ilE ur

A STIIDY OFREPROD IICTlV EPHYSIOLOGYIN TilE MALEOC EANPO

ur

MACROZOAR CES AMERICANUS

by

Zhilnu WallB

A thes issubmitted totheSchoo lof GraduurcStudies

inpartialfulfilmentoftherequirements forthedegre e of Masterof Science

DcparnncmofBiology Memorial UniversityofNewfoundland

19%

51.John' s Newfoundland

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Nal:onalLlbfary

01Canada SiblkllhCqvenatcoae

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ABSTRACT

To providemoreinformation on thereproductivebiology of an intern ally fert ilizing marinespecies,atwo-year study of thereproductive physiologyof the male ocean pout(Mar.rowarcesa1llericalllls)wasundertaken.The physiologicalcharacteristics ofthe spawning season andsperm physiology, including the changes inthe gonadosomatic index (GSI),plasmaandrogenicsteroids,the timingof spermiation,spermmotilityand spe rmconcentration.and the variationsof pH,osmoticpressure and thebiochemical compositionof seminalplasma during thespaw ningseason were investigated. The spermiationresponsebegan in June,and by July allmaturemales sperrniated.

Spermintlonended inlateSeptember orearlyOctober.Spermmotilityrose andfell in asimilar manner fromJuneto September. Compa redwiththemilt of mostinvestigated male tcicosrs. oceanpoutmillhadalow spermconcentration (0.91-6.81x 10"spz/ml), andwas correlated withspermatocrltvaluesduringmostof the spawningseason(r¹=0.79, p<O.OOOI),The GS(ofthe maleswasrelativelylow,ranging between minimum values inSeptemberandOctober(0.26•0.31%)and a maximum in July (1.9%). Plasma testosteronelevelsroserapidlyseven weeks inadvanceof spermiationand peaked one weekbeforethe onsetof spermiation,anddecreasedthereafter, Plasmalevels ofII·

ketctcstosterone paralleledthose oftestosteroneduring the spawningseasonand reached u peakcoincidentwithonsetof spermiation.

Biochemical compositionof seminal plasmaalso varied through thespawning

seas o n. ThepHofseminalplasmaincreasedfrom7.4107,t)duringtIll'periodIll' spermiation.and the averagepH(7.71HO.OJ)forthe spawning seasonremain...xlcloseII}

anexperiment allydeter min edoptimum pHrangefor0Cl:3npoutsper mnlOtilily(1111~_ 9), Allhoughthevaluesforseminalplasmaosmolalityfellfrom41(110 HI}mmtlll\;g.

duringlhereproductive season.theaverage osmolalityvalue(3~b.tJmm(lllkg)waswithin theoptimum foroceanpoutspermmotility(300•4001ll1l1(1Ilkg) Incllmparin!:\

fluctuationsinsperm motilitywiththebiochemical CUIl1 11ositill1l(IftlCC,1l1ptllll Sl,.'lllillill plasma duringthe spawningseason,thisstudyshowedthat increasedMg"levels were correlated withthesummerperiod ofmaximumspermI11tllilily.A scnsona! declinein Na'and C1" ionlevels WOlSreflectedinlower seminalplas maoSlIIo!lllilyvalues

ACKNOWU;n GMENT

Thisthesis work was carriedout underthe supervisionof Dr,Laurence (rim,I wishto express my special appreciationto Dr.Crimfor providingme with academic guidance,valuablecomments, and many usefuldiscussions.Iwouldalsolike to thank him forhisencouragementandconfidence in me throughoutmy study.Itis all this that leadtothe completionof thisthesis.My appreciationalsogoes tothe othermembers of my supervisorycommittee,Drs.Joe Brownand GoverdinaFahraeusvan-Ree,fortheir valuablesuggestions andadvice regardingmyresearch andthesis.

The research for thisthesis wasconducted atthe Ocean Sciences Centre(OSC).

Valuable help has been received fromstaffas well<ISfellow graduate studentsatOSC.

In particular,Iwould like to thank Ms.Conniewllsouforhertechnicalsupport,and equallyimportant,herfriendship.Thediscussions withmy fellow graduate student Dr.

ZuxuYao on the reproductionof ocean pout were also beneficial. The OSCDiving Unit is acknowledged for collecting live specimens formy experiments.

Financialsupportfor this study was providedby a NSERC Research Grantto Dr.

LaurenceCrim (A (729)and a scholarshipfrom the Department ofBiology.

Iwould like tothankmy parents,especially my father,fortheircontinuous

encouragement10 me overthe yeaninpursuing 111)' study. ~Iyhusband lIungCh~'ll deserves my specialthanks for many usefulsuggestions anddiSC\l~siunsonsl,llisliloal analysisand graphicproductions,bothathomeand intheoffice Finillly,myIhal1k ~abu goes to my daughter Lulu for encouragingme 10finish my thesis~lthat shecanhave more compulerlime for her favouritegame,FreddieFish

TABLE OF CONTENTS

ABSTRAcr.

ACKNOWLEDGEMENT

LISTOF FIGURESAND TAUL.E

....ii

.... ... . .. ...iv

CII APTlm I.GENEI?'A L.INT RODUCTION.

1.1 THEREPRODUCTIVEPHYSIOLOGYOF MALE TELEOSTS 2

I.[.1Environmentalregulationoftheseasonaltesticular cycle 3 1.1.2Endocrineregulationof spermatogenesisandspermiation 5

I.UTesliculurslrucfureandfunctiol1 10

1.2 A COMPARISONOF SPERM PHYSIOLOGY BETWEEN EXTERNALLY

ANDINTERNALLY FERTILIZINGTELEOSTS 13

1.2.ISperm morphology 14

1.2.2 Spermactivatlonand motility 17

1.2.3Sperm metabolism 20

1.3 A REVIEW OFOCEAN POUT REPRODUCTION. . 22

1.3.1Distributionandsystematics. 1.3.2Biology ofreproduction

. 22

2J

1.J.3 Aquacuhurecapability. 1.4 OBJECTIVESOF THE STUDY.

15 APPROACHESOFTHISSTUDY.

. ....26 . ....27

CHAPTE R1.PIIYSIOL.O GICA L.CIM R,.\CT ERI ST ICSOFSI' AW NINGINTi l t:

MAL.EOCEA NPOUTMarmUlfJrt·(!.,11II1l!riC'III11U• 2,I INTRODUCTION

2.2MATERIALS AND METHODS. 2.2.1Exporimer nale nimals.

2.2.2The gooadosomaricindex(GSI).. 2.2.3Spermiation.

2.2.'1Spermmotility. 2.2.5Spermatocrit. 2.2.6Spermconcentration. 2.2.7Androgenic steroidsin bloodplasma. 2.2.8Statistic:alanalysis

2.3RESULTS.

2.3.1Seasonal variationsinthcGSI 2.3,2 The patternofspermiation. 2,) ,)Seasonalvariationsinspermmotility. 2.3.'1Variationsof'spermatocrit.

vii

... .21)

2') 29

;'Ill

.10 . .... .11 ..31 ... .32

..l2 ...J.'i

.35 .... ..15

....16

. .. .J')

2J5 Sperm concentration. 2.3.6I'la5ma androgenic steroidprofiles. 2.4DiSCUSSIO N.

. 4'

. 41

. 42

CIIt\ PTE RJ.SF.ASONALASPE(.-rSOFSPERMPIIYSIOLOG Y ANDSEMINt.L PLASM AIIIOCm;l\IISTRYINTilEOCf.ANrOUT 49

3.1INTRODUCTION. . 50

3.2MATERIALSANDMETHODS.. . 51

3.2.1Collection offishand milt. . 51

3.2.2 Determinationofsperm motility.

3.2.3Seminalplasmaanalysis..

. 52

. 52

12.4 Determination of tile effects of pH and osmolalityon spermmotility ...53 3.2.5Statisticalanalysis.

3.3RESULT S...•...

. ...•....54 ... . . ...•.... .54

3.3.1Seasonalchangesinspermmotility. . 54

3.3.2Seasonal changesinseminal plasmapHandosmoticpressure.. .55 3.13Seasonalchangesinbiochemical compositionofseminal plasma.58 3.3.4 pHandosmoticpressureeffects onsperm motility 59 3.4DISCUSSION.

viii

61

CIIAPTER-t.CO NCLUSION. 4.1 SUMMARYOFTH E FINDINGS.

4.2CONTRIBUTIONSTO FiSHREPRODlJ(-rIVEI'IIYSIOI.OGYAND AQUACUlTORE.

4.3IMPROVEMENT ANDFURTHERSTUDIES.

REFERENCES..

APPENDIX.

",

.70

LIST OFFIGURES ANDTABLE

Figure1.1Schematicrepresentationof thehypothalamic-pituitary-gonadalaxis in

tcleostea nfish 3

Figure1.2Schematicrepresentationoflobular(a)and tubular (b) testis in tcleosts.

Figure1,1Cross-sectionofa testicular tubuleshowing its lobularandinterstitial

compartments 11

Figure1.4Basicsperm morphologyin refccsts

.14 FigureI.SThedifferencesofsperm morphologyinexternallyandinternallyfertilizing

tclcosts:11)troutspermatozoa.40,000x;b) guppyspermatozoa.21,ooOx 16 Figure 1.6 Ocean pout.Macmzoarcesamericanus

....23

Figure 2.1Seasonalvariationsinthegonndosomaticindex (GSI)of the male oceanpout (Mcan+SE, ""'3 expectforSEr andJANwheren=2) Monthsassignedthe same letterarc notsignificantly different(p<0.05)

36 Figure 2.2 Seasonalpatterns of spermiation(a)andsperm motility(b) in maleocean pout

during199](n=lO) and 1994 (n=20).Week0represents the time ofonsetof spermiation (June22 in 199]andJuneI] in1994),(a) Changesof the spermiationrate(%)during the spawningseason. (b)Percentageof fishwith Class 4 motility(76%~100"10motilesperm)duringthespawning season.

