept in sepa-rate trays until the complete absorption of the yolk sac. The fastest maturation time (degree-day from the beginning of feeding trial until broodstock maturation) was belonged to 2 g Bio-Aqua kg-1 diet and significant difference were observed between this treatment with 0.5 g kg-1 and control group. There were significant differences in absolute and relative fecundity between treatments and the highest absolute and relative fecundity was observed in 2 g kg-1 treatment. The maximum egg diameter and minimum number of eggs g-1 was observed in fish fed with 2 g Bio-Aqua kg-1 diet. Furthermore, results showed significant differences between 2 g kg-1 treatment with 0.5 g kg-1 and control group in fertilization rate, eyed eggs survival, alevins survival, time (degree-day) from fertilization to eyed stage, from eyed stage to hatching and from hatching to complete absorption of the yolk sac. The results of the present study showed that Bio-Aqua probiotic complex improved reproductive performance in female rainbow trout broodstocks and the best performance was observed in fish fed 2 g Bio-Aqua probiotic kg-1 diet.

Keywords: Feeding, Fecundity, Probiotic complex, Propagation, Salmonids

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DOMINANT-NEGATIVE REGuLATION OF OOCyTE HyDRATION IN MARINE TELEOSTS MAy BE MEDIATED By AquAPORIN SPLICE VARIANTS

Alba Ferré1, François Chauvigné1, Cinta Zapater1,2, Roderick Nigel Finn3, Joan Cerdà1*

1Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain; [email protected] of Aquaculture Torre de la Sal (CSIC), Castellón, Spain3University of Bergen, Bergen High Technology Centre, Department of Biology, Bergen, Norway

Previous studies have shown that aquaporin-1 water channel genes are arranged in binary clusters (aqp1aa-aqp1ab) in teleost chromosomes, and that the Aqp1ab channel mediates the temporal hydration of pre-ovulatory oocytes in marine pelagophil species. Based upon a deep phylogenomic analysis, here we report that while all teleosts retain the aqp1aa paralog, an additional aqp1ab-type gene comprises a ternary cluster (aqp1aa-aqp1ab2-aqp1ab1) in many, but not all species. For example, gilthead seabream (Sparus aurata) and Atlantic halibut (Hippoglossus hippoglossus) retain both aqp1ab2 and -1ab1, while Atlantic salmon (Salmo salar) and zebrafish (Danio rerio) only have the aqp1ab1 paralog. By contrast, Senegalese sole (Solea senegalensis), stinging catfish (Heteropneustes fossilis) and European eel (Anguilla anguilla) only have the aqp1ab2 paralog. Unlike to freshwater spawning species, such as zebrafish and Atlantic salmon, in marine pelagophil species such as seabream, halibut and sole the aqp1ab-type transcripts are always more accumulated in the ovary than in any other tissue. RT-PCR and cloning analyses further revealed the existence of different aqp1ab1 and -1ab2 splice variants, which encode C-terminal truncated isoforms and one variant in sole bearing an extended extracellular loop. Co-expression of the variants with wild-type Aqp1ab1 or -1ab2 in Xenopus oocytes showed that the Aqp1ab1 and -1ab2 variants can inhibit wild-type-mediated water transport in a dominant-negative fashion. In Senegalese sole ovarian follicles, two of the aqp1ab2 variant mRNAs are expressed in similar titres to the wild-type mRNA throughout oocyte growth and maturation. Subsequent immunohistochemistry analyses using isoform-specific antibodies revealed that while the wild-type is trafficked to the oocyte plasma membrane in postvitellogenic oocytes, the splice variant with an extended extracellular loop is retained in the cytoplasm. These findings suggest that the aquaporin-mediated hydration of marine teleost oocytes may be regulated through the alternative splicing of aqp1ab genes.

Keywords: Aquaporin, splice forms, marine fish, oogenesis, dominant-negative

Acknowledgements: The study was supported by the Spanish Ministry of economy and Competitivity (MineCO) (Grant no. AGL2016-76802-R to JC), and the Research Council of norway (Project 254872/e40 to RnF). Participation of FC and AF was funded, respectively, by a “Ramon y Cajal” contract and a predoctoral fellowship from Spanish MineCO.

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A MuLTIPARAMETRIC APPROACH TO ASSESS THE DEVELOPMENTAL SuCCESS IN TELEOST DuRING EMBRyOGENESIS

Maud Alix1, Daniel Zarski1,2, Yves Le Roux1, Dominique Chardard1, Bérénice Schaerlin-ger1*, Pascal Fontaine1

1UR AFPA, University of Lorraine – INRA, 54505 Vandœuvre-lès-Nancy, France; [email protected] of Warmia and Mazury, Department of Ichthyology, ul. Oczapowskiego 2, 10-719 Olsztyn, Poland

The developmental success could be defined as the ability of a zygote to survive, develop and grow into a normal individual. It is particularly linked to gamete quality, mainly oocyte. The various oocyte qualities leading to different developmental suc-cesses can be considered as a continuum, where it appears really difficult to define the frontiers of different developmental profiles. In the literature, despite of the large panel of parameters used to characterize the developmental success in several species, most of studies focus their attention on only one parameters appearing crucial to the authors (e.g. survival at hatching). However, this practice doesn’t take into account different nature of the impairments (e.g. early lethality, deformities), what can be cru-cial in understanding the mechanisms underlying the egg quality. As a consequence, most of studies compare only two groups of spawn defined arbitrarily with different qualities ignoring many aspects of the development and lack the opportunity to study the continuum previously defined. The present study aims at defining frontiers of sev-eral developmental profiles characterizing variable egg quality in the Eurasian perch, Perca fluviatilis. To do so, we determined the most biologically relevant parameters to characterize the spawn development and allow the proper cluster definition. In that context, parameters characterizing the survival and deformities are the two obvious counterparts to define the developmental success. In addition, the hatching period appears also crucial even if its role is poorly understood. In total, 12 parameters charac-terizing the embryonic survival rates, hatching phase and deformities occurrence were studied in 45 spawn of Eurasian perch. Using a principal component analyses followed by a hierarchical clustering analyses, three variables, better characterizing each of the counterparts appeared sufficient to define spawn clusters according to their develop-mental success. In total, 4 clusters of spawn could be defined from the good quality with high hatching rate (83.9%) and low deformities occurrence to worst ones with a high mortality (>95%) before the organogenesis period. The 2 intermediate clusters presented lower survival rate at 72h (56.5% and 48.2%) and hatching rates (47.8% and 26.8%) and with various deformities occurrences (14.8% and 2.5%). Further investiga-tions are still needed to understand the origins of these impairments. Nevertheless, this simple, quantitative and multiparametric analysis could be generalized to diverse species. Defined spawn clusters could allow us highlighting new mechanisms involved in the developmental success.

Keywords: Developmental success, eurasian perch, survival, hatching, deformities

Acknowledgements: We thank the French Ministry of Research for M. Alix fellowship and inRA for their financial support of the project. We also thank LUCAS PeRCHeS, the GAeC Piscicole du Saulnois and the GFA du Kuhweg for the fish.

