INFORMATION TO USERS
This manuscriptbasbeenreproducedfrom themicrofilm master. UMI films thetext directlyfromthe original or copy submitted.Thus.some thesisanddissertationcopies areintypewriter face, whileothers maybe from anytypeofcomputer printer.
The quality ofthisreproductionisdependentupon the qu.uty or the copysubmitted. Brokenorindistinctprint, colored or poor quality illustrationsandphotographs, print bleedthrough. substandardmargins, andimproperalignment can edversely affect reproduction.
IntheunlikelyeventthattheauthordidnotsendlJMIa complete manuscript and thereare missing pages. thesewillbenoted. Also.if unauthorized copyrightmateriaJhad toberemoved, anotewillindicate the deletion.
Oversizematerials (e.g.,maps,drawings,charts)arereproducedby sectioningtheoriginal. beginning at theupper left-hand comer and continuing fromlefttorightinequalsectionswithsmalloverlaps. Each originalisalsophotographedinone exposureandisincluded in reduced Connattheback of thebook.
Photographsincludedinthe original manuscript have been reproduced xerographicaUyinthis copy. Higherquality6"x9"blackandwhite photographicprints are available foranyphot ograp hs or illustrations appearinginthis copy foran additional charge.Contact UMIdirectlyto order.
UMI
ABeD.t.HowelllnfimnlltionComp:my 300NortbZOebRoad,AnnA1boI" MI 481(l6..1J46 USA
3131161-4700 800JS21-D600
AN lNVESTlGATION OF BATCU-8PAWNING REPRODUCTIONINCAPTlVE YELLOWTAll. FLOUNDER.Plellrotlectes!errugin eus.
BY
ANTHONY 1. MANNING.B,Se
Athesissubmittedtothe SchoolofGraduateStudies inpartialfulfi1mentoftherequiremen ts
for the degreeof MasterofScience
DepartmentofBiolo gy&OceanSciencesCentre Memorial UniversityofNewfoundland
Sept1996
St.John's Newfoundland
ABSTRACT.
The batc h-spawnin grep rodu ction offemaleyeUowtail flounder ,Pleuronectes ferrugineus,was examinedina captivesetting overthespawni ng period s of1994 and
1995. Individual female yellowtail were examineddailywiththeobjectives of determiningtheinter-ovulat ory periodofthis species.as wellas,theannualegg productio n,mean batchfecundityand number of batches perfemale.Eggqualitymeasu res (fertilizationandhatchingrates) were alsomade onbatchesfrom females toquantify maternal differences and inter-bat ch variat ionwithin females in egg survivalrates
A one-dayinter-o vulatory period waspredominantin this speciesand the frequency of this period increase din1995duetoreduction s inirregularovulatoryactivity detectedin certainfemalesin1994.The meandurationofspawningforindividualfemales ranged betwee nJI to48days,over which time eggdiameterand dry weight were observedto decreasewithinindividualfemales
Female yellowtail flounde rdemon strate d considerable individual vari ation in seaso naleggproductio n.In1994,the mean femaleproductio nwas550000eggs (7.9x10leggs'kg" spawni ngfemale). in1995this increased toI187 000eggs(1.5x 10"eggs'kg"spawning female).The numberofovulationsper femalewas high, amean batchnumber of14wasseenin1994.rising to22 in1995.SmaIlbatchsizes ( 100000 eggs) dominatedbatch fecunditydistributions,andthe data suggestedthat females increase the numberof ovulationsratherthanincreasebatchfecund itywhen egg
Egg qualityinyellowtail flounderwasfound tobesubjecttomaternaldifferences In addition.femalesdemoostratedsignificantinter -batch variationinfertilizAtion and hatchingrates.Aninter·annua.Iincreaseineggquality was observed.wheremean fertilizationratesrose from] 8%10 60%.Thisincreasewasnotdue toslowero....er- ripeningrates since spawningtemperatures were notsignificandydifferen t betweenyears Theeggqualitydataalsodidnotdemonstrate anyperiod withinan individual femal e's spawning season in which the batches withthe highest qualityeggswereproducedA relation ship was seen betweengametepotential rates.determinedby eggmorpho lo gical characteristics,andfertilizationrates.However,since individuals had theirown separa te relationships.gamete potential ratescan only serve asarough estimate of egg quality, where fertilization rateweregenerally25%lowerthangametepotentialrates.Hatchin g rateswere.overall,higher than fenilizationratesandshowednorelationshipwith feniliza.tion rates
Investigatio nsonever-ripening indicatedthat adaily examina tio nprotocol is requiredto avoid reductio nsin fertilizationratewhich were greatestduring thefirst24 hoursaftercollection.Hatching ratesdec lined lessdramaticallythanfertilizationrateswith over-ripe ning
"Theinter-annualincreaseinthe reproductiveperformanceforcaptive yellowtail flou nder mayhave beenbaseduponreductionsinstressfroman additio nalyear of acclimatio ntocaptive conditions or theintroduction ofa commercial feed in 199 5.
Eitherfactor may havebeen responsiblefor thereductionoffemales withpooreggquality andthe observed increases ineggproductionforspawningfemales.
iii
ACKNOW LEDG EME~
I wouldlike to givemymosthean-fd tthanks and appreciationto my supervisor
Dr.Laurence Crim whose advice and supportwas alwaysavailabletomeoverthecourse
ofthe project.Iwouldalsolike to thankthe membersofmysupervisory committee Dr.MargaretBunonandDr.JohnGreenfor theirhelpItld guidance.
Myappreci a tionisalsoextendedtoDr.PierrePepin forallo wingme tousehis image analysis system,Dr.RayThom psonandMs.ElizabethHatfield for giving me access to amicrobalance.Ms.ConnieWilson fo rhersupportinthe laborato ry.Mr.HongChen fo rhisstatisticaladvice.Mr.RossWilson for his advice onfishcare,and all thesuppo rt staff attheOceanSciencesCentre.
Aspecialmentio n ofthanksis duetomy fellowlab- mates and graduatestudents, Stephen Bettles. Susan Clearwater.JoakimLarsson.Burnin Shangguan. Zhi1anWang. and Dr.Zuxu Yac,asweUasDana TaylorandRomaOsmond.fortheirhelpand comradeshi p.
last but not least, I wouldlike tothank the CanadianCe ntrefor FISheries lnno vation (CCFI)andFisheriesProductsInternational(FPI)for theirfinancial supportof theproject,inadditionto the SchoolofGraduate Studies ofMemorialUniversity of Newfoundlandforitsfunding contribution
TAB LE OFCO NTE NT S
ABSTRACT...
ACKNOWLEDGEMENTS...
UST OF TABLES. LISTOFRG URES .
Part1.Il'ITRODUcnON...
Part2.METHODOLOGY...
Part3. BATCH-SPAWNINGAND EGGPRODUCTION . 3.1. RESULTS ...,
3.2. DlSC USSION...
Part4.VARIABILITYIN EGGQUALlTY.... 4.1. RESULTS...
4.2. mSCUSSION...
CONCLUSIONS...
REFERENCES...
page
vi viii
18 19
56
68 69
100
108 [II
TABL ES Pan3:
USTOFTABLES
page
Female spawningrecordsfor-the1994and1995spawning seasons... 20 Meanmrer-ovulatoryperiodsandmost frequentintervalsforindividual
femalesforbot hseasons.. 34
Meanvalues for prespawninglengthandweigh t.numberofbatchesovulated.
spawning duration. eggproductionandweightlossforspawningfemalesin
1994... 35
Mean values forprespawning lengt h andweigh t.number of batchesovulated.
spawni ngduration.eggproductionand weightlossfor spawningfemalesin
1995... JS
Prespawninglengthandweight,numberof batch es ovulated, spawningduration.and eggproduction informationforindividualfemales
inl~. . 37
Prespawni nglengt h and weight,numberofbatches ovulated.