.. ... . ... .. ...38 Figure 2.3 The variation ofspermutocrit duringthe 199]spawningseason (Mean±SE,

""'10),Week 0 representsthe time of onset ofspermiation (June22).

.39 Figure2.4 A microscopic view (400x) ofa miltsample (maleE8) collectedin June199].

40

Figure2.5Amicroscopicview(400x) of amiltsample(lIIaleES)collectedinAlIg.lI~1 1993 .

.4 () Figure2.6Therelationshipbetwee n spermatoc rit(~.)andspermconccntralin..(10"fml)

foroceanpoutmilt.The8samples(circles)contaiuing~OO/.or higherof spermatldswereexcluded fromregressionanalysis

·12 Figure 2.7Changesin plasmaandrogens in the11lo11eocean pootduringthe 11)<)4

spawningseason (Mean:i.SE.n=6).Week0representsthetime,,!onsctuf spermiation foreachmale;negativevaluesindicated weeksbefore spcrmintiou

Figure3.1 Changesof'spcnum ctiluy (weighted mcan)during thespawningscusonill 1994, Theweightedmeanis the mean spermmotilityclassforeachseason weighted bythe number ofIlah in eachmotilityclass.. S5 Figure3.2pHvalues(Mcan+SE) of seminalplasma litvarious times ofthespawning

season.pHufthecarlyseasonwassignificantlylowermanIh.1Iort he middleand

lateseason (p=O.OOOI.n=I I). ...56

Figure3.3Acomparisonofosmotic pressure(mmol/kg;Mcan+SE) ofseminalplasrnaat various times overjhe spawningseason.Osmoncprcs.'lUreofthe earlysea'!"ll was significantly higherthan that of the middleandlate season (p=0.0005,n=b)

..57 Figure3.4The effectsof pHonspermmctility(class).

. :'i',

Figure J.5Theeffectsofosmotie pressure (mmollkg) onspermmotility(class).

....MI Table 3.1 Biochemicalcomposinonofoceanpoutseminalplasma throughout the

spawning season.Values(mmnllL)are mean

'*

^SEM.Means withthesmileleiter arenot significantlydifferent(p<O .O~ .n=6)

CIIAP TE RI

GENERAL INTR ODUCTI ON

1.1THEREPRODUCT IVEI'II\,SIOLOGY OFMALt:TH.EOSTS

Reproductionin maleFishes.as in females.isregulatedhythebrain-pituitarv- gonadal axis (ReddingandPalino 1<)9J) ,Sincemuch of'the existin,!;knowledgeIl l'fish reproductionis formedonthe basis ofthestud y of tclcusts,,1IIUthe oceanpmlt is classifiedas teleost, the followingliteratu rereviewwillfocus nilthetcleoste;1l1 spcclcs Asinall vertebrates,externalcnvironmcmulcues influence thetclcostennbrain- piuntary-gonndal axis by the releaseof gonadotropin-releasingbormouc(GnRII)fromthe hypothalamuswhichreachesthepituitaryviadirect penetrationof Fibres,or asinuthcr vertebratesmay access thepituitaryby11specializedportalbloodsupply(Redding and Patino1993). After reachingtile pituitary,GnRI'1bindstogo nadotropic cellmembranes stimulatingthereleaseof gonadotro pichormone(Gtll )intothecirculation Viathe circulationGtH reachesthetestiswhereit binds tospecific receptorsinLeydig cells or someother somaticcellsofthetestis,resultingin the synthesisorsteroidhormone s.

Androgenicsteroid hormonesfromthe testis inducedevelopmcm, growthlind1'111011 maturationof malegermcells andultimatelyspermiation.In addition,steroidhormones alsoregulate GnRHand GtI-Isecretionby eitherpositive ornegative feedbackucthmat hypoth alamicandpituitarylevels to regulatethespe rmatogeniccycle (Figure1.1)

Environmentalcues

sensory organs

o 0

~

electrochemicalsignals

GtHlI ~GtHI

-

---~~-<- ---

S(lCrmQlogcncsi~spcnnialion

~~h,nnon"

Figure1.1Schematic representation of the hypothalamic-pituitary-gonadal axisin relcosteanfish(basedonReddingandPatino1993)

1,1.1Enviro nme ntalreg ulati on or thescasunnltesucutn r cycle

Thereproductivecycle inteloosts,beginningwiththerecrudescenceof thegonads and eventually final marunuion of gametes,usuallyoccurs at a particulartimeof a year,

particularlyfor teleosts ofthetemperate zone(Munrol"/II.19(0),Since thclim ing(11' reproductionforeachspeciesis relatedto conditionswhichoptimizesurvival andgrowth of the progeny.each species preparesfor breedingby undergoingseasonal-specific gonadaldevelopment. Certainpredictable external environmentalfactors.suchil.~

photoperiod andtemperaturecuesare important inthecontrol{If gonadaldevelopmclll, activating specific sensory organs,c.g. photoreceptor(pi neal),which produce electrochemical signalsthataretransformed bythe central nervous system intohormonal signals.

The effects of such environmcntalcuesdillerfront speciestospecies(Billard 1990a;Redding andPatiilo 19CJ3),anddifferent stagesof gonad developmentmayhe affected by differe ntenvironmentalcues, Photoperiodisa majorfactorinllucncing spermatoge nesisin salmonids,Lo ng photoperiodin thespring results inincreases(If gonadotropin (GtH) in bothpituitary and plasma, followedby the onsetIII' spermatogenesis. Thecomp letionof spermatogenesisinrainbowtrout(OIl('tJrhYllf'llfu mykis.s)normallyrequires a decreasing photoperiod regime from16L:8Dtn BL:16Din6 months.However,photope riodhas noeffect onspcrmintioninrainbow troutwhen the temperatureisbetweenIS''C and18"C and in this specieslowtemperaturesfavour spermiation(Bretonand Billard 1977).In goldfish(Cal1Luitf.f(/urallls)wheretemperature also plays a majorrole in spermatogenesis interaction withphotope riodisdifferen t fmm thatfoun din the trout,as long days arc more favourablethanshortones The

spermatogenesisin goldfishnormally occurs between10· 24"Cfrom Februaryto May and cannotbe initiatedifthe fish arereared at 30"e.GtHcont ent inpituitaryislower in goldfish heldat30"Candhigherwhileat lower temperatures.Furthermore,goldfish donot spermiatc undercons tant rearing temperatures (10, 17,24"C),but theydo under naturalcond itions,suggestingthat fluctuating temperatu res favourspermiation (Gilletct al.l?i8)

In additionto photoperiodandtemperature,socialenvironmentalcues arc alsoan importantfactor,especiallyforspermiation. Ithas been demonstratedthatfemale pheromones, e.g. rainbow troutand pocciliids,attract and stimulatethemales during spawn ing season Miltproductio n,and plasma le vels oftestosterone (T). II·

ketotestosteronc (I I.KT).17a.hydro xy-4-prcg nen-3-o n e, and 17a,20P-dihydroxy -4.

pregncn-J-onc(170:, 20P-OH P). increaseinmalerainbow trout whenpairedwithovulated fenmlcscomparedwithisolatedmales(Olsenand Liley1(93). ln goldfishand carp (Cyprilm.scarpio),GtH levelsare increasedandspermiationstimu lated after the malesarc placedwithfemales(Billard1986)

1.1.2Endocrineregulationofsperrnatogenesisand spermiati on

Spemuuogenesisand spermiationareregulatedby theendocrine (brain-pitui tary- gonadal) axis.Experimental disruptionof anypartof this axisinte rfereswiththe process

ofspermatogenesis andspermianoe.

GnRHis animpcrnntneuralmcdarc rofspermntogenesisandspermiation in teleosts,beingaprimaryr~latororGtHrelease.Biochemica landimnmnocytllC!lCmicai investigations demonstrate thatthe hypothalamusof males and fema lescontainasmany asthreedifferent formsof GnRH(CrimandBettles19') 6). Seasonal changesinthe hypothalamiccontentofGnRH areassociatedwiththerelea seofpituitarygonadrrtmpic hormones (GtHs),testicular recrudescence and maturation(YIIetot.1991;Amann ("ut.

1993) . GnRH treatmentofmalereleostsincreasescirculatorylevels of GIl lnudsex steroidhormone(e.g. testosterone lind Jt-kctorcsrosteronc) Slimul,lting spcrmiatiou (Donaldson andHunter1983;Ngamvon gchcnet1//.19 8 7;HarmiuandCnm 1993;

Harmi netul.1995).