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LIPID CLASSES AND FATTy ACID COMPOSITION PROFILE DuRING in ViTRO OOCyTE AGEING IN TENCH, TinCA TinCA

Azadeh Mohagheghi Samarin1*, Azin Mohagheghi Samarin1, Sabine Sampels2,3, Anna Krzyśków2, Tone-Kari Knutsdatter Østbyec4, Miroslav Blecha1 , Jiri Kristan1, David Gela1, Tomas Policar1

1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic; [email protected] 2University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, Husova tr. 458/102, 370 05 Ceske Budejovice, Czech Republic3Swedish University of Agricultural Sciences, Department of Molecular Sciences, PO Box 7015, 75007 Uppsala 4Nofima (Norwegian Institute of Food, Fisheries and Aquaculture Research), P.O. Box 210, NO-1431 Ås, Norway

Oxidative stress has been proposed to be the initiating factor for the progress of oocyte ageing. To identify the role of oxidative stress during oocyte ageing in Tench Tinca tinca, possible changes in the fatty acid and lipid class composition were evaluated. In addition, the level of malondialdehyde (MDA) as an indicator of oxidative stress were measured. Stored ova of 6 females at 20 °C were fertilized at 0, 2, 4, 6, 8 and 10 hours post-stripping (HPS). Embryo survival rates were assessed as an indicator of the egg quality. Fatty acids were analyzed with gas chromatography equipped with a flame ionization detector and PVT injector. Quantitative analysis of the separated lipid- and phospholipid classes was done by scanning the plates after derivatization with a CAMAG TLC Scanner 3. The results indicated that Tech eggs could be successfully stored in vitro for 4 hours after stripping at 20 °C. Thereafter, the embryo survival rates decreased significantly and dropped to 5% at 10 HPS. The level of malondialdehyde showed an upward trend during the progress of oocyte ageing indicating the possible role of oxidative stress in unfavorable outcomes of oocyte ageing. However, the lipid composition of the eggs were not affected by ova ageing as well as fatty acid composition. The RNA content of the eggs with non-fertilizing ability were drastically lower (84 ng/ul) than the fertilizable eggs (140 ng/ul) regardless of ageing. Lower quality eggs exhibited lower levels of cholesterol but higher levels of triacylglycerol in a minor degree.

Keywords: Tinca tinca, fatty acid composition, oxidative stress, lipid composition, oocyte ageing

Acknowledgements: This study was financially supported by the Ministry of education, Youth and Sports of the Czech Republic – projects „CenAKVA“(no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii“(no. LO1205 under the nPU i program), Grant Agency of the University of South Bohemia in Ceske Budejovice (no. 060/2016/Z), nF-CZ07-MOP-3-184-2015, nF-CZ07-iCP-3-185-2015, GAJU projects (no. 060/2016/Z and 085/2017/Z) and nAZV projects (no. QK1710310 and QJ1510117).

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A NOVEL METHOD FOR RAPID ELIMINATION OF STuRGEON EGG STICKINESS uSING SODIuM HyPOCHLORITE

Martin Pšenička*

University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic

Reducing egg stickiness is a crucial step in artificial culture of sturgeon eggs, with commonly used methods being time consuming or interfering with hatching. Sodium hypochlorite (SH) at varying concentrations and exposure times was tested on ster-let Acipenser ruthenus eggs, with 0.03% SH for 40 s effectively eliminating stickiness without impairment to eggs, embryo development, or hatching when compared with other de-adhesion methods (clay; NaCl, urea, and tannic acid). Field tests using sterlet, Siberian sturgeon Acipenser baerii, and Russian sturgeon Acipenser gueldenstaedtii showed similar numbers of larvae hatched using SH and clay. Immunohistochemistry revealed a protein carbonyl group only on the surface of eggs treated with SH, evidence of oxidation as the mode of action and indicating that oxidation did not affect inner egg layers or cytoplasm. Treatment with SH is a rapid, simple, and inexpensive method for de-adhesion of sturgeon eggs.

Key words: egg de-adhesion, sturgeon, artificial reproduction, sodium hypochlorite, protein carbonyl

Acknowledgements: The study was financially supported by the Ministry of ed-ucation, Youth and Sports of the Czech Republic – projects CenAKVA (no. CZ.1.05/2.1.00/01.0024), CenAKVA ii (no. LO1205 under the nPU i program), by the Czech Science Foundation (no. P502/13/26952S). Special thanks to Zuzana Linhar-tová for preparation of histological samples.

SeSSiOn iii. GERM CELLS:

FROM BASIC SCIENCES TO APPLIED BIOTECHNOLOGIES

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GERM CELL ISOLATION OF RAINBOW TROuT uSING DENSITy PERCOLL CENTRIFuGATION

Sude ATMACA1, Aygül EKiCi 2*

1IstanbulUniversity,InstituteofGraduateStudiesinScienceandEngineering.DepartmentofAqu-aculture2IstanbulUniversity Faculty of Fisheries,Department ofAquaculture,OrduStreetNo:200 34130Fatih/İstanbulTURKEY;[email protected]

Germ cells of both genders have functional gamete differentiation abilities and due to this unique combination, which enables gamete production types. The topical of germ cell technology, as well as the protection of genetic sources for sperm of large sized fish along with egg cells for smaller sized fish, have shown to assist development of the fish gonads and for the reason of breeding, these applications are important for the study. Despite the various methods of germ cell isolation used, the preference to use the percoll gradient application was undertaken in this study due to it being an inexpensive, easy and practical method. Rainbow trout which was produced in Sapanca Inland Water Product Production Research and Application Unit depend on Istanbul University Faculty of Fisheries was used in the study. Germ cell isolation; it was obta-ined from male rainbow trout which have 14–16 months old and 130–150 g weight. Testis tissue was incubated with % 0.4–0.5 Trypsin in PBS buffer for 1–2 hours for enzymatic seperation. At the end of this time, 5ml of sample passed through 50 µm filter with 40 μg/ml DNase and 1% BSA was provided to stop enzymatic activity. After filtration, Percoll gradient step was applied. In this step, Percoll gradient application of % 45–10 and % 30–10 was applied and upper, middle and bottom layers in Percoll were taken after centrifugation (4 °C,500g,30min.).Andthen,thereceivinglayerswasdilutedwithPBSandexaminedunderamicroscope.

Keywords: Rainbow trout, Oncorhynchus mykiss, germ cell, percoll gradient

Acknowledgements: This study was supported by Scientific Research Projects Coordi-nation Unit of istanbul University. Project number 59902. We would like to thank to Menekşe Didem eRCAn, ege GÜnGÖR and İlker KeSKİn.

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Poster presentation, Session III. GERM CELLS: FROM BASIC SCIENCES TO APPLIED BIOTECHNOLOGIES

METHODS OF SPERM MICRO-INJECTION IN uNFERTILIZED EGGS: COuLD BE uSED AS ASSISTED REPRODuCTION TECHNIquES IN STuRGEONS?

Effrosyni Fatira1*, Martin Pšenička1, Taiju Saito1

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Laboratory of Germ Cells, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected] 2Ehime University, South Ehime Fisheries Research Center, 25-1 Ainan, Japan

Sturgeons considered being one of the most critically endangered animal group due to over-harvesting and illegal fishing for their roe, and habitat degradation. In that sense, the current project tried to investigate methods of assisted reproduction by utilizing single or multi-sperm injection into unfertilized egg and monitor the develop-ment of the resulting embryos.

A single-sperm from Sibirian sturgeon injected in unfertilized and non-activated beluga eggs, by placing a drop of sperm (1.5 μl) in a petri dish with PBS. In addition, activated single-sperm injection performed by activating the sperm for 10 s and then de-activation by placing it in PBS. Also, multi-sperm injection performed by filling the microneedle and injecting each egg in the animal pole. After the transplantation, incubation for 30 min took place, while followed washing with tannic-acid for 30 s (to remove the egg-stickiness) and activation with water. The transplants successfully hatched with 15%, 19%, 3% success in non-activated single-sperm, activated single-sperm and multi-sperm group, respectively, while the control group exhibited 32% success. In addition, non-activated single-sperm and multi-sperm experiment performed using as sperm-donor the sterlet and as egg-re-cipient the Sibirian sturgeon. The transplants from non-activated single-sperm group could successfully hatch (4%), while the transplants from multi-sperm group couldn’t surpass the blastopore lip formation in gastrula stage (10%). The control group could reach the hatching stage (74%). Also, non-activated single-sperm and multi-sperm experiment per-formed using as sperm donor the sterlet and as egg-host the Russian sturgeon. Both groups reached hatching stage with 6%, 7% success in single-sperm and multi-sperm group, respectively, while control group exhibited at the same stage 75%. Besides the above transplantation experiments with fresh-stripped sperm, cryopreserved sperm from beluga (~40% motility) has been used by performing activated single-sperm and non-ac-tivated single-sperm transplantation into sterlet eggs. Only one transplant from non-acti-vated single-sperm group developed the blastopore lip in gastrula stage (3.6%), while the control group at the same stage exhibited almost the same percentage (3.8%). A group of non-injected eggs (n=76) that kept in PBS with sperm and treated as mentioned above, has been used as the negative control group and showed no development.