spawningduration.andeggproduction informationfor individualfemales
in1995.... 38
Batch numberandmeanbatchfecun dity informa tionforindividualfemales
in 1994 and 1995... . 41
Anova and randomizationtestresultsexaminingforaninter-ann ual effecton
the batch fecundity dataofrepeatspa wners... 44
Decreasesinegg diameteranddry weight overthe spawningseasonsof
1994females... 49
10 Totalestimated dryweight ovulatedby individualfemalesin1994... 49 II Compariso nofthe1994eggproductiondatawithfecundityestimates
forwildyellowtailflounder females.... 67
Part 4:
Meanvalues forgamete potentialrate,fertilization rate,hatching rate, larval production,[he differencebetweengametepotentialand fertilizationrates(GP-FR )and the difference betweenfertilizationrate and larvalproduction(FR·LP)for the spawningfemalesin1994.. 69 2. Meanvalues forgametepotential,fertilization,hatchingand larval
productionratesfor individualfemalesin 1994(N=number ofbatches
which werefertilized)... 70
Mean valuesforgamete potential rate,fertilization rate,irregu lar cleavage rates,andthe differ encebetweengamete potentialandfertilizationrates
(GP-FR) forthespawning femalesin1995.... . . 71
Meanvalues forgametepotential,fertilization,and irregular cleavagerates for individualfemalesin1995(N=numberofbatches which werefertilized). 76 Anovaand randomizat ion test resultsexaminingforan inter-annualeffect on [he fertilization rate data of individualrepeatspawners(N=samplesize
per season).... 85
6. Anova results examiningforan inter-annualeffectontemperature conditions experiencedbyindividualrepeatspawners... 86 7. Mean number of daysofegg storag e,priorto fertilizationrates decreasing
to<10%,lin ed for eachinitialfertilization rate class... 97
FIGURES Part2:
LIST OFF1GURES
pag'
Phot ograph of one ofthreetankscontaining1994 spawners showing the standpipeguard whichwasplacedovertheelevat ed standpipetoprevent
theloss of any spontaneouslyspawned eggs... 17
Part 3:
Meanbody weight(g)changesforthesix malesandten femaleswhich
survived from Sept199310SepII99S.. 23
2. Dailyeggyield and temperature reco rds for the 1994 spawningseason... 26 Daily egg yield and temperatu rerecordsfor the199 5 spawning season.. 28
Comparisonof theseasonaltemperatu re pro files forthe 1994and1995
spawrVngperiods... 30
5. Frequencyhistogramsrepresentingtheintcr-owlatoryperioddata observed fromthe brood stockduringthe1994 (n"146)and1995(n:209)
spavwningseasons.. 33
Frequencyhistogramsofbatch fecundity dataobserved among
the broodstockduringthe1994and1995 spawningseasons.... 43 lme r-annualcomparison ofbatch fecundity distributionswitruntworepeat
spawners..., 46
Relatio nshipbetweeneggdiamet er dataandcorrespondingday inthe
spawningseasonofthe group in 1994 SI
Relationshipbetweeneggdryweight and corresp onding egg
diameterduringthe1994 spawning season... 53
10 Relationshipbetween eggdry weight andcorr es ponding dayin the
Part 4:
Variabilityin fertilization rate(%)data between and within females
during 1994... 73
2. Variabilityin hatching rate('Yo) data between and within females
during1994... 75
J. Variability infertilizationrate(% )databetweenand withinfemales
during1995.. 79
4. Temperaturedata andfertilizationrate(%)data of individual femalesasthey
were recordedover the1994spawning season... 82
5. Temperatur edata and fertilization rate(% )data ofindividual femalesasthey
wererecorded over the1995spawning season.... 84
6. Fertilizationrate('Yo) variabilityin the early.middleandlateperiod sofegg
production ofseven1994spawners... 88
7. Relationshipbetween fertilizationrate ('Yo)andgamete potentialrate('Yo)
forthe 1994 season... . 91
Relation ship betw een fertilizationrate ('Yo)andgametepotentialrate(% )
forthe1995season... 93
Relationship betw eenhatchingrate ('Yo)andfertilizationrate('Yo)for
the1994seaso n... 95
10 Meanchang es ineggquality(% )occurringwith over-ripeningforbatches
stored in vitroin an incubato r(n=21)... 99
Part1.
INmOQUCTION
Theyellowtail flound erisa batch-spawnin gflatfishof the familyPleuron ectidae Describedasanoffshorespeciesinhabltingthecontinentalshelfof theWestern North Atlantic.itsgeogra phic rangeextends from Labrador toChesapeakeBay(Walsh. 1992).
Thisspecies iseurythermi c. tolerating temperatures of-1-4 °C to 16°C across its geographic range (Colton.1972;Walsh.1992).Theyellowtailflou nder has aprotracted spawningseason which ischaract eristic of batch-spawners.Thespawning seasonmay begin in mid-Mar chandlast intoAugust depending onlatitude (Royceet al.,19S9~
Coltonet01..1979).Information fromtheyellowtail tlounderstocks of the Grand Banks of NewfoundlandindicatedthatspawningcanoccurinlateMay withpeak activity during thesecond halfofJune(Pitt.1970)
Batch-spawning(serialor multiplespawning)isarep rod uctive strategy where portions of the late-vi te llogenicoocyte populationofthe ovary are owlatedperiodically over a female'sspawningseason.Histological evidencefor batch-spa wning in yellowtail flounder hasbeendetectedbyHowell (1983) andZamarro (1991)wherelowpercentages of hyaline oocyteswere observedintheovariesofspawningfemales.Ovulato ryaetivily within a batch- spawn erfollowsatem poral rhythm.whichisdefinedbyan inter-o vulatory period.Zamarro(1991) proposed thatyellowtail flounderhad aninter-ovu lat oryperiodof oneday.Thisproposal waspartlybased on histologicalevidenc ewhichindicatedthai matu rationof ooeyteswascontinuous;asthe ovulation of one batchoccurs,the oocytes of thenextbatch in theserieshave alreadycompleted nuclearmigration.Howell(1983) revealedthatyellowtail flounderalso show the groupsynchronous pattern tooocyte developmen t,whereatleast: twopopulationsofOOC}1esatdifferentstages of development
canbedistinguished in the ovaryat anyonetime(Wallace&Selman,198 1).
Smigielski(1979) injected femaleyellowtail Rou nder with pitu itary extract prep arationswhichinduced maturationandovulatio n.Adosage of2mg of pituitary extract perkg ofbodyweightwassuccessful onlywhenthe gonadosomaticindex measurementsreached 20%.whereGSI was determined fromasubsampleof control females.Inducedfemal es ovulatedoneto fivetimeswith aperiodicity of oneor rwcdays Theinduc ed eggsdemon strated fertilizatio n ratesranging fromSO%to 90%and hatchingrates of40 0/.to80%.
This projectisthefirsttoexaminethe femalereproduction ofyello wt ailflo under witho ut anyartificial stimulatio nof maturationandovulatio n.Yellowtailflounde rhasbeen commer ciallyimportantinthe Newfound landfishery and is a poten tial candidatefor aquaculturedeveJopment.Information onthereproductivebiology ofthis speciesis thereforerequired forpro per broodstock management in aquacultureTheaimoft Nstwo year studywas toexaminethe batch-spawningreproductionofyeUowtail flounderina captivesetting.
Thethesisisdividedintotwo sections, the firstisconcernedwitheggproduction Of part icular interestis the frequency of ovulatoryevents andthe pane mof egg produ ction associa tedwith thebatch- spawnin g strategywhichcanbedescribedbythe number and fecundity ofafemale ' s batches. Theseco ndsectio nconcentrate s on egg qualityinyello wt ail flound er.Eggquality as generallydefinedbyKjersviketal.(1990 ) is
descriptorsmay includeegg size,egg mechanical strength.biochemicalcomposition.
egg survivalrates or the rate ofsynunetrical cleavage oftheearly blastome res (Kja rsvik et al,1990)
Studyingegg quality isimportant as itindicates the efficiency by whichthe diversion ofmatemalenergy resources tothe gonad results in thesucce ssfulproduct ion of progeny.Theobjective of theexaminationofeggquality inthis studywasto assessthe successwithwhich the eggs of yellowtail flound erareableto complete the fertilization andhatchingprocesses.Oftenneglectedtopicsinthe studyof batch-s pawn ersare the matemaldifferenceswhichexist ineggquality,in additiontothe inter-batc hvariation which may bepresent withinafemale.Theseissues were oneof theprimary concernsin theinvestigation ofegg qua lityinyellowtail flounder.
Thespecificobjectivesofthis studyofthe batch-spawningrepr odu ction of the yellowtail flounderare listed below.
A:Egg production
(I)report the timing of reprod uctio nandtheenvironmentalconditionscoinciding withovulatoryactivityincaptivity.
(2)determinethe inter-ovu latoryperiodforfemaleyellowtailflounder.
(3) estimate theannualeggproducti onand thebatch fecundityvariation forindividual females.
(4)examinethechangesineggdiameterand dryweight over a female'sspawni ng
B:Eggquality.
(I)determinetheoverall performance ofthe groupineggqualityparameters (2)test the use ofeggmorph ologicalcharacteristics aspredictors ofeggquality.
(3)determineifsignificant maternaldiffer encesineggquality occu r.