Gonadotropinsregulale spermatogenesisandspermialionviasteroidbonnoncs synthesized inthe testis. M;mystudiesassoci ateincreasedpiluitary and blIK)({

concentrationsofGtHwithtileonsetofspcrmaogencsb andspermia tioninmaletd euslS (Crimet(,1.1975; EscalTreandBillard 1976; Ordon,,'(fl.1c)80;Dillard IlJK6) ExperimentallrealmentofmaleteleostswithexogenousGIHprerara tionsorwithGnRl1 whic h induces GlHsecretion,increasessex steroidlevelsfrorntestis an dstimulates spermiation (Donaldson and Hunter198];UcdaI!I Cli.19K5;Ngarnvo ngcllO netIt/.19K?;

Harmin andCrim1993;Hannin ,,'al.19( 5).Studies of hypophysectomizedmale tclcosts andinvitrostudiesof testicularsteroidproductioninrespon seIo Gtl lhave shownthat

thedegreeto whichspermatogenesisand sper miation depend onthepituitary varies accordingtothe dev elopmentalstage of testisand spec ies.In general, removingthe pituitary afterfullmaturationofthetestis causesra pidtesticulardegeneration and inhibitionoftesticularsteroidogenesis (Billard 1986;Billard1990a). Such treatment restricts spe rmiationinsomespeciessuchas goldfish and winterflounder,buthas no effectonotherspeci es suchassalmo nids andplaice,Pleuronecusptatessa(Billard I990a).Therelative sensitiv ityoftesticular steroidprod uction inresponsetoGtHwas foundtobe minimum at theonsetof spermatogenesisand maximalduringthe sperm iation period(I.eGac andLair 1988; SakaiIffal.1989).Spermatogenes isandspermiatio nare restored after replace mentho rmonetherapy withhomolo gousGtHorpituitary extracts

Recentstudiesindicate thatthemale teleos teanpitu itarycontainstwotypes of GtH cells.Two distinct glycoproteins,GtH-IandGtH-lI,havebeenisol atedandcharacterized fromthepitu itaries ofchum(Ollcorh yllcl1lls keto),coho(Ollco r hylichuskisUICh),and masu salmo n(Oncorhynchus II1QSOII),rainbowtrout,carp,andAfrican catfish(Cla nas gariepilllls)(Kawauchietat, 19&9; Swansonefol1991;Van derKraaketal.1992;

Am anaet(II.1993;ZamlbergenetaJ.1993). Bothtypes of GtHaresteroidogenic diff ering onlyintheirrelativepotencies(Suz ukietal.1988) . GtH-1isfoundto predominatein pituitar y andbloodduringsperm atogenesis, decliningduring thetim eof finaltesticul ar matura tionandspermiation.Incontrast,the levels

or

GtH-1iwhichare low duringspermato genesis. increasedramaticallyto be comedominant duringthetime

offinaltesticular maturationand spermiation.Onthis basis, itissuggestedthatGIU·I is primarily invo lved inspermatog enesis whileGIII · IIactionsarerese rved fortheIinal aspects ortesticular maturationandspermiation

Severalsteroidsfrom the testis,includingtestosterone{T],II-kckltcstush..'rune (II-Kn,17a·hydroxy-4-p regncn-)-()ne,and 17«. 20p -dihydrm c:y-4.prcgn cn-] -o ne( 17(l, 2OP.QHP~areinducedby GlHandare importantinthedevelo pment andnHilu ralion of theteteoseentestis.Anumber ofstudiesof the steroidprofilesduring the reproductive cycle demonstrated that plasmalevelsofT and11·KTarchighduringthe laterstag~'lltil' spermatogenesisand declineefler thebeginn ingofspe rmiation (FosticrotIII.19 K];Ucdn

e tai.1983a;Sco tte lt i l. 1980;Huntetod .1982;KimeandManning11)82;DaYIICc'land Scott1985).Basedonin vitroand in

vn'l:l

studies.ithas beensuggested thatTslimulates andmaintains spermaogenesiswhilebothTandII·KTinduce spermiation(Fustier('I(/1.

1983) ahhough II·KTmay also playarole in the regulationofthelatestagesof spermatogenesis85well asinitiationofmilt production (FostierrIul,1987).Thetcst i~

of som especies alsopro duces17«, 2Op·OHPandplasmalevelsofthissteroid peak duringspermiationfollowingthedec lineof TaOOII-KT IUedacllf /.19111b;Kobayashi ctd.1986 ).In therainbowtrout,thechangesin plasma concentrationof17a,20ri-()111' were associated withspe rmprod uction andtheK':Na'ra tioin thesemina lplasma suggested that17IX,20P-OIlPmight be involvedinthc processofspcrmilliinnby regula ting the io niccompositonof the seminalplasma[BaynesandScoll 1985)

Inadditionto loca lactiononthegonads,steroidsalso act on theendocrinecells of the hypothalamus andpituitary,reb'IJlatingtherelease ofGnRHandOtHand ultimately controllingthe spermatoge niccycle(Billard1986;Goos 1987). Sex steroidscan exert

b

Figure1.2Schematic representation of lobular (a)and tubular(b)testis inreleosrs (modifiedfromBillardI990a ).

botha negativeandpositivefeedbackaction onGnRHand GtH secretio n, depending on the stage of testiculardevelopmen tinteleosts. In adult tcleostsactively undergoing spermatogenesis,testicular steroids exertnegative feedback on thehypothalam us-pituitary sincecastration initiates and stero idreplacement inhibitsGtHrelease(Billard 1986;Schulz et at.1993).However, in immatureorsexuallyregressedteleosts,testicularsteroidsexert positivefeedbackon thehypoth alamus-pituitarystimulatingGnRH and GtH production (Crimand Evans1979;Crimet al.198 1;Crimand Evans 1983;Billa rd 1986; Weiland Marcuzzi1990).

1.1.3Testicular str ucturean dfunelion

Thetestisof teleosts,whichis usuallya pairedelongatedorganattac hedto the dorsal body wall,containsboth germ andsomatic cells(Sertolicells,boundarycells, Leydigcells; Billard1990a).Gametesare produced bygermcells.whilesomaticcells su pport, nourishandregulat ethedevelop mentofgermcells.Based on thedistribution of spermatogonia,the teleost testicularstructurecan be assigned to eitherthelobular (u nrestricted)orthe tubular(restricted)type(GrierI!Ial.1980;Nagahama1983; sec Figu re1.2,page 9). Intheformer,the mitoticallyactivespermatogoniaare found ran domlyalongthe entirelengthof the testiculartubules,usuall y immediatelybeneath the tubular basement membr an e (Figure1.2a), asinsalmonidsandcarp(Grieret al.

10

198 0), In the laller type.found inthe guppy and goodeidteleo s ts, sperm atogoniaare completely restricted totheblind end(apex) ofihetubules,immediatelybeneaththe testicularcapsule{Figure1.2b).

The teleesrean testisconsistsoflobular (tubular)andintemi tial compartments (Fib'Ufl:1.3;Nagahama1983;Billard1990a).The tubularcompo nentconta insgerm and

Figure1.3 Cross-sectionof atesticular tubule showingits lobular and interstitial compartments(modifiedfrom Billard1990a).

11

Sertolicells, which form the germinalcysts.Germ cellsdevelopwithincysts with cytoplasmicbridgeslinking germ cellsundergoingsynchronousmatu ration. Evidence suggests that Sertoli cells notonlyprovidethe physical supportandnurturingof germ cells,but they arealsoinvolvedin phagocytosisof residual bod iescast offby developing sperma tids.as well asthe formation

cr

spemutczcugmeta(Grier1( 8 1).A basement membran elayer liningthetubula rsurfaceand a discontinuousboundary-celllayer separates thegerminalcysts from interstitialtissue.The interstitiumofthe teleosttestis containsvarious interstitialcells,including bloodandlymph vessels, andLeydigcells.

Leydig cells areconsideredthe maintesticularsteroidogenicsite (Nagahama 1983;

Fostierel al.1983).Sertolicells,andsome epithelialcellsofthesperm ductandseminal vesiclesofsome teleosts arcalso capableof producingandrogenicsteroids(Nagahama 1983;Posneret

at .

1987;Schoonenetol:1987),the mostimportant beingtes tosterone (T),ll-ketotestosrerone(II.KT),andandrostenedione (Fustier(!/al.1983;LairIlJlJO; Bourne1991).Thetestisofsometeleosts alsoproducesprogesterone,17n· hy drOlty·4 - pregneo-a-cne,17(1" 2op·d ihydroxy-4 -pregnen.3-one,ll-dcoxycc rticoste ronc,andperhaps smallamountsofestrogens{Fosticretol.1987;Barry1:1III.I'){))).Take ntogethe r,these steroidsarc involved in a varietyof physio logicalactivitiesincludingthereg ulation of spermatoge nesisand spermiation, secondarysex characteristics and reproductive behaviours,secretoryactivityof thehypothalamusand pituitary, and generalmetabolism (Fostierot ol.1983;Billard 1990a).

12

1.2ACOMPAR ISONOFSPERMPHYSIOLOGY BE1W EENEXTE RNALLY AND INT ER NALLY FERTI L IZINGTELEO STS

Spematoeoa' in externallyfertilizingtcleosts are immotilein themalegenital tract oraftermilt^lcollectionuntilmotil ity,whic hisnormally ofbriefdurat ionuptoseveral minutes(Billard1986),isinitiated byreleaseintothe externalaqueo usenvi ronment.

Usually,the concentrationof spermatozoa (s pz)inmiltofexternally fertilizing teteosts is high(IOv•1011 spdml;DoieI

at.