The current project proves to be the first evidence of several sperm-microinjection techniques among sturgeon species and the results proved to be promising. It pro-vides with a great hope for future assisted reproductive techniques that reconstruction of endangered species can be generated even by a single cryopreserved sperm after transplantation into an egg.

Keywords: Single-sperm microinjection, multi-sperm microinjection, activated single-sperm microinjection, beluga cryopreserved sperm, Sibirian sturgeon, Russian sturgeon, sterlet

Acknowledgements: The study was supported from Grant Agency of the University of South Bohemia in České Budějovice; GAJU Fatira 068/2017/Z 2017.

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COLD SHOCK ANDROGENESIS IN COMMON CARP

Roman Franěk1*, Katsutoshi Arai2, Vojtěch Kašpar1, Martin Pšenička1

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]. 2Hokkaido University,Faculty and Graduate School of Fisheries Sciences, 3-1-1 Minato, Hakodate, Hokkaido 041- 8611, Japan.

Androgenesis is a mode of uniparental inheritance with only paternal genome trans-mission to offspring. The basic principle of androgenesis is inactivation of maternal ge-nome in order to produce androgenetic haploids. Embryos are subsequently exposed to shocks blocking the first cell division and doubled haploids are produced. Conventional methods for maternal genome inactivation are using UV or Gamma radiation, thus there are certain requirements for equipment and standardization of the irradiation process. A low cost method was introduced using immediate exposition of embryos right after fertilization to cold shocks in order to inactivate maternal genome. This method was successfully applied in loach (Misgurnus anguillicaudatus), Japanese flounder (Para-lichthys olivaceus) and zebrafish (Danio rerio). Here we reported successful application of cold shock androgenesis in common carp (Cyprinus carpio). Haploid production was optimized firstly. Eggs obtained from Ropsha strain females (green, scaled) were mixed and fertilized with sperm from blond (recessive colour pattern) males. Eggs were trans-ferred within 10 sec after fertilization on baskets into styrofoam boxes with water and milk (9:1) (temperatures 0; 2; 4; 6; 8 °C) and remained there for different times (15; 30; 45; 60; 75 min). Embryos were then washed with a dechlorinated tap water and kept in an incubator until hatching. Proportion of pigment less and haploid like fry (bended and shortened body) was counted. Ploidy of haploid like fry was confirmed on a flow cytometer. Highest haploid rate was found in eggs incubated for 60 min in 2 °C. This conditions were used again with subsequent diploidy restoration. Cold shocked eggs were transferred to water bath with milk (24 °C) for 30 min. Then a heat shock (40 °C) starting 30 min after the cold shock and lasting 2 min was applied. Achieved yield of swim up and eating androgenotes was very low (˂1%), however it is very usual, without respect to method used for genome inactivation. Many androgenesis resulted in zero yield from our experience and commutations with others, thus any survivals are regarded as a success. Only paternal inheritance was confirmed by microsatellites covering different linkage groups. We intend to use produced androgenetic offspring for second round of uniparental inheritance to establish isogenic lines in common carp.

Keywords: Androgenesis, chromosome manipulation, cold shock, common carp, ho-mozygosity

Acknowledgements: This study was supported by the Ministry of education, Youth and Sports of the Czech Republic: projects CenAKVA (no. CZ.1.05/2.1.00/01.0024) and CenAKVA ii (no. LO1205 under the nPU i program), GAJU Franěk 034/2017/Z, GAJU Kašpar125/2016/Z.

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EXPLORING THE POSSIBILITy OF uSING TIGER TROuT (SAlMO TRuTTA × SAlVElinuS fOnTinAliS) AS A RECIPIENT FOR GERM CELL TRANSPLANTATION

Ákos Horváth1*, Jelean Lujić1, Zoran Marinović1, György Hoitsy2, Nataša Cvetković3, Jo-vana Lovren3, Béla Urbányi1

1Szent István University, Faculty of Agricultural and Environmental Sciences, Institute of Aqua-culture and Environmental Safety, Department of Aquaculture, 2100 Gödöllő, Páter Károly u. 1., Hungary; [email protected] 2Hoitsy & Rieger Kft., 3517 Miskolc-Lillafüred, Erzsébet sétány 55, Hungary3University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia

Gonad histology was performed on subadults of the tiger trout followed by trans-plantation of gametogonia into alevins. Tiger trout is an interspecific hybrid of the brown trout (Salmo trutta m. fario, ♀) and the brook trout (Salvelinus fontinalis, ♂). Tiger trout is generally considered sterile with severely impaired gametogenesis mak-ing it an ideal recipient candidate for germ cell transplantation. Our objective was to explore the possibilities of using this hybrid for transplantation of gametogonia from other salmonid species.

Gonads were collected from 1+ individuals of brown trout and tiger trout. According to our histology studies, the gonads of tiger trout showed evidence of severely dis-rupted gametogenesis as compared to those of the brown trout. Tiger trout ovaries contained very few vitellogenic oocytes and the majority of ovarian follicles were filled with a multitude of highly basophilic cells, while those of the brown trout were char-acterized by oocytes in early vitellogenesis. Testes of the tiger trout contained either mostly spermatogonia or more advanced stages of spermatogenesis without the char-acteristic features of spermatocytes. Very few spermatozoa were observed in the tes-tes as opposed to the advanced spermatogenesis in brown trout testes. Prior to these studies, females of tiger trout were observed to ovulate very few and highly irregular eggs at this farm in exceptional cases, however, no spermiating males were found.

Transplantation of isolated rainbow trout spermatogonia and oogonia was attempt-ed into tiger trout alevins approximately 5 days post-hatch. In total, 371 alevins were microinjected. A high mortality rate was observed both among the injected larvae as well as the control. At the age of 3 months, 15% of the transplanted larvae survived (55 fry) as opposed to an approximate 25% survival in the control. The transplanted fry is currently grown until sexual maturity.

Although tiger trout seems to represent a good candidate recipient for salmonid germ cell transplantation due to its impaired gametogenesis, high mortality rates should be taken into account when using this hybrid for transplantation purposes.

Keywords: Tiger trout, hybrid, histology, gametogenesis, transplantation

Acknowledgements: The study was supported by the project nKFiH Snn 116912 and the Stipendium Hungaricum Scholarship Programme (grant 106360 to ZM). We would like to thank to Márton Hoitsy for his support during the transplantation experiments.

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MuLTI-PHOTON AuTOFLuORESCENCE LIFETIME IMAGING MICROSCOPy (MP-FLIM) FOR NON-INVASIVE AND LIVE METABOLIC CHARACTERIZATION OF TyPE A SPERMATOGONIA IN MEDAKA TESTIS

Mario Günscht1, Cornelia Wetzker2, Klaus Reinhardt2, Alexander Froschauer1, Frank Pfennig1*

1Technische Universität Dresden, Institute of Zoology, Chair of Zoology and Developmental Biol-ogy, D-01217 Dresden, Germany; *[email protected] Universität Dresden, Institute of Zoology, Chair of Applied Zoology, D-01217 Dres-den, Germany

Throughout the life of a male, haploid gametes arise from undifferentiated type A spermatogonia from testis. Type A spermatogonia (SgA) are unipotent in a strict sense and their fate is to produce sperm. Beyond that, it has been proven that they can serve as a source for adult pluripotent stem cells as well. Increasingly, intra- and interspecific transplantation of SgA are employed in livestock breeding and have great importance for research and species conservation. In order to carry out such sophis-ticated techniques on living cells, SgA need to be reliably and swiftly identified in the donor tissue. For that purpose we have established stable transgenic medaka reporter lines. Early germ line cells are labeled in these lines by oct4-promotor driven EGFP fluo-rescence or vasa-promotor driven Histon2B-mCherry fusion protein. Using the oct4:EG-FP-line we determined the proportion of SgA in the testis to be 1–3%. This pool of SgA must be regarded as heterogeneous and includes undifferentiated SgA capable of self-renewal, as well as more advanced SgA committed to differentiation and cycling but with unknown potential for self-renewal and stemness. To shed light into that ex-pected heterogeneity of SgA, we applied a phasor MP-FLIM approach to medaka testis. Phasor MP-FLIM is known from non-invasive characterization of several kinds of stem cells. This approach is label-free and uses the difference in autofluorescence lifetimes of free and protein-bound NAD(P)H in a cell, a measurement of the metabolic state of the living cell. A high ratio characterizes the glycolytic state of a cell whereas a low ratio is found in cells with mainly oxidative phosphorylation. A correlation between a high free/bound NAD(P)H ratio and the stem cell potential of cells has been shown. In our measurements, among hundreds of type A and B spermatogonia and spermatocytes, 161 cells were classified as SgA. One of them showed a clear glycolytic metabolic signa-ture. All other cells were characterized by an oxidative state. We verified our measure-ments by targeted chemical induction of glycolytic or oxidative condition at medaka testis cells. In the future, we aim to explore the potential of this metabolic mapping as a label-free method for live identification of gonial stem cells of fish gonads.