(4) determineifindividuals showconsistencyintheir egg quality betw eentheir batches (5) evaluate whetherindividual femalesproduced theirhighestqual ityeggs duringa
particular time period intheirspawning season.
(6) assessthedeclineineggquality withover-ripeningintheeggs ofyeUowtail flounder
Part 2.
MEmopoloey
Brood stpckmanagement
Agroupof seventeen females (labelled A-Q) were hdd in captivityfortwoyears priortothe startof thisexperimentwhichwasconductedattheOceanSciences Centre (OSC)of MemorialUniversity of Newfoundland.lbe6sh wereheldin an exterior SOOOI tankwhen theyexperienced naturallight conditions.Thetank was aeratedandreceived a water supplywhich was heatedduring the winter months to ensure a minimum water temperatureof approximately+1"C.Temperature data forthetank were record ed daily FromSept 1993tomid-May\994,the experimental fish were feda shrimp dietthree times a week,where over the nine month feeding periodfemalesreceived a meanration of 1.47kgfish".The rationlevel for the group was based onthe reportby Collie(1987) which estimatedanannualconsumptionrateof 1.4-1.6kg yr":fish"·for wildyellowtail
When thefirstovulating female wasdetectedinMay1994,the brood stockgroup wastransferred tothreeinterior4001tanksThetankswereaeratedandhad an approximatewater nowrateof 10 Imin".Lightconditionsincluded indirect natural daylightandartificiallight baseduponthenaturalphotoperiod .Duringthesummerof 1994,chilledandambient water supplies weremixed and provided a water supplywith mean daily lanktemperatureswhichranged from4.1
"c
to12.8"c.
Seven males werekeptamong the seventeen femalesto providepossible pheromonal cuesforreprodu ct ivedevelopmentand ovulation.Six to eight fish were allocatedtoeach tank. which corresponded to a stocking densityof approximately4.5to
The tanks used in 1994 had outflowstandpipeswhichdrainedwaterfrom the surface.Thus,acylindrical tube.taller thanthestandpipeand with a perforatedbottom, was placedover the standpipe to ensurethatspontaneously spawned, floatingeggs could bedetected (Figurel).The surfaceof the waterand the bottomof thetankwereskimmed daily withafine meshednet for evidenceof naturalspawning.Thetanks used in1995 had abottomdraindesign,however.sinceno spontaneo uslyspawned eggswerefoundin 1994.a bottom drain design wasconsideredtobe acceptable
Followingthe1994 spawning season,fifteen survivingfemales andsix surviving maleswere transferredtoan interior2000Itank.Thebrood stock experiencedartificial light ingwhich reflectedthe natural photoperiod cycle.Feeding ofthe shrimp diet.three timesaweek,was resumedinSeptember,until the middle of December when a formulated moist pellet.manufacturedby Connors Bros.,was introduc edwith the aimof giving thebroo d stoc k a morenutritionallybalanced diet. Food consu mptionby the experimentalgroup between Sept 1994and thestartofthe 199 5 spawningseason in June averaged1.42 kgfish".
When ovulationwasdetectedin1995.feedingceased andthirteenof theoriginal females available forstudy in1995weretransferr ed tothree 400I tanks.Asin 1994.
males were keptwithfemalesto provide pheromonalcues.Anambientwatersupply was available overthespawningseason where mean dailytemperaturesrangedfrom 2.6°Cto
Examinationprotoco ls
Femaleswhich had notyetovulated andhadswollen,finn ovarieswere chec ked
once or twice a week.Checking females involveda brief handling at the water surface,if theovary region felt soft or spongy,the female couldbestripped.Strippingofeggs requiredthe removal offemales fromthewater. whereupontherepea ted applicationof gentle pressurealongtheovary towards the gonopcreproduced freelyflowing eggs.Egg batche swere collected in prechilledtOOmIbeakers,labelled and thensetonice.Ovulating femaleswere chec ked(and/o r stripped) daily,usuallybetweentoam andIIam.until their ovaries werethin and Oat, and eggshad not been obtained for one week-lnitially in 1994,netswereused toexamine femalesbut their usewas discontinuedafter tail infections appearedinthree females.Fish were thencaptured and handledwithgloved hands sincenets could notbe used
Miltfrom twoorthreemales was collectedalong with the egg s.Males were remo vedfrom thewater and the area aroundthe urogenitalporewas driedofsea wate r Milt, whichwasexuded fromeach male by gentlepressureaboveand posteriorto the urogenital pore,was immediately aspiratedfrom the poresurfaceinto separate prechilled onecc syringes.Care wastaken to avoidcontamination of themiltsample with seawater or urine.The miltcontaining syringes were carefullycappedand placedinice.
After collectio nofthegametes,eggand miltcollections wereimmediately processed, with eggquality procedur eshavingpriorityin theproces s schedule Estjmatjo ogreg g prodyctioo
For thisstudy a batch was defined as a5mIor greateregg volumewhichwas
1995).Aftercountingeggsunder adissectin gmicr osco pe. a mean of me triplicates was calculated andwasconvertedto aneggconcentrationvalue(eggspermI).Batch fecundity wasdeterminedbymultiplyingeggvolumebythemeaneggconcentration. Totaleggproductionwas determined fromthesumofthebatch fecundities Eggqualityassessment
Every eggbatchin 1994underwentanassessment ofeggqualitywhichwas quantifiedaccordingtothe foUowingparameters:gamete potential rat e,fertilization rate, and hatchingrate.In1995, although gamete potential ratewas again detenninedfor every batchovulatedfroma female,theeggquality assessmentworkwasred uced,inorder to conserveincubatorspace,byonlydeterminingthefertilizatio nand irregular cleavagerates of a subsam pleofeggbatchesprodu cedbyany female.The two criteriafor determining which1995 egg batcheswouldundergo fertilizationtrialswere:
I)only batches withavolume~10mJwere fertilized;
2) providingthevolume exceeded 10mleverythirdbatchovulatedby a femaleunder wentevalua tion,otherwi se thenextbatch~10mJwas used
Some additional batches underwent fertilization trialswhich did not meetthese criteria.eitherbychanceorbecausecertainextra batches werevery large andrepresented a significantproponionof.female'sprod uction.Thirteen extrabatchesweredistributed amongsixofthe199 5 femalesasfollows ,wherealetteridentifies anindividual female: A (2).0(2).H(2).I(I).M(2).Q(4).
Gamete Posennat Rate -Threesamplesof 100unfenilized eggswere visuallyassessed undera dissecting microscopeandthemeanpercentageor"goodquality"eggswas
10
recorde d.Good qualityeggs hadatransparent cytoplasm,appearedsphericalwithno damageor dimples. and hadno prematureperivitellinespace.There appearsto be agreementon thesecharacteristicsof"ge ed quality"eggs among authorsof studiesof several other species,including. goldfish,Carassivsauratus( Fcrmacionet aI., 1993);
turbot.Scophthalmusmaximus (McEvoy,1984;Fauveiet al.,1992;Omneset al.,199 1);
andJapanese flounder,Pleuranectesyokohamae (Hiroseet al...1979) FertilizationRate-The fertilization techniquewas modeUedafterthe protoco lof Harrnin&Crim (1992).Fresh milt collectedfrom2-3males was combinedonlyif motility was verified undera compoundmicrosco pe.Three aliquotsof200-300eggs (volumeof 125~1)wererandomlysampled fromeach egg batchusingan Eppendorfpipette.The aliquots were placedinpet ridishesset onice.andfive microlitresofundiluted milt were then addedto eachaliquotof eggs. Sperm activationwas facilitatedby stirringin125~Iof chilledseawatersolutionfollowed byanadditio naltwomillilitres after two minutes.The seawatersolution consistingof I urn filte red,UV sterilized seawaterwith concentrations of 0.1gl-l'streptomycinand0.06gl-1.penicillin.Afterten minutes,20-25m1of seawater solution was addedto thedishes whichwere thenplacedin anincubatorset at 5"C.The fertilization rate was determinedfoUowingincubation of the eggs tothe 4.8 or16cell stage ( approximately6 to 8hours)under an Olympusdissecting microscope at10- 15X magnification.
In1995,thefollowingrevisions weremadeto thisprotocol:1-becauseeggsize
volume.2-Given that the durationofyelJowtail sperm motility islessthantwominutes the 10 minutepostaetivationperiodwas shonened to SminutesThis reductionintime was notconsidered to have an effecton the results of fertilizationtrials Hatching Rate- After the fertilization rate hadbeendetermined theeggswerethen returnedto the incubatoruntil hatchingoccurredwhere the incubator temperature was monitored daily.Renew al of the seawatersolutioninthepetridishes occurredevery two daysas was doneinChambers&Leggett(1987).The numberof larvaethathatched was det erminedandexpressedasa per centage offertilizedeggs.