1982;Kruger etcd.1984;ClcreszkoandDabrowski 1l)94;HamlinandCrim1993; Suq u etetcII.1992). This characteristic isalsoreflected by the highspe rmatocrit([thevolumeof packedspermatozoa/the volume of total milt}xIOO%),usua llyrangi ngfrom 40% -80"10. By contrast,thespermat ozoaof internally fertilizingteleo st!are alreadymotile inseminalplasma(Koyaet01.1993;

PavlovandRadzikhovskaya1991)andarc transferreddirec tlyintothe femalegenital tract. Insomecases, thespermare grouped together into spermatop horesor spcnnatozcugmata (Billard1978;Grier1981)which becomemotileupontransferintothe femalegenitaltract.Inthesespecies,theconc entrationof sperm atozoa and spermalocrit

'spcnuatozonesperm -mlhespcnntseminalplasrua

13

T "'"

1 _•

mickl\o-pjcc;~

,

1 T

^{IIllj- - - - f1agc1!um}

Figu re1.4 Basicspermmorphologyin teleoststallerJamiesonandLeung \lJ(1) arcgenerallylower(10" - 10- spzlmlandI•10-/. ,respectively)COllIpared withtill:

externally fertilizingtelecsts(Pavlovand Radzikhovskaya 1991) andtheduration ofspcml motilityininternallyfeni lizingteleost is Ion3 cr.up10severalhOIJ~or days

1.2.1Spe r m mor phology

In a review of teleost spermmorpholog y Jamiesonand Leung(1991)stated that teleost spermcanbestructurallydividedinto a head.the mid-piece. andan elongatedtail {Figure 1.4).Typically,fish spermhead s are sphericalorovalshapesinthe absenceIl(

an acrosom e characteristic ofteleosts which is probablyrelatedtothepresen ce ofthe

14

micropyleinteleost eggs.The mid-piece consists of a mitochondrialsheathanda central lIagcllum.Inmost teleost sperm,the mitochondriaarefew in number,not modified, and situated in alow collarimmediately behindthe nucleus.The flagellum ofmost teleost spermconsistsof atypical 9+2axoncmalpattern (ninepairsofperipheralmicrotubules and one pairofcentralmicrotubules) Aflagellatcdspermand biflagellatedsperm are foundin some species

Generallyspcrm morphologyseems tobe related to the biologicalmode of fertilization, For exampic, the spermatozoafrom external fertilizersexhibitsimple structures and do not appearmodified (Figure1.5a; Billard I990a).The nucleus ofsperm generally isround or sphericalshapeandthechromatin is less condensed,because of limitedreplaceme nt ofhistones by protamines. Areduced mid-piece contains few mitochon dria, which probably resultsinshortdurationmotil ityafter spenn activation Thesesperm cells,withoutanymodified structure (no sperm bundles), are freel yreleased fromcysts. thenshed into water (BillardI983a),Inteleostswhich have evolvedinternal fertilization,spennare complexandgreatlymodified (Billard1990a). Thespermnucleus is elongatedasIIbladebyIIseriesof microtubulesduringspermiogenes is(FigureI.5b).

Meanwhile. thehistonesof the chromatinare replaced byprotamines, resulting in high nucleuscondensation.Themid-piece is extremelywelldeveloped,and contains numerous largemitochon dria, which is probab ly relatedto the longduration of sperm motility.

Glycogenparticles arc visible in themid-piece areainsomeinternallyfertilizingspecies

15

Figure 1.5 The differencesof spermmorphology inexternally and internallyfert ilizing teleosts:a) troutspermatozoa.40.000x;b) guppy spermatozoa,2l,OOOx(ancrBillard 1990b).

(guppy,Poecili a reticulata}. Inmanyinternally fertilizing telcosts, spermare packed togethereitheras unencapsulated(spermatozeugmata;see Figure! ,2b) or encapsulated (spermatophores)balls.Sperm transfer in bundlesis coupledwith the evolutionof the appropriate anal fin(gonopodium)and behavioralmechanismsto ensureefficienttransfer of malegametesinto the femalereproductive tract

16

In summary.thespermmorphologyof externallyfenilizing teleosts is primitive with around nucleusandshort mid-piece.In teleostsevolving theinternally fertilizing mode,spermmorphology ismorecomplex.withelongatednucleusand ....'elldeveloped mid-piece

1.2. 2Sl~r mactivaliolland llIolility

Itissuggestedthat thestimulationofspermmotilitycouldbeowing toachange ofmembranepotential.sincespermmotility doesnot arisein thepresenceofdesmcthoxy verapaniel,a specialinhibitorof voltage-dependentcalciumchannels (Billard1990b) Osmoticpressure,ioniccomposition and pHmaydepolarizethe sperm cell membrane ar.d are themostimportant factorsdeterminingtheactivationand motilityofthe sperm in external fertilizers (Stoss1983).

Changesiiiosmotic pressureinitiates sperm motilityinexternally fertilizing releosts.Hypotonicitygenerally activatesspenn motilityinfreshwatertetecsts, especially in carp (Redondo et(I/.1991).Ontheother hand.the motility of spermatozoainmarine Ie1COSI$.suchas cod(Gadus moThuo),flounders(Ullumda yoko1lamQeandKarei/ls bicolofQ(/ls)and halibut(HippoglossllshippogloSSILf).isinduced byhypertonic suspensio n media (Billard etC11.1993). Motility occurs ina widerrange ofosmotic pressures in marineteleoststhan in freshwaterteteosts

17

Besides osmoticpressure.ionic compositionisalsoinvolved ininhibitingOf

stimulatingthe motility ofspermatozoa in bothfreshwaterandmarinetcleosts. Ionic regulationof spermmotilityin externallyfertilizingreleossis very complex, and is mainlydeterminedby potassium(K').calcium(Ca")and magnesium(Mg").Although themechanismof ionic regulation of spermmotilityisstillunclear.potassium inhibits.

andcalciumandmagnesiumactivatesperminsalmonids(Morisawa andSuzuki1980).

In contrast.potassiumionincreasedthemotility ofcarpspeno (DillardandCa sson1992 ) Inmarineteleosts,potassiumionhad noinhibitoryeffect onspcnn motilityin the slimmer whiting.Siflagocii ima(Goodallet 01.1989),flounder,P/1IIicllthysflesus.andcod.G{/(fIlJ marnua(Morisawa1985). The studybyGoodalletal.(1989 )showedthatboihcalcium andmagnesium ions increasethe durationofspermmotilityinthesummerwhiting Clearly.ionic regulationofspermmotility varies from speciesto species.

ExtemalpHalso affectssperm motility.Generally,alkalinepHfavoursincreased motilityandfertilityofsperm. whileacidic pHinhibitssperm motilityinmanyex.1Cfl1i1l1y fenilizing teleosts(exceptinafew telcosts whosespermisacid-tolerant,e.g.chain pickerel[Duplinsky1982}). Anactiva;ingsolutionbelowpH7.8failedto inducethe mocility ofrainbow troutsperm. andthe optimummotilityof seabass sperm wasreached inpH9seawater (Stoss1983).Thehighestmotility ofhalibutspermoccurcdat piI~

ranging between7.5to8.5(Billardetul.1993).

Knowledgeoftheregulationofspermmotility inthetefeostswithinternal

18

fertilization is limited. Sperm motilityis achievedintheguppy.gcodeid and lJomidl1hidaeteleosu,afterthe breakdownofthe spermatozeugmata orspermatophores in the female genitaltract.Billard (1978) demonstrated thatmotilityis initiatedwithout dilution immed iatelyafter the spermbundleof the £uppyis dissociated.Accord ing to MorisawaandSuzuki(1980).however,breakingup the sperm bundleand initiatingsperm motilityof b'UpPYandthelopminnow(Gambllsi aajfinls)occurred in electrolytesolutions sucha.s NaCI or KCIsolutions at concentrationsbetweenSOand300mmollkg, especially inKCIsolutio n,butnot ina nonelectrolyte solutionsuchasmannitol(100 -700 nllnolikg) They suggested thationic concentrations, especially potassium concentrations.

mightinitiatespermmotilityin these internallyfenili zing teleosts.The spermatozoaof the elkhornsculpin [internal insemination).Alcichrhysauicomis(Koya1:1aJ.1993) and wollftsh(AllarllichasII/plu)arealreadyactive in freshly collected miltwithout apparent initiation of activation.Koyaet at.(1993) showed that thegreatestmotilit yofelkhorn sccfpinsperm occurred in Na'-supplementedmediumrather than other ionsand thatthe optimum ranges for pHandosmoticpressurewerepH7-9 and 300•400mmo1Jkg, respec tively. Therefore, osmolality.pH and ionic compositio nappearto beimportant factorsregulating spet1Tl motilityin internallyfertilizing teteosts

19

1.2.3 Sper m met ab ol ism

The duration ofsperm motilityis veryshortin extern allyfertilizingte h.'ostswhere osmoticshock may be one factor (Billard 1983b).For example.thespermatozoa

o r

trout (Salmo lflllt a)andcar pare quickly damagedafter dilutioninfreshw ater. Onthe other han d,someteleost spermfro m externalfertilizers arccapa bleof withstand ingoSlllotic shock andstill display short-te rmmotility, e.g.tilapia(Oreocuromu1Il0,fS(lII lb i , /lJ) Interestingly. the duration ofmotility is notextended if spermarcactivated in a isosmotic saline medium(from45sec.to 90 sec.: Billard1( 78). Thelimited supply ofmetabo lic energyduringmotilitymay beanimportant factoraffecting theduratio nofmotility.The ene rgy,mainly provide dbyAl P(Billardetd.1995a),isobtainedfrom entnbolizing both endogenousandexogen ous substrates.