Keywords: Spermatogonia type A, spermatogional stem cell, metabolic fingerprint, Phasor-FLiM, free/bound nAD(P)H, fish testis, transgenic medaka lines, oct4, vasa

Acknowledgements: The study was financially supported by the Zukunftskonzept of the Technische Universität Dresden awarded by the Deutsche Forschungsgemeinschaft through the excellence initiative.

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GENERATION OF GERMLINE CHIMERA IN STuRGEON

Martin Psenicka, Hilal Guralp, Kseniia Pocherniaieva, Zuzana Linhartova, Viktoriia Iego-rova, Taiju Saito

University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]

To make a germ line chimera, primordial germ cells (PGCs) or early-stage germ cells such as spermatogonia (Spg) or oogonia (Oog) has to be isolated from donor Siberian sturgeon (A. baerii) and transplanted into sterlet as a host. Generation of a germline chimera by transplanting PGCs requires a visualization technique. We used synthesized green fluorescent protein (GFP)-nos3 3’UTR mRNA or FITC to observe PGCs. We injected synthesized mRNA into the vegetal pole of fertilized embryos at the 1- to 4-cell stage to visualize and isolate PGCs during somitogenesis. Isolated single PGC transplanted into the host goldfish embryo and traced the donor-derived cells under the fluorescent stereomicroscope. Isolation of Spg/Oog was performed from juvenile (2–4 years old) sturgeon males and females by enzymatic dissociation with 0.3% trypsin in PBS. Next, dissociated cells were purified by Percoll gradient. Then, purified cells were labeled with the PKH26 red Fluorescent Cell linker Kit for general cell membrane kit according to the manufacturer‘s instructions. Labeled donor cells transplanted by a glass needle into the recipient sterlet larvae. The larvae were kept at 18 °C and fed by tubifex. Ten larvae from each group were examined by the fluorescence stereomicroscope at 6, 30, 50, and 90 days post transplantation (dpt). To produce only donor-derived gametes, we used some sterilization methods such as UV irradiation of germplasm or gene knoc-kdown agents such as the morpholino antisense oligonucleotide (MO). We irradiated embryos by 254 nm UV for 90 sec. until first cleavage stage. Or we used dead end (dnd) gene to knockdown by MO. The donor PGCs or Spg and Oog were identified, labeled, isolated, transplanted, and traced in host embryos and larvae, respectively. The gonial cells were found to proliferate in about 60% of recipients. We developed, for the first time, a series of techniques of enzyme dissociation, Percoll concentration gradient sorting and transplantation of sturgeon testicular and ovarian cells. Sterlet, one of the most common and smallest sturgeon species with shortest reproductive cycle (about 5 years), was used as the recipient and Siberian sturgeon, bigger and en-dangered species with later maturation (18–28 years), as the donor of cells. Because these two species can hybridize with each other, it is suggested that they are closely related and could be used for the standardization of transplantation experiments. To-day, we generated and keep about a thousand of sturgeon germline chimera for further analysis and offspring production.

Keywords: Germline chimera, sturgeon, transplantation, oogonia, spermatogonia

Acknowledgements: The study was financially supported by the Ministry of education, Youth and Sports of the Czech Republic - projects CenAKVA (no. CZ.1.05/2.1.00/01.0024), CenAKVA ii (no. LO1205 under the nPU i program); by the Grant Agency of the Uni-versity of South Bohemia in Ceske Budejovice (018/2014/Z) and by the Czech Science Foundation (no.P502/13/26952S).

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TARGETING dnd1 IN STERLETS (ACiPEnSER RuTHEnuS) By CRISPR/Cas9 GENERATES PHENOTyPIC ABNORMALITIES

Abdul Rasheed*, Roman Franěk, Martin Pšenička


Sturgeons are commonly known as living fossils and are famous for their caviar. These ar-chaic giants are facing several threats for their survival due to overfishing and interference in natural habitats, that in-turn make them “more critically endangered than any other group of fishes”. Sterlet (Acipenser ruthenus) is common sturgeon species with fastest reproductive cycle among sturgeons; thus this species can potentially be used as host for surrogate produc-tion for huge and IUCN red-listed sturgeon species. Ablation of germ cells or germ cell free host is pre-requisite for surrogate production, and to achieve this, knockout and/or knockdown of dead-end gene (dnd1) should be done in order to disrupt the migration and survival of primor-dial germ cells (PGCs). Previously we have successfully used antisense morpholino oligonucle-otide (MO) in sterlets to knockdown dnd1; however, due to cost-intensiveness, low efficiency and toxic to cell, we decided to use cutting-edge genome editing technology called CRISPR/Cas9 that presents several advantages over other genome editing technologies. CRISPR con-sists of two important components i.e., guide RNA (gRNA) and CRISPR-associated endonucle-ase (Cas9). Acipenser ruthenus dnd1 (Ardnd1) mRNA was used to select the target sites, and oligonucleotides were annealed and cloned into px330 plasmid (Addgene #42230) according to Cong et al., 2013. Purified plasmid was then diluted with KCL prior to injection into eggs at 1–2 cell stage. Due to fact that PGCs are generated in vegetal pole of sturgeon eggs, we inject-ed fluorescein isothiocyanate (FITC)-biotin-dextran into vegetal pole to label them as a marker for dnd1 knockout; whereas purified-diluted plasmid was injected into animal pole. To validate injection method and PGCs labelling, we injected only FITC in control group. Visualization of PGCs was done at 21 days post fertilization (dpf) under fluorescent stereomicroscope, and significant difference was recorded in number of PGCs in px330 plasmid injected embryos and in control group (P<0.05). Intriguingly, px330 plasmid injected embryos exhibited strange abnormal developmental patterns and ultimate mortality. In order to authenticate whether this was due to plasmid toxicity, we injected un-ligated plasmid into animal and vegetal pole of eggs, surprisingly, no abnormal developmental pattern was recorded in any injected embryo. To compare CRISPR/Cas9 methods, we also injected sgRNA and Cas9 protein (Eupheria Bio-tech) into sterlet eggs from two different females, and similar results were obtained. Mutation in dnd1 causes loss-of-function in mice and rats where it leads to testicular germ cell tumor. From obtained results in sturgeons, we presume that knockout of dnd1 in sterlets could have a strong/novel correlation with other important genes that significantly contribute in somatic development of embryos. However, further studies are suggested to determine the biological mechanisms behind correlation of Ardnd1 with other genes.

Keywords: Sturgeons, dnd1, Sterilization, CRiSPR/Cas9, PGCs

Acknowledgements: The study was financially supported by the Ministry of education, Youth and Sports of the Czech Republic-projects “CenAKVA” (no. CZ.1.05/2.1.00/01.0024) and “Ce-nAKVA ii” (no. LO1205 under the NPU I program), by the Grant Agency of the University of South Bohemia in České Budějovice (No. 125/2016/Z), by the Czech Science Foundation (No. P502/13/26952S), by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 and GAJU 079/2017/Z.