Larval Producti onRase- This term was calculated bymulliplyingtheaveragefertil ization
rate andtheaverage hatchingrate ofabatch(lP'1.-FR%x HR %/100).Thisrat e indicates thepercentageoftheeggs in a batchthatsurvived to hatch.
Irregu larCleavage Raie-The percentage offertilizedeggsinthe earlycelldivisionstages (ie4 to 16) whichshowed asymmetrical divisionof ceUs(orirregular deavage) Ovcr-riOOl jOgSD.!dy
InI99S.theeffects of ageingoneggquality were determined on batcheswith gamete potentialratesexceeding 70%,lmmediatdyaftercoUeaionof batches.eggswere sampledforthe determination ofeggqualityparam et ers including gametepotential.
fertilization.irregularclea vageandhatching rates.Afterwards.a100mIbeaker.covered with perforatedParafilrn,wasusedtoholdaonecentimetredeepsampleof eggs froma batch.whichwasthenstored in an incubator wheremeandailytemp er atur es rangedfrom 6.9°C to 8.8°C.The eggsinthe beaker underwent samplingforeggqualitytesting after havingbeenstoredinthe incubator for 24hou rs.48hours. 72 hours.etc.untilthe storage
12
periodhadexceeded 96hou rs.lftheeggfertiliza tion ratefell to<100/.before the 96 hour limit. samplingwasterminat ed.
Eggdiamet er andeggdryweightmgsureroents
Asampleof fresh unfertilizedeggswerepreserv ed inasolution of5%formalinin Bo rax(sod ium tetraborate)bufferedseawater (Pepin,per'S.comrn).Themean diameter was obtainedfrom20eggsusing Optimus Image Analysis software wit ha Hitachicamera at2S0Xmagnificati on.Addit ion alimageanalysis softwareincludedFrameGrabber, Imaging Techn ologyOFG.
Theuse offormalinas afixative and preservative cancausechang esin theegg size.Changes indiametersof fresh eggsafter preservati on wasdetermined fromsamples oftwentyeggsfromfour different females eitherfreshly measured ormeasured after 30 daysof storageinthe formalinsolution.Measurementsof eggdiametersin this investiga tio n were made usingan ocularmicrometer on adissecting microscope at40X magnification.
Eggdry weight was determined forsome batchesfromfemales.Generally very smallbatchesor batches withverypoor gametepotential rateswerenotused.Small pieces ofaluminiumfoilwereplaced overnightinamum e furnaceat 450°Candthe averag e weightobt ainedfrom twomeasurem ent s on aMenlermicrobalanceto 0.1ug.Eggs were first water-harden ednolon ger thana dayafter collectio n.A sampleofeggsfromthe floatin glayer of wat er-hardened eggs was poured into avacuumfilter apparatu sunder
surface of thefilter.Next.theeggswerewashedofsurface salts with a0.9%ammonium format esolutio n(WhyteetaJ.•1987),followedby distilled water.Threereplicat es of 6ve eggsrepresen tedabatch.After placingfiveeggsonaweighedfoilusingsmallforceps.
the foil sampleswerele ft overnight in adrying ovenat 100"C.Foileggsamples were storedin a desicooler until theyreturned toroomtem pera tu re andwer e readyfor weighing. Again, eachfoil-egg sample wasweighedtwiceon themicr obal anceandthe meaneggdrywei ght wascalculated from the weightdata forthethreereplicate fo ils Stat jst ical Ana ly sj$
Statistical analyse swer eperformed using the Sta tistic al AnalysesSystem (SAS).
Residu alsfromallanalyseswer e test ed fornormalityandtransfo rmat ionsoft he datawer e done when deviat io ns from normality were indica ted.Linearrelatio nshi psunderw e nt regression analysis., if normal ityproblems were seen onlya Iogarithm.ictransformationor squaring the data was attempted.
Statisticalanalyses wereundertakenusing the fenitization rateand hatching rate datain orderto examine matenW differencesinegg quality (maternal effects)and the degree of inter -ba tchvariationwhich occurredwithin females (batch effects).Maternal effects weredete rmin ed by nestedanovas., where batc heswithinfemales served as the nest ed variableinthe model. Nested anovas weredone wit hthe Gene ral Linear Models (G L M) procedurewhich isro bust10the effects of unbalanced data sets.Unplanned paired comparisontestsincluding theDuncan' sMultipleRangeTest, theTukey's Stude ntize d RangeTest(HSD),theBonferr oniT-test and theLeastSquare Means testwere don e in conjunctionwithnest edanovastodetermine whichfemales couldberesponsiblefo r
14
significan tmaternal effects.Batch effects withinfemaleswereanatysed byone-wayanovas as were comparisons examining inter-annualeffects.Forallanava situations, randomizationtests werealsoperformed asanon-parametric alternative..whereeach test was represented byI000randomizationsof the data.
In1995,duplica teanalysesweredone fortheeggquality data.The first analysis examinedthedata set which strictly adheredtothesamplingcriteria for batchesto be testedforeggquality.The second analysis tested datasetswhichincluded the extra batchesco llected outsid e thecriteria. If differencesbetweentheduplicateanalyses occurred, the resultsofboth analyseswerereported.
Sincetheselection of batchesforeggqualitytesting was modifiedin 199 5.
differencesbetweensamplingofbatches betweenyearshad toberemovedprior to testing for inter-annual effectsineggquality.Therefore, datasetsfor1994females were reconst ruct ed, wherea sample of the data was selectedusingthecriteria usedin199 5 Balanced datasetsforinter-annualcomparisons were ensuredbyrandomlyselecting additionalbatchesof~10mIfrom the 1994 data set when needed
Figurel.Photograph ofoneof threetankscontaining 1994spawnersshowin g the standpipe guard whichwasplacedover the elevatedstandpipe[0 preventthe loss ofany spontaneo usJyspawned eggs.Ifspontaneous spawning hadoccurred floating eggs could be ccllectedwith afine- meshednet hoursafterthespawning event.
Part3.
BA TCH- SPA W N ISG ANDEGGPROP U CDON
3.1.RESULT S Spawnjng reco rd
Prior to eachspawning season.the spawningconditionofthe females was assessed by examining thedegree of swellingof thegonadalregionofeachindividual (TableI).In 1994,threefemales(N,P,Q) werenon-reprodu ctive.a statecharacterizedby thin.flat ovaries.The 1994groupspawningseason began onMay 13andended Aug29, a durationof 109 days.Elevenofthe fourteenpotent ialspawners ovulated.seven ofwhich initiatedovulatio nwithintwoweeks of thesummer solstice.The remaining three potentialspawnersincludedfemales1andK.,which dieddue to tailinfection sbefore ovulating, andfemale G.which remained swollenthe entireseasonbut did not ovulate.
In1995,thirteen females of theoriginal groupremained in the experiment.Asin 1994.threeno n-r eproductive females were observedin thegroup(E.L.P).Theremaining ten femalesallovulated in1995,seven ofthese females wererepeatspawnersfrom1994 The three non-repeatspawnersincluded female G and two of the non-reproductive femalesfrom 1994(N&Q).The first ovulationwas detectedonlune14,andthe last occurred onAug30 (n'"'78days).Most 1995spawnersinitiated ovulationinmid-July (July (0-23),whichwas later thanin1994.In addition.fiveof the seven repeat spawners initiatedovulationten to eighteendayslater in1995thanin1994
TableI.Femalespawni ngrecords for the1994 and 1995 spawning seasons Spawning conditio npriorto the season is indicated foreachfemale.as well as the datesof thefirstandlast ovulatio nsforspawningfemales(Y-capableof reproduction ; NR-non-reproduct ive ;N/A-no t available;M - mortality;shaded areasindicate an absence of s awnin data
Spawning First Last condition batch batch
1994 1994 1994
A Y July 5 Aug5 Y
B Y July15 Aug16 N/A
C Y June 22 Aug 29 Y
D Y JulyI Aug' Y
y May13 NR
·F Y
G y
H Y
Y Y
K Y
Y
M Y
N NR
0 Y
NR
Q NR
20
Increasesinmean bodyweight for femalesoccurredoverthewinter monthsand peakednear spawning(Figure I).Large decreases inweightwere notedforfemales over the spawningseason. Malesalsoshowednotabledecreasesinaverag e bodyweightover the1994 spawning season.Theaverage femaleweightatpostspawninginSept1994 exceeded that of Sept199 ].Howev er.theaveragepostspawning weight ofthc same femalesin Sept1995was relativelyunchangedfrom thatofSept1994 (FigureI)
Examinatio nofthe length dataforindividual femalesrevealedthat somefemale didnot grow.while othersgrewat ratesof 0.5to ].5em per year.In bothyearsmean growthratesremained at approximatelyOneemperyear.In1995.length increases were notalwaysaccompanied byincreases in body weight.