For thespermofexternally fertilizing teleosts, thesupp lyofenergy from exogen ous substrates isvery poor,because the spermatozo a arc,releasedinanexterna l aqueousenvironment(SlOSS1( 83).Therefore,most ofthe energyformotilityisderived fromendogenous substrates, especiallythose substrateslocalized inthemid-piec e (Billard et aJ.I995a).Furthermore, the smallamou ntsofmitoc hondriaplayan importantrolein producingATP.In rainbow troutsperm,the intracellularAT Plevelsarc qu icklydepleted following theinitiation of motility (Billardetof. 1995a). Demcmbranat cd spermato zoa lnvitrodisplayflagellarwaves forextended periods of time (upto 30min.)atfrequencies

20

depending uponthe ATPconcentrationused inthemedium. Sincethedecreaseof Flagellar beat freq uencyduringrainbow trout sperm movement reflectsa decreaseof intracellular ATPconcentrations (Christenel(II.1987),theseresultsindicatethatsperm cellmetabolismis not fastenoughto cope with thehigh energydemand during motility andcannotcompensatefor the rate of ATP hydrolysisbythe dyneinATPase.Therefore, the energyavailableduring motility of rainbow trout spermis mainlyproducedby the accumulatedATPbefore motilityinitiation,

The spermatozoaofinternalfertilizers aredirectlytransferredintothe genitaltract or thefemaleandthemetabolic energy duringlong-term sperm motilitymay beprovided bybothendogen ous andexogenoussubstrates (ovarianandseminalplasma). Inthe presence of n welldevelopedmid-piece,thelarge amountsofmitochondriaenable sperm toquickly synthesizeATP tomeetthehigh energydemandduringextendedmotility.

Billard andJalabert (1973) suggestedthatglycogenpaniclesstoredin themid-piece of guppy sperm area majorendogenoussubstrateafter the initiation ofspermmotility.They also foundthatthe glycogenstores are much higher inspermatozoafoundin thetestis tha n in the ovariancavityofthefemale,andglycoge nstoresweredepleted when spermatozoa werediluted in a salinesolutionwithoutglucose.Insurfperchspermatozoa, Gardiner(1978) demonstratedthemetabolismof glucoseandnoticed thai the additionof glucoseto surfperchandthe b'UPPYspermconsiderably increasedthe durationof motility.

Foll owing the cessationof spermatozoa movement,they were reactivated by a saline

21

solution enriched withglucose.

In conclusion,thesperm ofexternalfertilizers displaya shortduration ofmotility, which is correlated with accumulatedATPbeforemotilityinitiation.Incontrast,sperm frominternal fertilizers aremotilefor long periods of time anddisplayrelativelycomplex metabolism andcatabolismprocessespossiblydependentuponbothendogenousand exogenoussubstrates.

1.3A REVIEW OF OCEAN POUT REPRODUCTION

1.3.1Distribu tion nndsystemntles

The oceanpout(Macrozotlrtes ameticanuscdistributedon both sidesoftheNllrth Atlantic Ocean, is acommon species intheNorthwestAtlantic,whereit can befound from BattleHarbor,Labrador,tofheGulfof 51.Lawrencelind alongthe coastsofthe Maritimeprovinces (Olsenand Merriman1946;Scaliand Scoll 1988), As a grou ndfish, the oceanpoutresidesindepths from 10m to over183m andismoreabundant onhard andsemi-hard bottomthan onmuddy substrate,Accordingto conditions atthetime of capture,itprefersatemperature andsalinityof6•9"Cand32 •34ppl,respectively, althoughitcan befoundintemperatures from O·lo.7"C.

22

The ocean poutis inthe familyzoareldce.thesuborder Zoorcoidei,theorder Gadifcrmes(Nelson1976).Commonnamesforthisfishinclude eelpout,'congo eel', mutlonlishand lampcreel(OlsenandMerriman 1946;Scottand Scou1988).Thiseel- like speciesischeracrcrizcdbynbroad andheavy bead, aterrnilllli mouthwith thick fleshylipsandconicalteethinIhefrontofeachjaw andoneserieson thesides.The small andshortpelvicfinsarclocatedin front of thelargepectoralfins.Thedorsal and analfinarc continuous10thepointedcaudallin(Figure1.6).Dorsalcolourvariesfrom muddy yellow to reddish brownmottledwithgrey orolive green, and the colour ofthe belly isfrom whitetodullyellow. Thesmoothscaleless skin is coveredwith mucus (Bigelow andWelsh1925;Scoll andScottt988).

1.3.1lliologyof reproduction

A detailedstudyoftbe biolo&.,v ofoceanpoutreproductionwascarried outby OISl.'I1andMerriman(1946).Theyandotbcrs(BigelowandSchroeder1953;Kohler1968)

Figure1.6 Dccnn pout.Macrozoorcesumertcanus(afterScott andScottt988).

23

reponedthat oceanpoetcarryouthmitedmigrations.areproductivemigration10shallow waterinspringand areturn to deep waterinautumn.Evidencefor aninshore-offshore migration was alsofound in the easternNewfoundlandarea.whichsuggestedtherl.Sh moveinshore to spawninthe spring(Keatsttl Cli.1985).

Olsen andMerriman(1946)repon ed that maleocean pout do not matureuntil reachingabody lengthof 25 em.and almost all metes of) 9em inlength(4-5 year old) aremature,Femalesmaturelater where the smallest maiuresizeisapproximately 45 em(5-6 year old),Spawningoccurs earlierin northernwaters and progressivelylater insouthernwaters. forexample,in theNewfoundland area,spawningOCCtlTSinlute Augus t (Keatse1ul:1985),whileinsouthernNewEnglandandtheGulfof Maineiltakes placeinlate September andOctober(Bigelow and Schroeder19 5] ).Multiplespnwnings areunlikely duetotheuniform slightly sphericaleggmasses(Keatsr:t"I.1985)which theocean pout depositsinrocky areas (OlsenandMerriman1946;BigelowandSchroeder 195];Keatset al.1985).Afierspawning. the egg mOlUCS areunder parentalprotection (BigelowandSchroeder1953;Olsenand Merriman1946)whichis usually providedby diefemale duringthe longperioduntil hatch (Anderson1985;Keatsetal.1985).Ittake"

about3 monthsforfertilized eggs tohatchinthelaboratory at 4•II~C(Mcthven and Brown1991)

Inrecentstudiesofmale andfemalereproduclionconducted at theOcean Sciences Centre (OSe) ofSt.John' s,Newfoundland,thespermofoceanpout havebeen foundto

24

bealready motileat the time of collectionofmiltsamples. Upon theadditionof seawater to the milt,sperm motility ceases immediately(Crimet al.1995; VaoandCrim 1995a, b;Yeo/!Ial.1995). During the spawning season,maturemalesdevelopeda papilla (protrusivegenitalpore),andcopulation hasbeenobservedbetween themalesand females prior to oviposition(Yeo andCrim 1995b).Artificalinjection of milt into the female oceanpout ovaryhasyielded fertilizedeggs,andthe females usually spawned theireggs within24hoursafter insemination(YeoandCrim1995a). Accordingtothese studies, itwassuggested that theocean poutisan internallyfertili zing species with biflagellated sperm and the eggshave atleasttwo-micropyles.However, detailed knowledgeofthe malereproductionandstudiesof spermphysiologythroughout the spawningseasonwere not available.

1.3.3Aquac ultu recapabihty

Theocean poutisrepon ed to havemanyfavorabletraitsforcoldwater aquaculture. Forexample,thefleshisdelicatewith lowfatand low cholesterol concentration (Sheenyet ol.1917).Market studies haveindicated that ocea n pout' sflesh is acceptable totheAmerican consumer (Sheenyet af.1977;BrownIf/al.1992). Since theocean poutcontainshigh concentrations of antifreezeproteinsin theplasma and tissues(FletcherIffal.1985;Hew»t al.1988) ,it is able tosurvivefreezingseawater

25

temperatures (-1.7 t'C:Kaoet01.1986;KingetIll.1911;9).Studiesalsoreportedthat newlyhatched larvae appeared adult-likewithfunctionaleyes andmouth,andthere was no pelagicstageorapparent metamorphosis.Therefore,the larval survival ratesarehigh (>90%)inthefirsttwo monthsof exogenousfeedinganddecreasedmoderatelyto 75• 80% inthe second year(Methvenand Brown1991;Brown1'101.]lJ92).

However, forsuccessful cultivationof the ocean pout,the supplyofseedwhich islimitedmustbeimproved through artificialreproduction.Because theocean pou tnnd wolfish(AnarllichasII/pus)sharesimilarreproductivebiologics(Kentset(If.1985;Pavlov andRadzikhovskaya1991),studiesof ocean pout reproductionmayalso contributeto our understandingof the reproductivephysiolog yof wollish,anotherpotential newspecies forcold wateraquaculture ineastern Canada

1.4 On.fECfIVESOFTilESTUDY

The purpose of thisstudy was to examine thereproductive physiologyofthe male ocean pout.Inparticular,the objectiveswere:

I) identifythereproductive season formale oceanpour:

2)invest igateifspermqua litychangesduringthe spawningseason;

3)determinethe seasonalvariationsin thetesticular index and the plasma

26

androgen icsteroidprofilesduringthe spawning season;

4) studythe relationshipbetween the biochemicalcharacteristics ofseminalplasma and sperm motilityinthe ocean poul.

I.SAPPROACIIES

or

TillSSTUDY

In accordance withthese objectives,two studies werecarriedoutonmaleocean pout in1993 and1994.

StudyI.This study focused on thephysiologicalcharacteristicsof thespawning seasoninthe maleoceanpout, including thetimingof spermiation.changesinsperm motility,spermatocrit,spermconcentration,thegcnedosornaucindex(GS1),and plasma androgenicsteroids.

St/ldy2,This study focused on theseasonalaspects ofsperm physiologyand the biochemistryofseminalplasma. includingthe seasonalvariations inpH,osmoticpressure and biochemical composition. Inaddition.the effects ofpHandosmotic pressure on spermmotility were investigated.