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STERILIZATION OF LOACHES (GENuS CObiTiS) By uSING ANTISENSE MORPHOLINO OLIGONuCLEOTIDE

Alena Zikmundová1,3,4*, Martin Pšenička2, Roman Franěk2, Lukáš Choleva3, Jan Röslein1,3, Karel Janko1,3

1University of Ostrava, Faculty of Science, 30. dubna 22, 701 03 Ostrava, Czech RepublicInstitute of Animal Physiology and Genetics, Academy of Sciences, Rumburská 89, 277 21, Libě-chov, Czech Republic2University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Wa-ters, Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic3Institute of Animal Physiology and Genetics, Academy of Sciences, Rumburská 89, 277 21, Libě-chov, Czech Republic4 Institute of Vertebrate Biology, Academy of Sciences, Květná 8, 603 65, Brno, Czech Republic

Sterilization of fish is an important technique in aquaculture and ontogenesis studies and may be achieved via various mechanisms, such as artificial polyploidization of hy-bridization.

Nevertheless, there are taxa where both polyploidy and hybrid transmission of ge-nomes occur naturally and do not affect the fertility of progeny. In such systems, po-lyploid clonal lineages exist that combine two or more divergent genomes and hence, other techniques should be applied. As a promising technique for embryo sterilization, one may use the antisence morpholino oligonucleotide (MO) targeting the dead end gene (dnd), which is specific gene encoding a RNA binding protein, which is decisive for migration and survival of primordial germ cells (PGCs). MO – treated embryos th-erefore develop normally but possess sterile gonads. To work properly, however, the MO must be fully complementary to the target sequence and hence and its application seems challenging in hybrids where two more or less divergent dead-end alleles occur in a single individual.

The genus Cobitis comprises hybrid polyploid fishes, which combine genomes of two parental species that diversified ~9Mya. In this study, we investigated the applicability of MO-anti-dnd technique to achieve sterilization of both diploid and polyploid as well as hybrid and non-hybrid strains of this complex.

We injected 100 μM Dnd-MO for deactivation of PGCs under the blastodisc of em-bryos at the 1–4 cell stage. PGCs were identified by injecting synthesized mRNA (5 nl 300 μg/ml mRNA), combining GFP and zebrafish nos1 3´UTR, under the blastodisc of embryos at the 1–4 cell stage. We also initiated the control group, where GFP-nos1 was applied but no MO was injected. We found out that PGCs were detected under a flu-orescent stereomicroscope only in the genital ridge of the control group, which proved that the depletion of PGCs by Dnd-MO was successful, because under fluorescent ste-reomicroscope no PGCs were detected in MO-treated fish. Moreover the body cavities of MO-treated and nontreated fish were investigated by histology. MO-treated indivi-duals had no germ cells in showing gonads. These results report that proper design and application of Dnd-MO enables complete sterilization even in hybrids that combine genomes as divergent as 9 Mya.

Keywords: Sterilization, polyploidization, morpholino, primordial germ cells, green fluroscent protein,dead end gene, loaches

Acknowledgements: The study was financially supported from project OPVVV CZ.02.1.01/0.0/0.0/15_003/0000460, GAČR 17-09807S, SGS 29/PřF/2016.

SeSSiOn iV. GAMETE “OMICS”

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EuROPEAN SEA BASS (DiCEnTRARCHuS lAbRAx) ANTI-MÜLLERIAN HORMONE: PRODuCTION IN A yEAST SySTEM AND FuNCTIONAL STuDIES

Cinta Zapater1*, Ana Rocha1, Gregorio Molés1, Soledad Ibáñez1, Silvia Zanuy1, Ana Gó-mez1

1Institute of Aquaculture Torre la Sal, CSIC, Castellon, Spain; [email protected]

In higher vertebrates, anti-Müllerian hormone (AMH) is required for involution of the Müllerian ducts during male sexual differentiation and for negatively regulating gonad-al development in both sexes. AMH signals through a transmembrane AMH type II re-ceptor (AMHR2) with serine-threonine kinase activity. Despite the absence of Müllerian ducts in teleosts, orthologues of mammalian AMH and AMHR2 have been described in some fish species, and a role of this hormone in sex determination and gonad dif-ferentiation has been demonstrated. In addition, it seems that Amh inhibits germ cell proliferation and differentiation, as well as steroidogenesis, in adult gonads of both sexes. However, the mechanism of Amh signaling and its implication in gonad devel-opment are poorly investigated. In European sea bass, a recent study showed higher expression levels of amh and amhr2 during early and final stages of spermatogenesis, and immunolocalization of Amh in Sertoli cells surrounding early germ-cell generations, in line with its role in germ cell proliferation and differentiation. Available information of amh and amhr2 expression during sea bass ovarian development shows an oppo-site profile for these two genes. Although this information may suggest a role of Amh in early stages of oogenesis, the specific mechanisms of Amh signaling during ovarian development still have to be investigated. As tool for this kind research recombinant sea bass Amh would be needed. We know from previous studies that this hormone is processed in a similar way to mammalian AMH, becoming a biologically active protein able to bind and activate the sea bass Amhr2. Here, we report the production of an His-tagged recombinant sea bass Amh in a Pichia pastoris expression system, where Amh is endogenously cleaved and secreted as a mature peptide into the culture me-dia. Bioactivity of this recombinant sea bass Amh has been demonstrated using COS7 cells co-transfected with the sea bass amhr2 cDNA and the BRE-luc reporter plasmid. Localization of Amh in granulosa cells of vitellogenic ovarian follicles was shown using a specific antibody. To investigate how Amh regulates sea bass ovarian development, we performed an in-vitro tissue culture of vitellogenic ovaries treated with recombi-nant Amh. First, we analyze steroid release in the medium by using a specific EIA. Final-ly, several candidate genes regulated by Amh were analyzed by real time qPCR.

Keywords: Amh, Amhr2, ovarian development, Amh signaling, paracrine factor

Funded by MineCO (AGL2015-67477-C2-1-R, AGL2011-28890), eU (LiFeCYCLe FP7-22719-1) and GV (PROMeTeOii-2014/051).

SeSSiOn V. GAMETE STORAGE

AND PRESERVATION

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EFFECT OF DIFFERENT STRAW VOLuMES, CRyOPROTECTANTS AND AVIAN EGG yOLK TyPES ON CRyOPRESERVATION SuCCESS OF NILE TILAPIA (OREOCHROMİS nilOTiCuS) SPERM

yusuf Bozkurt1*, İlker Yavaş2, Fikret Karaca2

1Iskenderun Technical University, Faculty of Marine Sciences and Technology, Department of Aqu-aculture, 31200, Iskenderun, Hatay, Turkey; [email protected] Kemal University, Faculty of Veterinary Medicine, Department of Reproduction and Arti-ficial Insemination, 31200, Antakya, Hatay, Turkey

Nile tilapia (Oreochromis niloticus) is one of the most cultured fish species because of its high resistance to poor water quality and diseases, fast embryonic development, maturity at early ages and year-round spawning. Egg yolk from different avian species has successfully been used as an additive for the cryopreservation of sperm in mamma-lian species. On the other hand, efficacy of egg yolk from different avian sources is still not clear. Hence, the objective of this study was to investigate the effect of egg yolks from different avian species, namely domestic chicken (Gallus gallus domesticus), tur-key (Meleagris gallopavo) and quail (Coturnix coturnix) on quality and fertilization abi-lity of frozen-thawed Nile tilapia sperm. Egg yolks from chicken, turkey and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken and quail egg yolks (p<0.05). Sperm was collected from 15 individuals and diluted at the ratio of 1:3 with an ionic extender containing 27.1 g/l Tris, 10 g/l fructose and 15% skimmed milk powder supplemented with different avian egg yolk types (domestic chicken, quail and turkey) and cryoprotectants (Me

2SO, MeOH and glycerol) at 10% separateley. Diluted

semen was equilibrated at 4 °C for 10 min and drawn into 0.25-ml or 0.5-ml plastic straws and sealed with polyvinyl alcohol. Samples were frozen 3 cm above of the liquid nitrogen surface and exposed to the liquid nitrogen vapor (≈−140 °C) for 10 min. After this, frozen sperm cells were kept into the liquid nitrogen container (−196 °C). The frozen sperm in different volume of straws were thawed in a water bath at 30 °C for 20 s (0.25-ml straws) or at 30 °C for 30 s (0.5-ml straws), respectively. Fertilization was conducted using 1 ×105 spermatozoa/egg ratio with each straw types. Supplementati-on of 10% glycerol showed better cryoprotective effect for sperm motility, duration of motility and viability (p<0.05) when compared to MeOH and Me

2SO. The highest ferti-

lization rates (52% and 60%) were determined with extender containing 10% glycerol and turkey egg yolk in both 0.25-ml and 0.50-ml straws respectively (p<0.05).