Regardingnon-reproductive females,inthefuseseasonthesefish demonstrated increasesinbody weight.However.in1995non-reproducti vefemales demonstrated decreases inweight.
FigureI.Meanbodyweight(g) changes forthesix malesand ten femaleswhichsurvived fromSept 1993 to Sept 199 5.Error barsrepresentstan darderro rs
~ -c
~ '" ~
~l'
::1I <
I
~
\ ::1\
\\
;
\ c
\
I 0
I I I
I -c
I I I I I
II
::1--
-c
\ ::1
\
\
\
\\\\
~
c\ Z
\
\\
o
DailyEggProductio n and Temperature
Peak egg productio noccurredduring midJulyto early Augustinboth1994 and 1995 (Figures2&3).Priortothedetection of ovulat ionineither season,temperature fluctuationsrising to 6"C wereobserved andmay havehada positive effect onthe initiationof ovulationin the brood stock.However.temperatures decreasing below4"C.
in 1994. or 3"C. in1995.hadnonegativeeffect onovulationin early spawners(Figures 2
&3).
The mean temperaturesovereachseasonwere similar.inthefirst yearamean of 8.1O:i:2.65 "C was recorded, while the second season hadameanof8.14:i:2.36
"c.
No significantinter-annual difference wasdetectedby statisticalanalysisintemperatu re variationbetween 1994 and 1995 (Figure4;P=O.8863).24
Figure 2. Daily egg yield and temperature records for the 1994 spawning season.
Thebar plot showsday today changesinpooled eggvolumes among the spawning females. Linear plotsare mean daily temperaturesforeach of the three holdingtanks the1994 spawning season
26
Figure3.Daily egg yieldandtemperature reco rds forthe1995 spawningseason.
The bar plot showsday to day changes in pooled egg volumesamongthe spawning females.Linear plots aremean daily temperaturesforeachof the holding tanks over the 1995 spawning season
28
Figu re 4.Comparisonof the seasonalternpera tueeprofilesforthe 1994 and 1995 spawningperiods.Daily mean temperatures are plonedfor1994 (do n edline) and 1995 (dashed line) from mid-Maytothe end ofAugust.
30
Inter -ovulatoryperiod
In1994 ,a oneday imer-c wlatoey period was themostfrequent. accou nting for 43 0/.of thepooledcases (0-146) (Figure 5).Atwodayintervalfollow ed,accounting for another 220/.ofthecases.Allother intervalshadrelative frequenciesbelow 10% Evidenceof irregularovulatory activitywasobservedbylarge inter-ovulato ry periods (maximum39days).Females demonstratingirregular ovulatoryactivityhad high mean inter-o vulato ryperiodswith large standard deviations (Table2).Ofthe1994females.
subjects Land 0wereresponsibleforthelargest intervalsobservedin the group(14to 39 days;Figur e 5).Sixofthe elevenfemales hadaone day intervalastheir most frequent period(Table2).
In1995, thefrequencyof a one-dayinter-ovulatoryperiod increasedto74.5% whilethetwodayinterval decreased to17.s%(FigureS).Irregularovulatoryactivitywas greatlyreduced amongthefemalesinthesecondseason.For nineofthe ten females.aone dayintervalwasthemost frequent(fable 2).Female N wasthe only femaleto demonstrate irregularovulatoryactivitywithintervals of6or7days.Allrepeat spawners showed decreases inmeanintcr-ovu!atory periodsin1995 (T able2)
Figure5.Frequencyhistogram srepresentingtheinter-ovulatoryperioddat a observed fromthebrood stock duringthe1994 (n=146)and 199 5(n=209) spawning seasons.
Table 2:Mean inter-ovulatory periods andmost frequentintervals for individual femalesforbothseasons shaded areas indicate an absence ofsoawnina dara}.
1994 Spawningseason 1995 Spawning season
,
mean s.d. Most frequent mean s.d. Most frequentinterval interval
A 1.7 ±1.0 I 1.2 ±OS
I
1B 4.6 ±2.2 5& 6
C 3.6 ±3.5 I 1.8 '1.1
I
1I
0 54 ±7.4 4 1.3 '1.1
I
1I
E 1.7 ±1.1 I
F 2.1 >2.5 1 1.2 ±O.4 1
G 1.2 ±O.4 1
H 2.2 ±1.7 1 1.6 ±1.0 1
1 2.0 ±1.7 2 1.1 ±O.5 1
L 7.9 ±7.3 2
M 2.8 >2.1 I 1.2 ±OS
I
1I
N 50 '2.5
I
7I
o
119.5 ±17.71 AlldifferentQ 1.6 ±O.7 1
I
34
Egg Pnx!uet ion
The rangeintotallengthforthespawni ngfemaleswasJJto44.5anin1994.The averagespawning femalemeasured 37anandweighed 694 g prior to spawning (Table]) In 1994.the grouppt"oducedan estimated6 047 316eggs..theavenge contributionper femalewas549 756eggs(7.9x 10'eggs"kg"spawning female ).This meanproduct ion was portio nedoutinto14batches. Individualspawningseasonslasted an averageof 48 days.the influenceof females wit hirregularovu lato ryactivityincreasingthemean.
Femaleslostanave rage of22%oftheirprespa wningbodyweightoverthe spawning
Table3.Meanvalues forprespawning lengthandweight.numberofbatches ovula ted.
s awninl.!duratio n.=I.!productionandweigh tlossforSD.llwn1n femalesin1994.
Season1994 Prespa wning Batches Spawni ng Egg %of
Length Weight Ovulated Duration Prod uctio n weight
(n-IIll) (an) (g) (days) log
Man 37 694
"
'8549756 22.0
s.d ±4.1 ±178 .7 '25 t:]53864 t:3.7
Table4. Mean valuesforprespawning lengthandweigh t.number of batches ovulated, awningduratio n.C22oroduetionandweieh tlo ss forsoa wnin females in 1995
Season1995 Prespa wning Batches Spawning Egg %of
Lengt h Weight Ovulated duration Prod uctio n weight
(n=lOll) (cm) (g) (days) log
Mean ]9.9 777 22 31 I186 881 2].2
s.d. ::1:].3 ::I:175
..
.10 ::I:596461 ::1:4.9In1995.thespawningfemaleswere larger.ranging from34to45em.witha mean total len gth of 39.9cmandmean pre-spawning weightof mg(Table4).Therewasan increased representatio nofmedium tolargesized femalesin1995.Females G.NandQ replacedfemalesB.0andEwhichweresmallfemales in 1994.The groupeggproduction increased nearl ytwo foldin1995toanestimatedII868811eggs,the averag eindividual contri buting I 186 881eggs(I.Sx 10'eggs.kg"spawning femal e ).The variation among thedifferent femalesineggproductionstillremainedhigh.Femal es . on averag e. ovulated 22batchesin lIdays.The averagefemalelost 23.2%of her prespawning bodyweight Female A.,a mediumsized femaleofl7em, had thehighestegg produc tionofl.3 millioneggs(Table 5).No tably.the productionvalues offe male s37cm or less. (C.H.I.
and A).frequently weregreater than the product ion of larger females,Furthermo re. the productionof female F exceededthatoffemale Eby3787eggs.yetfemaleFwas11.5 emlarg er thanE.Femal esB.0and0hadthelow est.eggproduct ion estimates .These femalesinterrupted ovulatoryactivity after five oreightbatchesand resorbed the remainingadvanced stage oocytesintheir ovaries.Thenumber of batchesovula tedby those femaleswhich completedtheirspawning seasons ranged from 8to27 (Table5).The highes t numbe r ofbatcheswasobserved from femaleIof3].5 cm.Alllarge femal es produced less than20batc hes.
The spawning durationforfemales was highly va riablein1994 (Table5).The first ovulatingfemale(E) completedher seasonin13 days.thesho rtest duration for1994.
Extre mely longspawnin g durationsof79 and 96 dayswere seen for females 0 andL.
respectively.Both cfthesefemales had demonstratedirregularovu lato ry activity intheir 36
inter-o vulatoryperioddata..