27

CllAPTER2

PHYSIOLOGICALCHARACTERISTICS OF SPAWNING IN THE MALE OCEANPOUT

Macrozoarces americanus

1.1 INTR ODUCTI ON

Oceanpout,MacTCJZOQrCt s americanus.is a benthicmarinefish in the Northwest AtlanticOcean(Ston and Scott1988),which migrates inshoreto spawnin shallowwater in thesummer. Despitethe knowledgethat males are summer spawners.the details ccnceming thetimingand durationofspermiation.changesinspermquality,andthe changesinplasma steroids duringthe spawn ing season havenot beenstudied.

Sincethelimingofspermiationandsperm qualityarean.ong the major factors inffuencmgthefertilizationrate ofeggs,a beucrunderstandingofmalereproductive biologywill improvemanagement ofbrood stockandassist withsuccessfulartificial reproduction incaptivity

1.2MATERIALS AND METIIODS

2,LI EJ.perimenl:d animals

Adull maleocean pout(2-4 kg) werecollectedfromNewfoundlandwatersby SCUBA diversduringthespawning season,fromJunetoSeptemberin1992 and1993.

Theyweretransported 10 theOceanSciences Centre,andmaintainedtogether with

29

females inflowing ambientseawater (from·1.9toIS.6"C) inindoorround (2 x2x OAm) fibre-glass tanks.Eachtank held ISfish.Theanimals were provide dwitha simulated naturalphotoperiodandfedchoppedcapelin twice a week.

2.2.2Thegcnadesc maucinde x (GSI)

From September 1993 10 August1994. samplesof thetestes were obta inedIrom males killedatI·2monthintervals(threemalesat eachtimeexceptin Septcmbe rJIJI)J andJanu ary 1994 whereonlytwomales were available).Seasonalchanges intcsucutnr development were determined according10 changesinthe gonadosoumticindex «gonu d weightIbody weight]x 100%).

2.2.3Spcr nuatlcu

Fortwoyears, during the reproductive seasonfromMay toOctober, spermiation wasdetermi nedatIor2-weekintervalsby collecting milt from a polyet hylenelube insertedthroughthe urinogenitalporeintothe spermduct. Tenmalesin 199 3and 20 malesin1994werestudiedandmaturatio nrateswerecalculated accor ding10the percentage ofspcrmiattng fish([spermiating fishItotalfish]x100%)

30

2.2.4 Sper mmotility

Miltsamplesafter collectionin borosilicatetubeswere brieflykept on crushedice.

Sperm motilitywas estimatedaccordingto thepercentageof motile spermatozoa.A 20 IIIdropofmiltwas placedbeneath a coverslip on a glass slide at room temperature (-20"C)for lightmicroscopic (400x)observationofthe percentageof motilespermatozoa.

Three slideswere preparedforeach miltsampleand three fieldsofview wereexamined oneach slide.After calculation of an average.the relativemotilityof spermwasranked accordingto 5 classesof motility:0) 0%,I) I - 25%,2)26~50%,3) 51 -75%, and 4) 76-1000/0 ofmotilesperrn,respectively.

2.2.5Spermatocrit

In1993,two smallaliquotsof freshlycollectedmiltsamples from10 maleswere transferred into microhematoerilcapillarytubes (length-75mm,10"'1.1-1.2mm).The lubes were sealedwith clay,centrifugedfor10 min.at12,000 x g ina Micro Hematocrit centrifuge(International EquipmentCo.•ADivision of DAMON,made inUSA;Model MB), and the spermatocritwas determined(packed spermatozoa volumeItotal milt volumex 100%).

31

2.2.6 Spermconcen trat ion

Thespermconcentrationof miltwasanalyzedin 37samplescollectedfrom 10 males in earlyJuly(July7),middle July(July19),earlyAugust(August I and2),late August(August31),andearlySeptember (SeptemberI)1993.Toinhibitmo ti litylind stillmaintainsperminteg r ity. miltWIISdilutedin 1:50DCSB 4 buffer (sucrose:150mM, MgSO/7HP:7mM.CaCl1'2H20:1.7mM.glycine:86 mM. and Tris-HCI:30 mM at pH8.0and osmotic pressure450 mmollkg[Billardetai.199])).Thesperma tozoa were counted usinga hemocytometer(AmericanOpticalScientific Instrume nt Division.1975) underlight microscopeat400xmagnificat ion,and the procedureWI'"repeated twice to obtainanaverag e.

2,2.7Androgeni c stero i ds in bloodpla sm a

FromMay to October 1994,six males wereana est hetizedeitherweeklyor biweeklyin100 ppm 2-phenoxyethanol(SigmaChemical Co.),anda1.0 mlblo od sample waswithdrawnfromthe caudal veinusing cool andheparinize d 23G needlesandsyringes. Blood sampleswereplaced in crushed ice after collection before beingcentrifu g edat 4"C for10min. at8.827 x g.Plasma samples were remo v ed and storedfrozen at-20"eunti l androgenicsteroid measurement.

32

The le velsof thetw o major male androgen s, testosterone(T )and II~

ketotestosterone(II.KT),weremeasuredinplasma samplesby radioimmunoassay(RI A ) acco rdingtomethods describedbyHarmi n andCrim (19 93) withsomemodificatio ns (appe ndixI&2).Briefly,100j.11samples of plasmaand 10 IIIetha nolconta ining1,000 CPMoftritia tedtestos terone(T)were mixedin borosili ca tedisp osableculture tubes Afte rincuba tionforI~2hou rs at roomtempe ra ture(20-2S"C),the mixtures were extrac tedtwicewith2mldicthylether befo refreezing theaqueousphase oversolidCOl ami combiningthe ethe rfractionsintoacleantub e.After evaporatingthe ether extracts undernitrogenwithgentle heating (40"C), the plasma residu es wereredissolved ioLO ml abso luteetha no land storedove rnight at4"CbeforetheRIAwasperformed .

Theplasmaextra ctionefficiencyfor the androg ensineachsa mple was determin ed in10 0 111of the eth ano lextractby mixingitwith10mlscinti llation cock tailina scintillationvial. Recoveryrate s(%) ofthetritiat ed testosteroneadded toeach sam ple were calculated from themixtu res inabeta counte r(Min ax i,~Tris Carb 4000series ).

Sinc erecover y rates of ll-ketotestosterone are similar tothat of testoste rone(±5%

diffe re nces; unpublished) , recove ryratesof testosteron e were usedfor ll-ketotest osterone.

Forthemeasur ementoftestosteron e,100IIIofthe ethano lplasmaextractsor standa rdtestosterone inethanol(I-1,000 pgltub e) werepip ettedintoduplic a te12 x75 mm borosilicatedisposable culturetubes.Followingeva po rationof the ethanolunder nitrogen,200IJ.Iassay buffer(50mMNalHPO~. 100mM NaCl, 0.1%NaN).0.1%

JJ

gelatin. pH7.4).100~Iiodinated T (10,000CPM),and100111dilutedTnmiserumwere ad dedto assaytubes.Artera primary incubationperiodof1 hr at22-2'tr,1,000111 of the separatingre a gent wasaddedto theassaytubes foranot her25min.incub ;'\tion periodprio r toeemnrugatic nof the tubes at1,000x g for15 min. Thesuper natant (containing freetesros tece e)was thencarefullyaspirate dandtheradioactiveprecipnates we recount edinaPackard Auto-Gam maCoun ter(secappend ixI).

Sim ilar methodswereused todetermine theII-K Tvaluesinplasma Again, 100 IJ.Iofetha nolextra c ted plasm a was evaporatedundernitrogen. A10011111li{jtlolIlf phosphatebuffered salinc(PBS:28mMNaH}'OlIlP,61mMNat ~lI'q,IS41l1MNne!.

O.I%gclali n,pH7.0 ), 100~ItritiatedIl· KT(6,000 - 8,000 CPM ),and a 100111 II- KT antiserum (1:70,000)weread dedto theassay lu bes.AOcranovernigh tincubationat 4°(', 600111ofa charcoa lsuspens ion(1.2 5 g charcoal,0.125gdextra n T-70in500mlIIIIS butTer) was added andthe assay tub es il...ubatedfor anotherhour at4"C.Following centrifugat ionof the tubesat1,000x g for 25min.,thesupenatamwasdecantedinto a vialaxltaining10 mlscintillation cocktailtodeterminethelevels of bou n dradioactivity ina betasci ntillatio ncounter(see ap pendix 2)

34

2.1.8Statisticalanalysis

Regression analysiswasappliedto37milt samplesto examinetherelationship betweenspermconcentrationandspennatoait Inaddition,thisrelationshipwas tested onIsubsetof29 millsamplescollectedfrom 10malesinmiddleJuly,earlyAugust,late August, and earlySeptemberfollowingthedisappearanceof therelativelylarge,non- motilespematids.

SeasonalGSIvariationfor males wasanalyzed using ANOVA.A GLM(General Linear Model)procedure(SASInstituteInc.,1989)WItSapplied, inviewof unbalance designdan.ALeast Sq uareMeans restfo r multip lecomparison wasutilizedto detect the diffe rencesbetween GSIvalues from September1993to August1994.

1.JRESULTS

1.3.1SeaSODalvariationsin the GSI

FromSeptemberto January.IheGSI of maleswaslow(0,26·0.31%;Figure2.1).

ByMarc h, howe ver.theGSI bega nslowly risingindicatingthatgonadalrecrudescence wasunderway. A major increaseinGSIwasdetected byJun ereaching apeak in July

3S

SEP OCT DEC JAN MAR MAY JUN JUl AUG

Figu re 2.1.Seasonalvariationsinthe gonadosomaticindex (GSI) ofthemale oceanpUlIt (Mean+SE.n2 )exce ptforSEPandJAN wheren=2) Months assignedthesameletter arenolsignificantlydifferent(p<O.OS).