Keywords: Cryopreservation, straw volume, cryoprotectant, egg yolk, tilapia

Acknowledgements: This study was supported by Mustafa Kemal University Research Fund (MKU-SRF-384).

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Poster presentation, Session V. GAMETE STORAGE AND PRESERVATION

CRyOPRESERVATION AND TRANSPLANTATION OF COMMON CARP SPERMATOGONIA

Roman Franěk1*, Zoran Marinović2, Jelena Lujic2, Vojtěch Kašpar1, Ákos Horváth2, Martin Pšenička1

1University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected] István University, Department of Aquaculture, Páter Károly u. 1, H-2100 Gödöllö, Hungary

Common carp is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. Howev-er, no method was developed for preservation of gonadal tissue congaing germ cells – bipontential precursors of gametes. An optimized protocol for slow rate freezing of common carp spermatogonia was developed using various factors. Testicular tissue obtained from juveniles (100–200 g BW) of common carp was frozen under different conditions. Firstly, 6 cryoprotectans were tested, the best survival was achieved in DMSO. Then cooling rates (0.5– 10 ˚C/min) and different molar concentration of DMSO were tested resulting in the best survival using 2.5 M DMSO and freezing speed -1 ˚C/min. Following trials were performed with different tissue sizes and incubation times with the highest viability in 100 mg tissue incubated for 30 min. Last trial tested dif-ferent sugars and their contractions with the best viability using 0.3 M glucose. The highest cell viability after first trial was 10 %, but subsequent optimization improved the viability up to 40.7%. Cryopr served cells were recovered by transplantation into sterilized goldfish. The exogenous origin of the gonads in goldfish recipients was con-firmed by fluorescent labelling and molecular markers. Results of this study can serve as an alternative way for long term preservation of germ plasm in carp which can be recovered in surrogate recipient.

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SuBPOPuLATION STRuCTuRE OF THE DANuBE BARBEL bARbuS bAlCAniCuS SPERM BEFORE AND AFTER CRyOPRESERVATION

Zoran Marinović1,*, Nataša Radojković2, Tijana Veličković2, Jelena Lujić1, Ákos Horváth1, Vladica Simić2

1Szent István University, Department of Aquaculture, Páter Károly u. 1., H-2100 Gödöllő, Hungary; [email protected] of Kragujevac, Institute of Biology and Ecology, Radoja Domanovića 12, Kragujevac, Serbia

The aim of this study was to determine the subpopulation structure of barbel Barbus balkanicus sperm, optimize the sperm cryopreservation procedure and delineate the effects of cryopreservation on the subpopulation structure. Motile spermatozoa hier-archically clustered according to their motility parameters (determined by computer assisted sperm analysis – CASA) displayed a four-subpopulation (SP1 – SP4) structure. Subpopulations were distinguished and classified according to two main motility pa-rameters: curvilinear velocity (VCL) and linearity (LIN). SP1 was defined by low values of velocity but high overall linearity making these spermatozoa slow linear. SP2 was comprised of fast non-linear spermatozoa which had high velocity values, but low lin-earity. On the other hand, SP3 was characterized by fast, but linear spermatozoa which displayed both high velocity and linearity values. Lastly, SP4 was comprised of slow non-linear spermatozoa which had both low velocity and linearity values.

By testing different cryoprotectants (methanol – MeOH, dimethyl sulfoxide – Me2SO,

ethylene glycol – EG, 1,2-propylene glycol – PG and 2-metoxyethanol – ME) and concen-trations (5%, 10% and 15%), we have determined that the usage of 5% Me

2SO yield-

ed the highest total motility of ~ 25%. Cooling rates influenced by frame height and cooling time in lN

2 vapor had a significant effect on post-thaw motility. Frame height

of 3 cm with lN2 vapor cooling time of 2 min seemed to be the most favorable method.

Additionally, supplementation of cryomedia with 0.1 M of sugars led to an increase in the total post-thaw motility (~ 50%), while protein supplementation lowered the post-thaw motility in all cases.

In fresh sperm, SP2 had the highest proportion with 33% while SP3 characterized by fast linear movement had the lowest contribution with 15%. After cryopreservation the subpopulation structure markedly changed where all subpopulations except SP4 had a significantly different percentage contribution. Most obvious was the increase in percentage contribution of SP1 at the expense of SP2 and SP3 indicating that sperma-tozoa lost in their velocity after thawing. This indicates that the cryopreservation most likely had a negative effect on the mitochondrial metabolism leading to the overall decrease in sperm speed (VCL).

Keywords: Barbus balcanicus, sperm subpopulations, cryopreservation

Acknowledgements: This study was supported by the Ministry of education, Science and Technological Development of the Republic of Serbia (project no. 43002) and the Stipendium Hungaricum Scholarship Programme (grant 106360 to ZM).

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FATTy ACID INFLuENCE ON PROCHilODuS linEATuS (CHARACIFORMES, PROCHILODONTIDAE) EMBRyO CRyOPRESERVATION PARAMETERS

Alexandre Ninhaus-Silveira1, Raphael S. Costa1, Fabricio Marçal S. de Souza1, José A. Senhorini2, Cristiane Bashiyo-Silva1, Diógenes H. S. Silva3, Rosicleire Veríssimo-Silveira1, Cristiele. S. Ribeiro1

1São Paulo State University UNESP, Ilha Solteira School of Engineering, Department of Biology and Zootechnics, Av. Brasil, 56, 15385-000 Ilha Solteira, SP, Brazil. [email protected] National Center for Research and Conservation of Continental Fishes, Instituto Chico Mendes da Conservação de Biodiversidade – CEPTA/ICMBio, Pirassununga, SP, Brazil, 13630-970.3Federal University of South And Southeast of Pará UNIFESPA, Faculty de Health and Biological Sciences. Folha 31, quadra 07, 68507-590 Nova Marabá, PA, Brazil

The present study aimed to evaluate the vitellogenic transference and incorporation of long-chain polyunstaurated fatty acids (LC- PUFA) into the membranes of Prochilodus li-neatus embryos, aiming to increase the permeability to cryoprotectants and resistance to electric fields. One hundred thirty broodstock of P. lineatus were kept in two excavated aquaculture tanks of 500m2, fed with control (C) or fish oil-supplemented (FO) diets for 12 months, and induced to spawn by intracellular injections of gross pituitary extract. Total lipids of the embryos were then extracted. The lipid extracts were separated into polar lipids and neutral fractions, and the fatty acids (FAs) composition was determined by gas cromatography. For the neutral fraction, the FO caused a decrease in the monounsaturated fatty acids (MUFA) (C18:1 and C20:1) and an increase in the n3 polyunsaturated fatty acids (PUFA) and n6 PUFA (EPA, DHA, and AA), generating an LC- PUFA increase with a magnitude of 16%. The polar FAs demonstrated a similar pattern to that observed in the neutral lipids. The FO caused a decrease of MUFA values (C18:1), while the PUFA showed an increase, mainly in the n6 PUFA (C18:2n6 and C20:4n6). Finally, a significant increase in LC- PUFA was observed for embryos from the fish oil-fed broodstock. To test for cryoprotectant toxicity, the embryos were exposed for 20 min to a cryoprotectant solution of 1,2-propanediol at concentrations of 5 and 6 M. Regarding the FO, a reduction in the survival of 33.1% was observed in the 5-M solution, but there was no survival in the 6-M solution. Samples of embryos were exposed to six polarized electric fields (0, 3.4, 11.2, 16.9, 25.8, 40.2, and 51.6 J) generated by an Ibramed Sonopulse II apparatus. At 11.2 J of energy, the survival of the C group was reduced by 98.3%, while the FO presented superior resistance, maintai-ning a similar survival from 0 J to 40.2 J. These results suggest that there was transfer of FAs between the broodstock and embryo of P. lineatus, with an increase of LC- PUFA, resulting in lower survival rates in the cryoprotectant test for FO and in greater physical plasticity of FO embryos to the electric field tests. This alteration of the embryonic lipid profile helps in cryopreservation studies, allowing to reduce both the time of exposure to the cryopro-tectants and the concentration of the solutions used in addition to develop organisms resistant to new techniques, such as polarized electric fields.