Table 5.Prespa wning len gthandweight.nwnberof bal chesovulated, spawningduration, d roducti inti . foindi·dualfemal . 994
ance. .., ucnoe crmancn r ~
'" ''I
s
PTespawning P'respawning Batches Spawning Egg1994 !.<ngth Weight Ovulated Duratio n Prod uctio n
(em) (g) (day»
A 37 739 19 J2 1324762
B 34 528
•
33 200454C 34.5 579 20 69 769990
D 40.5 882
•
39 206592E 33 444
•
13 489 792F 44.5 920 13 26 493579
H 36.5 7]6 19 40 75092 1
I 33.5 46. 27 52 723217
L 41.5
'64
13 96 34513 5M 42 880 17 45 671868
D 34 595 5 79 71011
In1995,the eggprodu ction ofbothfemales MandG exceededtwomillioneggs.
female M hadthe highest ofthegroupIt2as1304eggs(Table 6).Asin1994,the prod uctio n estimates ofsmaIlfemales,notably CandL weregreat erthanfemaleslarger than themsel ves.The smallestnumber ofeggsovulated byan individualin199 5was from femaleN,which ovula ted livetimes beforeresorbingheradvanced stageoocytes
Incontras twith 1994,six ofthetenfemales had eggproducti on estimatesgreater thanone million eggs(Table6).Whencomparin gthe results of theseven repeatspawners
five otherrepeat spawnersexhibitedlarge increasesinproduction.Female F,the largest femaleinbothyears, while demonstratingan increaseineggproduction. continuedto be exceeded in production by smaller femalesin1995.
The numberofovulatio nsperindividualwasgenerally higherfor 1995females (Table6).Batchnumberrangedfrom10to30 forthenine femaleswhich completed their spa wningseasons.Eightof the females had 20 or more batches.Female C had themost batches despitebeing the seco nd smallest femaleofthe group.This female also had the longest spawning duration of 52 days.Extremelylongseasonswere not presentin the 1995 group. The femalewiththeshortestspawni ng durationof15 days was femaleQ.the first to ovula tein 1995.Six femaleshad spawning durations between30and 34 days Table6.Prespawninglength and weight, number of batche sovulated, spawningduratio n, and eAAprodu cti oninfo rmationforindivid ual females in 199 5.
,
Prespawning Prespawning Batches Duration of Egg199 5 Lengt h Weight Ovulated Spawni ng Production
(em) (8) (days)
A 39 821 25 30 1088972
C 36 545 30 52 1622822
D 42 920 25 33 918936
F 45 988 26 31 1367185
G 43 971 28 33 2001472
H 38 712 20 31 7]8216
I 34 500 22 25 1255 91 5
M 42 925 28 34 2051 304
N 40.5 719 5 21 211736
Q 39 666 10 15 612 250
38
Pon jonjngofproduetjgn
Themean batc h volumefor the1994grou pwas20.9±8.3mI.whichwould have an avera ge fecundity of36807±16 353 eggs.In1995.themeanfemalebatch was 24.7±4.9mIwhich contained52 783:!::13 469eggs.Thebatchfecundity means of females ranged from14000 to70000in1994andfrom 36 500 to 71500in199 5 (Table7)
Batc h fecund ity isprimarilydetenninedbybatchvolume changes.ho wever, egg concentration (eggs/mI)wasnot constantfrom batchtobatchwithin females. Egg co ncentratio nwas seentochange erraticallyover thespawningseasons ofindividuals Fact orsaffectingegg concentratio n withinafemale include theamountofovarian fluid andthechanges inegg size whichoccurover afemale'sspawningseason. Six ofthe repeat spawners hadina-eases inmeanegg concen tratio nin1995.Femal eChadthe largestincrease in meaneggconcentration,from2090eggslmJin1994to2899 eggsImIin 1995
Da ta collected onbatch feamdityindicated that generallytherewasIinlechange betweenyears in batchfecundity(Figure 6).Thetop plou sho wthat batches of 10 000 to 20000eggswerethe most frequentin bothyears.although a decreaseoccurredin the relativefrequency ofthis class in 1995(Figure6).The secondmost freq uentbatchsize was20000to 30 000eggsin 1994 ,ho wever,thischangedto 40000to50 000eggsin 1995.Examiningthedata forrepeatspawners(thebottomplo ts Figure6)showsthat,
classinbothyears. The introduction of large batchsizes in 1995 was themostsignificant inter-annualdifferenceinbatchfecundity distributions.Batchesexceeding 200000 eggs appearedin 1995,where nonewereseen in1994 (Figure6). Thetotalrelative frequency of batchesof thissize wassmaU,lessthanJ%.
Sincemostrepeat spawners increasedtheireggproducti onin1995.the batch fecu nditydata for repea t spawnerswas examined todetermineifinter-annealdifferences occurre dinthese females,andwhetherincreasesinbatch sizeaccompanied increase sin eggproductionwhen they occurred.The results of statisticalanalyses comparingthe balch fecundity datafor repeatspawnersshows that fiveof the seven repealspawners didnot haveasignificant inter-annual differenceinbatch fecundity(C,D.F.H.M; Table8) Female Ahada significant decreasein batchsizein1995.although thedecreasein total eggproductionin1995was not large.Female I had a significant increase in batch fecunditywhichaccompanied theincrease in her production. Notab ly,only femaleI had fewer batchesin1995 thanin 1994.All otherrepeatspawners had an increase in the number of batchesbetwee n years.
Regardingthe changes whichoccurin batchfecundity within afemale'sovulation sequenc e,an irregular patternwasobserved10 betypical of femaleyellowtail in both seasons.Examining the batch fecundity recordsof femalesCand Iasexamples,the irregular pattern isclear,where batchsize can increase or decreasequicklybetw een batches(Figure7). In addition.no reductioninthe irregular pattern occurredbetween years within theserepeat spawners.
40
Table7.Batchnumberandmeanbatchfeamdityinformationforindividual femalesin 1994and1995
•
1994 BatchNumber 1994FecundityM~
(<ggSlBatchI
1995Numbe<Batch 1995Fecundity (_1MeanBatchA I"
oon4 I
2' 43 559B 8 25 056
C 2. 38500
I 3.
54094D 8 25824
I
2' 36757E 8 6122 4
F 13 37968
I
2. 52 5S4G 28 7148 1
H I" 39522
I
2. 36911I 27 26785
I
22 57 087L 13 26549
M 17 39522
I
28I
73261N
I , I
42 ]4 70
I ,
14200I
Q
I I. I
61225Figure6.Frequencyhistograms of batc hfecundity data observed among thebrood stock duringthe 1994 and 1995 spawning seasons.The toptwoplots comparethe data from allspawners in each year.The bottom two plotscompare the data fromrepeat spawners only.
35
1994 199 5
30 n""157 n:219
l
25i
20'"
15.~
::!
10100 200 300 100 200 300
35
1994 199 5
30 n=123 n"176
~25
e s
20I
~ IS.~
~ 10
100 200 300
Batchfecundity(x 10J)
100 200 300
Batch fecundity(x103)
Table8:Anovaandrandomizationtestresultsexaminingfor aninter·annuaieffect on the batchfecundity dataof reoeat SDawnetS
Repeat
-
F·value P< Randomization Significant SeasonalSpawner (>- increaseIdecrease
in batchfecundity
A 44 4.47 0.041 0.045 Yes.decrease
C '0 2.83 0.099 0.078 No
D J3 1.97 0.171 0.178 n.s.probincrease
F
,.
1.86 0.181 0.188 No,H
,.
0.11 0.741 0.742 NoI
4 .
21.0] 0.0001 0.001 Yes. increaseM
4 '
2.95 0.093 0.091 No44
Figure 7. Inter-annual comparison of batch fecundity distributionswithintwo repeat spawners.The top plots show the batchfecundity records offemaleCfor1994 and1995.The bottom plotsshow the batch fecundity records offemaleIfor 1994and199 5
160 , - - - - -- - -- C 1994 140
120 100
C199 5
10 IS 20 25 30
100
to 15 20 25 30 0
~--- 80
60
( 1994 11995
10 15 20 2S 30 0
Batchnumber
46
10 IS 20 25 30
Batch.number
Egg diameterandcxgdry wejght
Egg size amongthe1994captivefemalesranged from0.983mntin diameterto 0,829mnt.The mean fresheggwas0.901 :t0.033 mmindiameter.Eggdryweight ranged from 34.6ug of dry manertc21.9ug,the meaneggweighin g 26.9±2.9J.l8-Bothegg diameter andeggdryweightdecreased assuccessive batches wereovula ted bya given female(Ta ble 9).Data were notobtainedforfemaleE andfew datawereavailable fo r females L and O.Decr eases ineggdry weightover thespa wning seasons offemaleswhich comple tedspawni ngranged fro:n3.2 ug to (0,8ug.The largest dropwas seenin female A whichalsohad thehighest egg pro d uctionofthe1994 females.Resorbingfemales 8 and0had changesin eggdry weight ofless thanI ug.