(GSI=1.92% ) togetherwith the onsetof spermiation.The 051declinedsignifican tly dur ingthe spawning seasonfromJulyto August

2.3.2 Therallern of spl'rmia l ion

A two-year studyolihc oceanpoutspermiation response yielded a similarpattern

30

for both years. In1993spe rmiationwas detectableinsomemales (40%)byJu ne 22 while allthe maleswerein spermiatingconditionbyJuly 18(Fig ure 2.2a ) .Sperm iation term inatedby Septe mber24,conclu dingaperiod of13weeks. Duringthe1994 spa wningseason, miltwasfirst collectedfromsome males(400/0)onJune13andall males were spermiating byJune30.Spermiation wasterminated byOctoberJ(Figure 2.2a)with the period ofspermiation lasting17weeks.Allmales that spermiatedin1993 remamredan dprodu ce d miltagain in1994.

1,3,3Seaso n alvaria t ionsinspermmotility

Throughoutthe spawningseason,thepercentage ofmotilesperm,rose and fellin amanner sim ilartothepaucrn ofthespermiationresponses(Figure2.2b).Initially,only half of the spermiatingmalesin1993andnoneofthemal esin 1994 producedspermof highmotility(percentageof motile cells >75%,).Astheseaso nprogressed,sperm motility improved,reaching apeakbyJuly andAugust. By September,however,sperm motility fellagain

37

100

60

~

i ,g . .

[j;

~

0 10 0

(b)

i

E

" ^{/ \}

^~

~

" ^\

~ ^\ \

~

^~

fi

~

^{20 ,(}

^\

\

f / ^h-.,

~

^{10 12 14 \,.}

Week

Figur e 2.2.Seasonalpatternsofspermialion (a) end sperm motility(b)inmaleocean pout during 1993(0:=: 10)and1994(0=20).Week 0 represents thelimeof onsetof spermiation (June22in1993andJune13 in 1994),(a)Changesof thespermi atio nrate (%)duringthe spawning season,(b) Percentage offish with Class 4motility (76%.

100%motile sperm)during the spaw ni ngseason

J8

2.3.4Vllr illotions ofspermllotOtr it

In 1993, thespermarccdtdeclinedfrom>4%to<1%asthe season progressed (Figure2.3).Initially,the spermatoc ritwasrelativelyhigh duetothepresence ofa high percentage (>50%)ofbig,non-motilespermatidcells inthe early seasonalmilt samples (Figure2A),Asthespermatocritfelltolower values(IA-1.8% )duringthemiddle of spawningseason, thelarge spermatids had nearly disappeared(Figure2.5). Finally,the spermatocritfellto thelowest values by theend ofthe spawningseason.

10 12

Figure2.3.The variationofspermatocritduringthe199] spawn ingseason (Mean*SE, n=IO).Week0representsthetime of'onsetofs penniation(June22).

39

Figure2.4 A microscopicview (400x)ofamill sample (male E8) collected inJune1993

Figure 2.5 A microscopic view(400x) ofamiltsample (maleE8) collected in August1993.

40

2.3.5 Sperm eencentrsu en

The spermconcentratio nofoceanpoutmillranged betw een0.9 1 ·6.81x 10"

spzl ml(n"'"37).Ahhou gh nolinearrelationshipWallfoundbetweenspermconcentration andspermatocritfrom theanalysisof allJ7millsamples.linearity was eslablishedif samplescontainingahighpercentage(>50%)ofspermatids wereexclude d(Figure2.6;

~=O.79.P<O.OOOi.""'29).Thelinea r relationship is described by the equation.

Y""I.46H.48 X, whereX isspe rmatocrtt midYis spermconcentra tion (IO' /ml)

2.3.6l'l:nma llndrogenic sler oidprofiles

A studyofthe plasmalev elsoftesoserone (T)andu-ketotestosteroee(I I-KT) during the spawningseasonrevealed similarpaUems forboth hormones(Figure2.7).

Plas ma Tlevelsincreasedseveralweekspriorto theonsetof spermiation.brieflyreaching

•maximum approaching30nglml justbeforetheonsetof miltprod uction. AsTrapidly decreased. maximallevels ofIl-KToccurred at thelimeofonset ofspermiation.While the period ofspermiation contin ued.Tand II-KTlevels beganto decline, reaching low levelsforthe durationof ihe spawningseason (July10September).

41

6-

..

3 4

Spermatocr it(%)

Fib'llfe 2.6.Therelationship between spcrmatocnt(%)lin t!sperm conccnrreuonf1O"ml) for oceanpaulmilt TheS samples(circles)containing50"/.or hight...Ill'spcrnmlicbwerrc excludedfromtheregressionanalys is.

2.4 DISCUS SION

Thistwo-yearstudyclearlyshowedthatthereproductivecycleorthemale\lCe,1O poutischaracterized bydistinct seasonalvariationsin thegunudusomaticindex,SI'CI Ill

42

50

10

·10.{j -Q -4 .2. 0 2 4

Week

-+-Testosterone

m

-0- 11-keloleslos!erone(11-KT)

Figure2.7.Changes in plasmaandrogensin male ocean poutduringthe 19 9 4 spawning season(Mean±SE.""'6).Week0 representsthetime ofonsetof spermiat ionforeach ma le;negativevaluesindicateweeksbefore spermiatio n.

mot ility,spennatocrit,spenn concentrationsof milt, andplasma androgen levels.Atthe beginning ofreproductive development inMay, GSIandplasma androgens(TandII.KT) werelow.In June,a significantriseinGSIwas accompanied by increasesofbothTand 11·KTintheplasma. Just beforespermiation,T reached itsmax imumlevel before rapidlyfalling. The onset ofspermiationcoincidedwith maximallevels of II-KT and low spermmotility inmiltsa mplesch aracterizedby ahigh spermatoc rit and alarge

43

percentage ofspermatids.Astheseason progressed, theaSIvaluespeakedinJulyand sperm motilityimproved together withadecliningspermetccrit.and lowerlevels of T and II·KT.Inthemiddleof thespawningseason (Julyand August),sperm motilityreached its max.imumalongwitharange of[owspcrmatoeritand spermconcentrationvalues.and thedisappearanceofspermetlds inmilt. Spermmotility,spermconcentrations in themilt, spermatocrit,GSIandplasmaandrogen valuesall declinedlatein the spawning season (September and October).

BOyOkhatipoglu and Holtz (1984) notedthat sper mmotilityinrainbow trout improvesfromthebeginning tothemiddle ofspawningseasonbeforedeclining thereafter.

Asimilar patternwas notedforocean pout inthis study,with thebest sperm motility occurringinthemiddle ofthespermiationseason,insynchronywith the spawning

o r

females.Inhalibut, however,spermmotilityis betteratthe beginningof spawningbefore markedlydecreasing during the lasthalf ofthespawningseason,alongwithspcnn atocrit values reachingnearly100% (Methvenand Crim1991).Incontrast,thespermatocrit of rainbow trout(Billardetal.1971;Munkittrickand Moccia1987;Buyukhuti pcghrund Holtz1984),brown trout(Billard 1983c) andoceanpoutdeclinesasthespawningseason progresses.Inthe oceanpout,a highspermatccritvaluedocsnot indicate a highsperm concentration atthebeginning of thespermiation,butratherthepresence of a high percentage ofimmotile spermatid cellsin thesemen'.

'S e rnen~mi lt

44

Alt houg hthe spermconcentration of milt canbe accurately determ ined by hemocytometer methods, this techniqueis too timeconsuming for routinemeasureme nts.

Alternatively, the spermatoerit has been used to measure sperm concentration infishmilt by manyauthors(Baynes and Scott1985;Munkhtrickand Moccia 1987; Garcia199 1).

Onthe otherhand,Suquet»tal.(1992) used spectrophotomete rmethods,reportingthat the spermatocrittechniqueisinaccurate for estimatesofthe spermconcen trationin the turbot.Afterthedisappearance ofspermatidsfrom ocean pout miltby themiddleof July, spermatocr itis anaccurateand convenientmeasureof sperm concentrat ion,whichis us efulfor accuratelydetermining theminimumnumber of sperm required for artificial insemi nationtechniques

Both sperm concentrationoffish miltand theaSIvaluesinteleostsarcrelated to themode of malereproduct ionand the spawningbehaviour(Billard 1986; Billar dand Cosson1990), Billard(1986) suggestedthathigh sperm concentrationscouldcompensa te fo rrelatively short spermmotilitytimesranging from 30 sec.to 30 min.and arcneeded for externa llyspawningspecies where fertilizationof eggs takes place inanopen environm e ntresultingin sperm beingquick lydiluted. Forexample.hig h sperm concentrationsrangingfrom10 • 55X10⁹spzlmr arcfound inthemiltof salmon ids (Clcrcseko andDabrowski1994),thecommoncarp(KrugerItt01.1984), the winter Flounder (Hamli nand Crim1993), andtheturbot (Suquetetat.1992), and evenhigher concentratio nsofsperm, 16.5 xlOwsptlml in semenof thebluefintuna(Doilital.1982)

45

and halibut (Crim

I!'

al.unpublished). In contrast,the interna l fcnihzarionof eggsin the ocean pout is associatedwilhbotha prolongedsperm motili tytime(days)and relat ively low sperm concentrat ions in the milt(I.7 x W spzJml),a range alsoreportedfo rthe internally fertilizing wolffish (Pav lovand Radz ikhovskaya1( 91).Also notedwasthe highGSI foundin males releasinggametesinopenor running water without parental care (B illardandCasson 19(0). Forexample, GSls varyingfrom7•16% werefoundin maletroutand carp (Billard1986),the winterflounder(Harminet al.19( 5),andAtla ntic cod(Trippeland Morgan 19(4).However,insome speciesthe malesrelease gametesin protectedareas or directly intothe female bod yandinthesecases theaSI islow.For ins ta nce.theGSIrangesfro m 0. 1·2%inma leTilopiaspec ies (Billard1( 86) and the male wolffish(Pavlovand Radzikhovs kaya 1( 91).Like wise.the rela tively lowGSIin male oceanpout is related tothe copulativespawningbehaviour(VIto and CnmIQ9 Sb) Prev ious studiesof the relationship betweentheseasonal plasma and rogenprofile and reproductivedevelopmentof maleteloostshave usuallyfocusedon testo steron e and ll-ketotestostero ne.Between thesetwo androgens, II· KTis the predominan t androge n insalmonids(ScotteIal:1980;Kime and Man ning1982;FostieretClI.1982; ), winter flounder (Hann in

erat.