Keywords: Biotechnology, cryoprotectants, fatty acids composition, membrane fluidity, curimbatá

Acknowledgements: This project was funded by Fapesp (Project. nº 2013/02588-5 and 2015/10115-5) and the facilities to reproduction experiments and fish were provided by CePTA/iCMBio.

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THE uSE OF DIFFERENT SEMEN POOLS DOES NOT GuARANTEE THE SAME SPERM quALITy AFTER CRyOPRESERVATION IN STEinDACHnERiDiOn PARAHYbAE (SILuRIFORMES: PIMELODIDAE)

Eduardo Antônio Sanches1*, Danilo Caneppele2, Tais da Silva Lopes3, Elizabeth Roma-gosa3

1São Paulo State University –UNESP, Aquaculture Centre - CAUNESP and Experimental Campus of Registro – CERe, R. Nelson Brihi Badur, 430, 11900-000, Registro/SP/Brazil; [email protected] 2Energy Company of São Paulo, Hydrobiology and Aquaculture Station, Paraibuna/SP/Brazil3Fishery Institute, APTA, SAA, São Paulo/SP/Brazil

The protocols of sperm cryopreservation need to be standardized for different spe-cies of fish mainly for species that are endangered, such as the surubim-do-Paraíba, Steindachneridion parahybae (Steindachner, 1877). However, to standardize the pro-tocols is very important established how to use the semen of males with different sperm quality, for this, the mixing of semen to form the pool of semen is a procedure employed and has the aim of minimize these differences. Thus, the aim was to realize spermatic cryopreservation of S. parahybae from the use of three different pools of semen in five cryoprotectant solutions (CS). Three pools of semen composed of four males were used in the pool “a” (272 mOSM kg-1) and “b” (283 mOSM kg-1) and three on “c” (278 mOSM kg-1) diluted in the solutions: GLU5 (5% glucose + 5% milk powder + 7.5% methanol); GLU8.5 (8.5% glucose + 0.25% milk powder + 10.0% methanol); SUC5 (5% sucrose + 5% milk powder + 7.5% methanol; FRU5 (5% fructose + 5% milk powder + 7.5% methanol; NACL (NaCl at 200mM). After dilution (1:3 semen:CS), the semen was freezing in nitrogen vapor (Dry-shipper, 22h) and transferred in nitrogen liquid for 27 days until the thawing (15s at 25 °C). The sperm parameters (MOT, VCL, VAP, VSL, STR) were evaluated by computerized sperm analysis (CASA/ImageJ) in two moments, dilution (before cryopreservation) and after thawing. It was observed differ-ences (P<0.05) in all parameters in the solutions and pool tested on the two moments evaluated. The values of MOT, VCL, VAP and VSL reduced and the STR increased in com-parison at fresh sperm (98.4±2.1% of MOT, 132.6±4.8 µm s-1 of VCL, 103.4±7.3 µm s-1 of VAP, 92.2±7.9 µm s-1 of VSL and 89.2±1.8% of STR). The better values of MOT after cryo-preservation were observed to GLU8.5 (72.3±3.9%) and to the velocities in the pool “a” in GLU8.5 (80.6±9.1µm s-1, 60.7±6.5µm s-1, 57.9±6.8 µm s-1 and 95.3±1.2% to VCL, VAP, VSL and STR, respectively). Thus, we verify different semen pools provide different values of sperm parameters and influence the results of cryopreservation. The solution containing 8.5% glucose + 0.25% milk powder and 10% methanol can be applied as cryoprotectant solution in sperm cells of Steindachneridion parahybae.

Keywords: Cryopreservation, neotropical catfish, semen mixing, spermatozoa, sperm conservation, surubim

Acknowledgements: The study was supported from São Paulo Research Foundation (FAPeSP) project number 2011/02818-5 and 2012/17083-3.

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EFFECT OF DIFFERENT CRyOPROTECTANTS ON MOTILITy AND FERTILITy OF MAHSEER (TOR KHuDREE AND TOR MuSSullAH) SPERMATOZOA

Sahana Shivaramu1,2*, Rupam Sharma1, Shashank Ogale1, Gopal Krishna1

1Central Institute of Fisheries Education, Mumbai, India2University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic; [email protected]

Present study was conducted to evaluate the viability of mahseer (Tor khudree and Tor mussullah) spermatozoa in presence of different cryoprotectants. Three cryopro-tectants were tested for their suitability viz., Dimethyl sulpoxide (DMSO), Glycerol and Dimethyl acetamide (DMA) at concentration of 10% v/v. This was achieved by diluting fresh milt with different cryoprotectants diluents with extenders at 1:10 ratio and motility percentage was observed until 24 hours at specific time intervals in re-frigerated conditions. Cryoprotectants were used at concentrations of 8%, 12%, and 15% with extenders and diluents were prepared, accordingly. Milt samples were diluted with diluent in ratio of 1:10 and equilibrated for 30 minutes and frozen at 5 cm above LN2 level for 10 minutes. Higher post thaw motility after 90 days was observed to be 68.16±1.48% for T. khudree and 70.07±4.71% for T. mussullah. Modified fish ringer solution with DMSO at 8% concentration was found best in terms of post thaw motility in both the species. Higher mean fertilization percentage of 78.11±1.59 was observed for 8% DMSO + modified fish ringer solution in T. khudree. Higher mean fertilization percentage of 77.55±1.59 was observed for 8% DMSO + modified fish ringer solution in T. mussullah. Out of this, best motility percentage was found in DMSO followed by glycerol for both species; however, DMA was found unsuitable for both species. DMSO and glycerol cryoprotectants were combined with modified fish ringer solution and Kurokura solution in concentrations of 8%, 12% and 15%, and diluents were prepared. Scanning Electron Microscopy (SEM) was used to determine morphometric character-istics of spermatozoa of both species and prominent spherical head with circular to elliptical nucleus and tail were observed in spermatozoa in both species.

Keywords: Cryopreservation, extenders, Cryoprotectants, Mahseer, Spermatozoa

SeSSiOn Vi. ANTHROPOGENIC

CONTAMINANTS AND FISH GAMETES

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THE POTENTIAL EFFECTS OF THE ORGANOCHLORINE PESTICIDES ALDRIN AND METHOXyCHLOR ON THE REPRODuCTIVE HEALTH OF THE MALE AFRICAN SHARPTOOTH CATFISH ClARiAS gARiEPinuS

Jamie Das Neves1, Helene Coetzee1, Irene Barnhoorn2, Ina Wagenaar1*

1University of Johannesburg, Department of Zoology, PO Box 524, Auckland Park, Johannesburg, South Africa, 2006; [email protected] of Venda, Department of Zoology, Private Bag X5050, Thohoyandou, Limpopo Prov-ince, South Africa, 0950

A field survey conducted in the Albasini Dam, Limpopo Province, South Africa showed a decline in fish sizes and numbers as well as the presence of the organochlorine pes-ticides aldrin (0.14 µg/L) and methoxychlor (0.23 µg/L) in the water. The presence of these pesticides were unexpected as aldrin is banned under the UNEP Stockholm Convention of 2004 and there is not sufficient information available on the registration of methoxychlor in South Africa and its effects on fish. The aim of this study was to de-termine the effects of environmentally relevant concentrations of aldrin and methoxy-chlor on the reproductive health of male catfish Clarias gariepinus under controlled laboratory conditions.