The effectof theformalinpreservativeonfresh egg diameterwasdetermined,The resultsfromthe fourdifferent batches indicatedthatfresh eggswhen preserved, experien ced a mean decrease in diamet er of 1.4%(±0.4%s.e.).Therangeinthemean was from0.53%to2.29¥•.Theeffea:of the formalin preserv ative on diamet ers offresh eggswassmall andwasnot considered to havehad a deleteri ous effectonthe results
Regr essionanalysesrevealed asignifican t nega tiverelation shipbetween thepool ed female eggdiame terdataandday in the spawning seasonofthe group(P=O.OOOI.
r
: I0.584; Figure 8),Whenregression s were done onthedata for individualfemales.seven significantnegativerelatio nship s were found amongthenine femaleswhich couldbe tested(0.000 1<P<0.05) .Theperc entage ofvariationexplainedbytheregr essionsfor
relat io nships.
Thenext set of regressions analysed the relationship between egg dry weight and correspondingeggdiameter.A significant positive relationshipwas foundforthepooled dataset (P=0.0001,
r ..
0.582;Figure9 ).Examining the datafoeindividualsseparately, only four of thesixfemales had significant rdationships(0.0001<P<0.02),The perc entageof explainedvariatio n ranged between 53%and 86'Y.,FemalesFandHdid nothave significantrelationshipsAsignifican t negat ive relations hip wasfoundbetweenthe eggdryweightdataof femalesand thecorr esponding dayinthe spawning seasonsof individuals(p=0,00 0\.
r -
0.309;Figu re 10).The pooleddata wasregressed against corresponding dayinthe spawningseasonofthe group,however, non-norm alityoftheresidualscouldnotbe corrected despiteasignifican tregressi on.Separate anaJyses of the data forindividuals revealed that allsixfemalestested hadsignificant negativerelationshipsbetweeneggdry weight andtime.The amountofexplainedvariance by an individual's regr ession ranged from33'Y.to90%The total matterinvolved in reproductionwas estimated by using the eggdry weight data in conjunctionwiththeproduction data (Table10).Missingdatawithin a female werefilledbyusing theprevious regressionrelationshipsorby averagingthe data ForfemalesE and0, where no datawasavailable, themean egg weightcalculated from all the femaleswasusedto estimate their total ovulatedegg dry weight.Femaleswhich underw ent resorptionhadspawnedlessthansixgramsof maner,whilefemales which completedtheirspawningseasons ovulatedbetween 10.0g 10 37.2g ohota!dry matter.
48
Table 9 DecreasesineIoUI:sizeanddrvweildttover thesoawnin seasons of individ 1994 EarlyEgg EarlyEggDry Late Egg Late EggDry
•
Diamet er(mm) Wci.ght bl g) Diameter (mm) Weight h l g}A 0.963 34.6 0.883 23.9
0 0.9 14 26.6 0.878 25.8
C 0.905 26.9 0.838 21.9
0 0.921 27.8 0.898 27.5
F 0.898 28.7 0.876 25.5
H 0.965 32.0 0.891 28.0
I 0.949 29.2 0.864 23.2
L 0.983 01. 0.868 24.9
M 0.888 26_6 0.829 22.3
0 0.967(n"l)
uals
Table10.Total estimated dry weightovula ted by .ndividualfemalesin1994
,
Fish TotalDry Weight(g)
A 37.2
0 5.0
C 18.4
0 5.9
E 13.1
F 13.2
H 22.6
I 19.1
L 10.0
Figure 8. Relationship betw eeneggdiameter dataandcorresponding day inthe spawning season of the group in 1994.Theregression linefor therelationship isshown (n=109).
Figure9. Relationshipbetween egg dryweightand correspondingeggdiameter during the 1994 spawning season.Theregressionlinefor the relat io nshipisshown (n=72)
3.
JS 0
34
33 0 0
J2 0 0
0 0
JI
g;
30 000&
0
-§,
298~
00-n
28•
0~
270<:0
<l!b€f\
1lI
2.w 00
25
o
0 '60 00
24 OOOJS
23
J5
0 022 21 20
0.82 0.84 0.86 0.88 0.90 0.92 0.94 0.96 0.98 1.00
Egg diameter(mm)
Figure tu.Relat ionship between egg dry weigh t andcorresponding dayinthe spawning seasons of females in1994.Theregression tinefor therelation ship isshown (n""72).
36
35
a
3.
33
a a
32
8 0 <0
1I
~ 30
a
(]l)8 0
-§,
29a CfJ
~
.~ 28
a
.a
~ 270 0 82 0 a a
~ 26
OO c:P°%O a
"'
25
a a [3 a
2.
a a ~
00 a
23
c§l OCb
22 21 20
10 20 30 40 50 60 70
Dayinthe spawning seasonsof females
3.2.DISCUSSI ON
Photoperiodandtemperatureare twoimportant environmentalroes for the timing ofreproductioninfish(Lam.1983 ).Since most yellowtailfemalesinthe current study initiated spawning neartheSUII1J'nCS"solstice. along phot operiodappearedtostimulate reproductioninthis group.WhilePin(1970) indicatedthat peakspawningofydlowtail on the GrandBanksOCOJr5inthesecondhalfofJune.the peakperiodofeggproduction forthecaptive groupinmystudywasin mid-Julyto earlyAugust.Thisdelaymayhave beenduetotheeffectsofcaptivity.
Regardingtemperatur e,a temporary increasetosixdegreesmaystimulate ovulationin captivity,butisnotnecessarytomaintaintheprocess.Evidence for wild spawnersontheGrandBanksshowedthatspawningtemperaturesremainedbetween one and fivedegrees(Pin.1970).althoughamaximumof8.6DChasbeenobserved (Walsh.1992).Apossible temperaturecueofsixdegreesmay onlybeassociated with captive situationsIncreasesintemperatur e may overcomestress associatedwithcaptivity which may delaythe initiationofovulation.Futureresearchshouldexaminetherole of photoperiodandtemperature roes incaptivity
Theoccurrenceof non-reproductivefemalesnotedinthis studyisa problem for themanagementofcaptivebroodstock.Turbot studieshaverecordedthat 40%to 50% of theirfemales werenon-reproductive(Howell&Scott.1989;Omneset al.,199 1) Non-reproductive femalewinterflounder. Pleuronectesamericanus,have been encountered in thewild.where thenon-reproduct ivestate was observedtobedependent onsomaticcondition. ratherthan age fact orsorsenescence(Burton&Idler.1984 ).
56
Thisstatewasinducedincaptivewinter flounderbylimitingfoodandreversedby increasing food availabilitythe followingseason (Bunon.1991 ).Asimilarlinktonutrition wasseeninturbot.,where 43%of females onalowration (0.5%wetbodyweight"day" ) werenon-reproductive(Horw oodet01.•1989).
Thecessationoffeedingoverthe spawningseason may havebeenrespo nsiblefor thefollowingobservatio nsongrowth.First,non-reprod uctive femalesin 1994grewover theprec eding year.while those of 1995 lost weight.Secondly.there wasno increase inthe average female'spost-spawni ngweight betw een1995and 1994.comparedwith1994and 1993.The factthatanon- feedin gpolicywasemployedfor a secondconsec utiveseasonin combinationwith high ratesof egg prod uctionin1995 may explain why,often.females had post-spawningweightslowerthanthe previous year.Otherstudies adopting a no n- feedi ng policy included Kjesbu(1989 )on spawningcod.Gadusmorhua.andSmigie lski (1979)onthe hormonalinduct ion ofspa wning inyellowtai l flounder.Accordingtothe ecolcgicallitera ture.wildyellowtail flounder feed during thespawni ngseason. however, spa wni ng fish showredu ced consumptio n ratescompar ed with othertimes ofthe year.On the basisof percentag ebody weighttheamountof prey consu med by wildspawning females was0.06%compared withpost-spawning orrecrudescingfemales (Langton.