1995).and halibut (Methven and Crim19(1),while inthis study, Tisthe predo minantandrogeninocean pout. Severa lstudies(ScoIIet(/1.1'180;HuntI!I al.1982; Kime andMann ing 19 82;BaynesandScott 1(85)have show n thatT peaks beforeII·K T which corresponds to the spermiatio nseasonin salmonids.According tothe

46

oceanpoutandrogenic hormone profiles,ihepeak ofTis alsoreachedprior10 spermiation,followedby a peakof11·KTin conjunctionwith the onset of spermiation.

Whilepeak levels oftheandrogens, particularlyII.KT, coincide withthe period of spermiation insalmonids(Scott~Iul19OO,Fostier('ICli.1982;Kime and Manning1982), mummichog (Cochran1987),andwinter flocnder(Hamlintfal.1995), interestingly, thereisa briefandrogenicpeakinthe oceanpout withspermiationcontinuingforseveral weeks,similartothehalibut(Methvenand Crirn19 91).Wingfield andGrimm(1977), lindScott('Irtl.(19HO)suggested thatTplaysamajor roleinthelatestages of spermatogenesis.A majorroleof II·KTcouldbe incontrollingspermiation (Scottetal.

1980). Fosticret

at.

(1982 and1984) alsodemonstrated thatthe levelof Il·KT in plasmawaspositivelycorrelated withsperm production inrainbow trout duringthe initiationandarrest ofspermiation,whichindicatesthatII· KTmight play arolein the processof spermiationorsperm migrationinto the vas deferens.On the other hand,there was norelationshipbetween II· KT levels and sperm production inrainbowtrout (Hunt etal.1982;Baynes andScott1985). Therefore,Baynesand Scott (1985) considered that amajor role ofll-K'Tcouldbe in controlling thesecondary sexualcharacteristics.The resultsof thecurrent study,suggest that II-KTmight playa rolein initiatingspermiation rather than stimulatingsperm productionduetotherapiddeclinein plasmaII·KTjust aftcrthe onsetofspermiation,andthatT couldbe involvedin thefinal testismaturation inthemale oceanpoll!'

47

Someinvestigatio ns {Scottand Baynes 1982;Ueda('Ial.11)8.1a.b:Baynes and Scott1985;Ueda~IcII.1985) suggest thatlrc-hydrcxy. 20p.dihydroprogt.'StCfo ne(I7« . 20flP)plays a majorrolein the process of spermiation infish.However.inthecurrent study,17ft.20pPwasnotdetectablein oceanpoutplasma during the spawningseason (datanot shown). Sincemaleocean poutcontinue(0releasemiltcontaininghigh concentrationsof motile sperm forseveral weeks10110wing theandrog endecline.further studiesare requiredtodetermine thepresenceofothersteroidsand iftheyareinvolved inthe controlofspermiation andsperm motilityintheoceanpout.

48

CHAPTER 3

SEASONAL ASPECTS OF SPERM PHYSIOLOGY AND SEMINALPLASMA BIOCHEMISTRY IN THE OCEANPOUT

Ma crozoarces tnn ericanus

3.1 INTRODUCTION

A betterknowledgeof sperm physiologyand seminal plasmabioch emistryarc essentialto improvingartificial fertilizatio n procedures for !ish. To date, most studies of fishspermiation have focusedonthespermof salmo nid,cyprinid andafewmarin e specieswhich are allexterna leggfertilizers(Scott lind Baynes\980;Morisnwat'l(/1 19 83; Stoss 1983;Krugeret at.1984;Billard 1986;Billardn

ot.

1l)9J ; Suquct et(II 1993;Harmin and Crim1993; Lahnsteineret al.1994; Billarder at.I995b).In contrast.

studies of sperm physiology and seminal plasma biochemistryininternallyfertilizing teleostshave been limitedto observationson guppy(Billardand Cossou1990 ),ami sculpinspenn(Koyaecat.19( 3),Because oceanpoutsperm aretransferreddirectly into thefemale (Crimet al.1995;Vao and Crim1995a),itisbelievedthatsperm physiology in ocean pout wouldbedifferent from speciesreleasing sperminto the extern al enviro nment.

In the past, few studies of fish sperm physiology andbiochemistry havefocused seasonal aspects ofsperm production, sperm motility,andchangesin milt characteristics. Buyukh atipoglu and Holtz(1984)reportedthat spermproductionlind motility in rainbow trout increasedfrom the beginningto themiddle ofspawn ingseaso n and declined thereafter.Therewere seasonal fluctuationsinsperm concentration,motility.

pH, osmolality,andsome biochemical constituentsofsemen inCyprinuscarpioand Oreochromu mossamotcus(Krugeretaf.1984). In rainbowtrout,Munkittrick and

50

Moccia (1987)noted thatspermatocrit,spermmotility and seminalplasmaion concentrations declinedas the season progressed. No seasonalstudiesof sperm physiology ininternal egg fertilizershave beenreported. Althoughsome information about male andfemaleocean poutreproduction hasrecently become available (Crimet at.1995;Yao and Crim1995a;Vaoet al.1995).detailedstudiesof thespermiation response andthe biochemicalandphysiologicalcharacteristicsof ocean pout sperm throughoutthe spawning season remain to bereported.Furthermore,the extremelylong periodof motilityexhibitedby ocean pout sperm provides an excellent opportunityto study variousbiochemicalparametersin relationto fishsperm motility (Vao and Crim 1995a).

In this study, changesin sperm motilitywere followed inrelation to pH, osmotic pressure and thebiochemicalvariationsoccurringin ocean pout seminalplasma throughout the oceanpout spawningseason.

3.2MAT ERIALSAND METIIODS

3.2. 1 Collectio n of fish anti mile

Sec Chapter2 (2.2.1Experimentalanimals) for collectionandmaintenanceof fish.

Throughoutthe spawning period,milt sampleswere collectedweeklyorbiweekly from

51

maturemales byinsertinga polyethylenetubeintothespermductviathe urinogenita l pore.Atotal of 98milt sampleswerecollected from11males in [994.

3.2.2Determination ofspe r m motility

Spermmotility was estimatedfrom freshlycollectedsamples of mlltnccording 10 thepercentageofmotilespermatozoa (Chapter 2). To determinespcrmmotilityforfhe early, middle andlate season.aweighted mean of sperm motility classforeachseason was used. The weightedmean isthe meanspermmotilityclassweighted by the numb er offishin eachclass.Itrepresents the average ofthesper m motility class for ;\11 fish in each season

3.2.3 Seminal plasmaanalysis

Afte rcentrifuga tionoffreshlycollectedmiltat 3000x gforISminutes at4"C, theseminalplasma was removed anddivided intoaliquots for immediate analysisorfor storageat-20"C. The pH and osmotic pressurevaluesofseminalplasma were immediately determinedbypH indicator stripsbetween therangeof pH5 and10 (EIlI Science,Germany),andbyFISKEONE-TEN osmome ter, resp ecti vely. Focusingon6 males,the tota l protein(TP)concentration, the levelsofcalcium [Ca'"] ,magne sium (Mg"),potassium(K'),sodium(Na'),chloride(Cl),glucose(GLU),andphosphorus(P)

52

were measured afterthawing theseminalplasma.Thesesamplesrepresentedthree different phasesof thespawningseaso n:I) early season;the first sa mpleof theseason colle ctedin Juneatthe initiatio n ofspermiation,2) middleseason;samples collected during themiddle of the spawning seasonin July andAugus t,and 3) late season;the last sa mple collectednearthe end ofthespawningseason in September.Thetotal protein concentrationwasdeterminedusingthePierceMicroBCAProteinAssay (Pierce1992).

The concentrationsof calciumandmagnesiumwere measuredcalorimetrically usingthe SYNCHRON CX) System;seminalplasma potassium, sodium and chloridewere measuredbyindirectpotcntio metryusingionselective electrod eshousedina flowcell Glucosewasmeasuredin a reactioncup bymeansofapolarograp hicelectrode and phosphoruswas determined using the Phospho rusSystemPack forHITACHI 705.

3.2.4Deternnnatlonof the effectsof ptllindosmolality011spermmotility

In the middle of the spawning season (August)afterpoo lingmiltfrom ) males containing highlymotile spe rm (>75%).aI mlsample wascentrifuged1000 x gfor10 min.at 4"C(noeffecton spermmotilityfollo wingsuch treatment) .The seminalplasma wasremove dandthespermpellet mixtureresuspendedin I mlof adiluent (183mM NaCI, 10.25mM KHCO"1.45mM CaCOl,0.84mMMgSO~'71-\0, and0.15 mM glucose)whichpreserve s ocean pout spermmotility (Yeoetat.unpublished),adjusted with NaOHor HCIat vario us pHsfrom 5-10and osmolality values of 320•350

53