Male C. gariepinus were exposed to aldrin and methoxychlor individually for 96 hours (27 °C; pH 8). A standard protocol was followed for the fish health assessment. Fol-lowing the exposure, each fish was weighed and measured. The fish were sacrificed, dissected and the testes removed, weighed and measured to determine the gonadoso-matic index (GSI) and the maturity stage. The right testis of each fish was fixed in 10% Bouin’s solution, processed and stained using standard histological techniques. The microscope slides were assessed and the changes quantified to determine the testes index (I

T). Milt was collected from the left testis of each fish and sperm quality param-

eters analysed by Computer Aided Sperm Analysis (CASA). These parameters included: progression as percentage motility (%); velocity percentage (%); curvilinear velocity (VCL µm/s); straight line velocity (VSL µm/s); average path velocity (VAP µm/s); linear-ity (Lin %) and wobble (WOB %).

The mean GSI value for aldrin and methoxychlor exposed fish was lower than that of the control and solvent control fish. However after statistical analysis, the only sig-nificant difference in the GSI values was between the aldrin exposed fish (n=12) and solvent control fish (n=6). Histological alterations were noted in the testes tissue of both pesticide exposed fish as well as the control fish, but according to the I

T scoring

scheme, this level of alterations can be considered as normal. No statistical significant differences (p>0.05) were found in the CASA parameters between the exposure and control groups. The results of this study showed that there was no clear hindrance to the reproductive success of these fish and the environmentally relevant concentrations of aldrin and methoxychlor did not have a negative effect on the reproductive system of male C. gariepinus during acute exposure under controlled laboratory conditions.

Keywords: Reproduction, pesticides, histopathology, staging, CASA

Acknowledgements (optional): This study was financially supported by the national Research Foundation of South Africa (nRF Grant no. 86056) and the University of Ven-da for assistance during the field sampling.

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COMPARISON OF THE SPERMATOZOA quALITy PARAMETERS AND FATTy ACIDS COMPOSITIONS IN CuLTuRED AND WILD BuRBOT SPERM

Miroslav Blecha*, Petr Svačina, Borys Dzyuba, Sergii Boryshpolets, Yevhen Horokhovatskyi, Hadiseh Dadras Asaybar, Oleksander Malinovskyi, Tomáš Policar


Burbot (Lota lota) is widely distributed throughout the Holarctic zone and lives in cold rivers and lakes. For the last few decades, it is considered to be one of the most endangered freshwater fish species in many parts of Europe for several reasons (envi-ronmental changes, inhibition of the spawning migrations, overfishing, water pollution and climate warming). Controlled culture and reproduction of burbot is highly import-ant as it is the endangered fish species and on the other hand, a promising candidate for culture in the intensive aquaculture. The aim of this study was to compare the spermatozoa quality parameters and fatty acids composition in sperm of intensively cultured burbot (RAS group; W= 133±49g; 9 fish) fed with commercial diet and wild burbot (WILD group; W=170±55g; 9 fish) which consumed the natural food only. The most important findings of this study was that only 7 males of RAS group were pro-ducing sperm and only 4 of them have motility higher than 5%. In WILD group, sperm samples were collected from all 9 males. Additionally, 6 of them demonstrated motility close to 100% (at the beginning of the motility period) and 3 others around 50-60%. In addition to lower motility in RAS group, different sperm behaviour was noted in this group. Not all the spermatozoa were activated during the first 10 seconds of sperma-tozoa activation and percentage of motile sperm have been increasing with the time (indicating delay in motility activation and prolonging motility in RAS group). Sperma-tozoa from WILD group activated motility at higher speed and demonstrated maximum velocity at 10 s. VCL and WOB performed a plateau in the end of motility period in WILD group. Fish from RAS group also produced significantly lower volume of sperm (1.8±1.2 ml) compared to WILD group (3.6±1.2 ml). There were found no effects of the feed on the final content of the major lipids in sperm of both examined groups (RAS group (PL=58.89±1.78%; Cholesterol=41.10±1.88%); WILD group (PL=58.27±2.38%; Cholesterol=41.73±2.31%). We can state that keeping of the burbot broodfish in RAS and feeding with a commercial diet may affect process of spermatogenesis and lead to decreased number of spermiating males, lower volume of produced sperm and lower spermatozoa concentration. Differences in diet composition may also affect the lip-id composition in spermatozoa membrane. It makes them less sensitive to osmotic pressure, causes a delay in their activation, decries the amount of activated (motile) spermatozoa and negatively affect their velocity.

Keywords: Lipid composition, Lota lota, motility, osmolality, velocity

Acknowledgements: The study was supported from following projects: CenAKVA“(no. CZ.1.05/2.1.00/01.0024), “CenAKVA ii“(no. LO1205 under the nPU i program), GAJU project (no. 060/2016/Z) and nAZV projects (no. QK1710310 and QJ1510117).

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A COMPARISON OF SuBJECTIVE AND OBJECTIVE ASSESSMENT OF FISH SPERM MOTILITy

Víctor Gallego, J. Germán Herranz-Jusdado, Christoffer Rozenfeld, Simone Pulsoni, Luz Pérez, Juan F. Asturiano*

Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Camino de Vera s⁄n, 46022 Valencia, Spain; [email protected]

Fish sperm motility is currently considered the best sperm quality biomarker in fish, and nowadays can be evaluated both by subjective and objective methods. Although subjective methods have been widely used over the history, several factors makes dif-ficult to compare the results both intra- and inter-labs. On the other hand, objective methods (carried out by CASA systems) have improved the sperm motility assessment thanks to an increase of accuracy and to the estimation of new sperm motion param-eters. This study aims to compare the accuracy and precision for subjective and objec-tive assessments carried out simultaneously by different technicians.

Twenty European eel males were weekly injected with 1.5 IU/g fish of recombinant hCG (Ovitrelle, Merck S.L., Madrid) during 12 weeks, and sperm samples were collected 24 h after every weekly hormone administration. Sperm motility of all samples was evaluated by triplicate by both subjective (directly by the microscope) and objective (by a CASA system) methods, and simultaneously by three different technicians with different degree of expertise on the sperm quality analysis. To perform an in-depth analysis, sperm samples were classified into three quality classes based on the per-centage of motile spermatozoa: C-I: 0-25%; C-II: 25-50%; and C-III: >50%; n≥12 for each class. Statistical dispersion parameters were estimated for assessing the accuracy and precision of the techniques and the influence of laboratory staff.

Accuracy. The ability of technicians to carry out an accurate subjective evaluation was measured as the difference between the CASA motility value and the subjective esti-mation. In this sense, experimented technician obtained suitable ranges, presenting subjective motility values only 10 points over or under the real value in all the sperm motility classes (C-I, C-II and C-III). However, although medium and low-experimented technicians had acceptable ranges in C-I class, overestimation of values was the com-mon thread in samples belonging to C-II and C-II classes, with overstatements of 25% from the real value.

Precision. In addition to accuracy, experimented technician was also more precise, showing the lowest dispersion parameters (standard deviation, SD; and coefficient of variation, CV) regardless of the technique used. In this sense, non-experimented tech-nicians showed generally highest SD and CV values, making less precise the motility estimations both by subjective or objective methods. Regarding techniques, subjective method showed similar SDs and CVs than CASA assessments in C-II and C-III classes, while in C-I class the objective method showed higher CVs than subjective estimation.

To sum up, the degree of expertise of a technician on the sperm quality analysis seems to be a key factor in order to reach accurate motility estimations, and non-ex-perimented personal staff can under- or overestimate real values, masking positive results in aquaculture research.

Keywords: sperm, CASA, motility, objective, subjective

Acknowledgements: Funded by the european Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement nº 642893 (iMPReSS) and the COST Office (COST Action FA1205: AQUAGAMeTe). VG has a grant from the UPV (PAiD-10-16).

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University of South Bohemiain České Budějovice

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