1983 ).Reduced appetite swere noted among the prespawningcaptive females in this study.Itapp ear s, however,thatfeedingduring the spawningseason mayberequire dto maintainsomaticcondition.maximizegrowth.and minimize the numberofnon-
Aone day inter-owlato ryperiodwasthepredominant intervalseeninthe captive group,whichsuppo rtsthe pro posal of Zamarro(1991)for adaily spawningstrategyfor yellowtail.Inadditio n,sinceearlyspawnen haveaone dayinter-ovuJat oryperiodastheir most frequen t interval . lh.isindicates thathightemperatur econditio nsseenlere- in both spawningseasonswere not responsiblefor theprevalence of theonedayinterval Whether a female spawnsdailyin thewild isuncertainsincebehavio ural factors are involved.
Nevert heless.the finding ofaonc dayinter-ovulatoryperiod indicat esthatfemales inthe wild havethe potentialof spa wning daily
Otherstudiesof batch-spawningflatfish reportedinter-ovu latory periods oftwo10 four days.three daysand 3.5daysforthe turbot.Scophrhalmus maximus,Atlantichalibut.
Hippoglossushippog lossus.and the barfin flound er.Verasper moseri,respective ly (Mc Evoy,J984;How ell&See n, 1989;Norberg et a1.•199 1;Meth venetal.,1992:
Ko yaetaL.1994 ).Scon(pen.comm.)revealedthat female plaice.Pteuronectespkuessa, had athreeday interval.Theplaiceiscomparablein size tothe ydJowtailRounder,unlike the otherlistedspecieswhichrange in size from2kgto 60 kg. Examinationofthc dataof Fauvd etcd.(1992)onturbotindicated thata onedayinter-ovulatoryperiod wasas frequentasthreeorfourdayintervals.Incontrast. daily ovulationcycles were seldo m observed in plaice (Scon.pers.comm.)
Inter-indi vidualandintra-individualvariability ininter-o vulato ry period was greaterin1994 thanin 1995forthecaptive groupofyelJowtailflounder.Inter- individ ual variatio nhasalso beennotedintheturbot studiesof McE voy(1984) and Howell&Scott(1989 ).Regarding intra -individual variation,McEvoy suggest edthat
58
female turbot were predictableintheirovulato ry rhythms,althoughoscillation between short andlongperiods were seen in some females. Conversely,alack of consistency within a female'sovulatoryperiods was observed inthe turbot andtheplaice (Howell&Scott,1989;Fauvcletal.,L992;Scott,pers.ccmm.)
Irregular ovulatoryactivity,seen in some 1994 yellowtailfemales.maybe indicativeof majordisruptionsinreproduction.Whycertain femalesdisplayedext reme irregularovulato ry activityis unclear,but mayrelateto individualvariability in stress tolerance or somaticcondition.Stress factorsmayhave caused irregularovulatoryactivity sincetheeffect oflong term stresshasbeenshowntoinhibit ovulationinthe batch- spawningred seabream,Pagrusauratus (Carragher&Pankhurst,199 1).Female0,one of thetwoindividuals displayingthelargest inter-ovulatoryperiods in1994,had a tail infectio n which mayhave affectedherovulationpatterns,as wellas,caused the resorption of hervitellogenic oocytes. Unfortunately,the 1994femalesdisplayingthehighest irregularitieswerenotrepeatspawnersinthe199 5 group andnointer-annualcomparison couldbe made
Inter-annualvariability in ovulatoryperiodswas clearlyshown bythereduction in irregularovulatory activityand theincrease in the frequencyof one dayintervalsamong the l995 females.Asa result,reductionsinspawningdurationsof individuals occurred, evenincases whereannual egg production hadgreatly increased. Thisledto a reduction in thespawningduration foran individual,even in cases where total production had
temperatureconditiol1S.However,an additional yearof acclimationtocaptive conditio ns mayhavebeenresponsiblefortheinter-annualdifferences via areduction instresslevels Twostudiesrelating potentialfecunditywithfemaJelength for yellcwt ail flound er offer anopportunityto comparetheperformance ofcaptivegroupswithestimatesfrom thewild.Howell&Kesler (1977)reporteda relationshipfor New Englandstocks.while Pin(1971)examinedthe stocksoftheGrandBanksofNewfo undland..Theregress ions found bythesetwo stu diesarelist edbelow:
NewEngland:Howell&Kesler(1977) :F-0.98 6L" 3.858(r'"0.784, P<O.OOI) Grand BanksofNFLD: Pitt (1971) .F:0.0355LA4.69(r"'0.558.P<0.01) The1994and 199 5 eggprodu ctiondataforfemalesin mystudywas compared withthe correspondingpotentialfecundityestimatesof both papers(TablesII&12). Pin(1971)may offera better comparison sincethecaptive femalesusedin thisproject werealso collectedfromthe Grand Banks.Itisevidentfromthe tablesthatHowell&
Kesler 's estimatesconsistentlyexceedthoseofPitt's.Sincefemalesfromthe New England areamatu reat a younger agethanthose oftheGnndBanks.dueto higher growth rates,theydemonstrat ehigherfecunditiesthanGnndBanks females ofthesame size (Royceetal..1959;Howell&Kesler.1977)
The comparisonindicatesthatonlyhalfofthe captivefemal eswere capableof matching orexceedi ng theeggproductionestimatedforfemalesof thesamesizeinthe wild.The femalesunableto realize their potentialfecundityestimatesincluded not only resorbingfemalesbutfemaleswhichhadcompleted theirspawning seasons.In1994,five females realizedtheir potentialprod uction estimatedbyPitt (1971).Thesewere allsmall
60
tomediumsizedfemalesof37aninlengthorless.Only femaleAexceeded both estimates.In1995,the five females exceedingPin'sestimatesincluded larg e females as well asthe smallestfemales. Four females, across thesizerange,hadeggprod ucti on valuesgreate rthan both potentialfecu ndity estimates.Theability ofsmaller femalesto real izeandexceedthe potent ialfecundityestimates ofeven larg efe malessho wed thatthe size-fecundi ty relatio nshipseeninwild populationswasno tpresentamon gthecaptive females of thisstudy.Hence, otherfactors outsideof sizeorage variableswereinvolved indeterminingeggproductionofyellowtailflounderin captivity
Thesefact orsincludediewyratio n.nutritio nalcomposition,spawning hist oryand stress.Regardingdie taryratio n.although group rationwascontroUedit can notbe assumedthatindividualsreceivedanequal pro porti onof theration.Indivi dualvariability infeed ingrates or aggressivenessplayaroleindeterminingthe ration afemal erece ives.
Conseq ue ntly,only a proportionof the females mayhave been ableto consumeeno ugh foodtorealize theirpotent ialfecundities.AccordingtoHorwoodelcd.(1989).female plaice.Pleuronecles platessa,fed a higherration (2-2.3%wet bodywrday·l)had 59% mor e viteUogenic oocytesthanthoseon alow ration (0.5%wetbodywtday·1increased laterto1.8%).Thedifficu lty oflarg erfemalesinreachingtheirpotent ial fecundities.
compared withsmaUer females.,mayhavebeendue toindividualratio nrequirements whichwere influenced by size.How ever.anyeffectrelatin g to sizemaybeinterrelated with spawni ng history.Kjesbu&Holm(1994) revealedtha t thefecundity ofrepeat
deplet ionof somaticenergy duringspawni ng,compare dwith recru it spawnerswhich have more thanoneyearoffeedingto contribute tothefecundit y of theirfirst season (Kjesbu&Holm. 1994)
Increases in egg productionin 1995 may have been aidedby a changein nutritional composition withtheintroduction of a commercialfeedbetween seasons The commercial feed mayhave had a greate renergy content than theshrimp dietpreviouslyused.Itmay alsohave had a greater concentrat ionof limiting nutri ents foregg production.The literature suggeststhatcertain dietarynutrientslimitegg productionin fish.Phosphorous deficientdietsnegat ivelyaffectedegg product ion in the red sea bream,Pagms auratus, (Watanabeet at.;1984)andin the ayu,Ptecogtossusaltivelis (Watanabe,1985) Diets deficientin a-tocop hero l,a majorformof vita min E,prod ucedfemaleslack ing vitellogenic oocytesin carp CJ1Jrinuscarpio (Wata nabe,1985)
Increase s in egg prod uctionmay also be explainedbyred uced stresslevels dueto anadditionalyear of acclimation to captiveconditions.St resshasdeleteriouseffectson reproduct ionwhicharemediated by cortisol,a corticostero idhormone.In "ivo and in vitrostudies haveindicated that cortisol causes a depression ofrep rod uctivehormones.
suchas,176- estradiol andtestosterone.This depress ion leads to decreasedvitellogenin levels,reduction s inoocyte growthand low gonadoso matic index values,as seen infemale tilapia.Oreochromis mossamtncus.brown trout,Salmotrutta,and rainbowtrout, Oncorhynchusmykiss (Carragheret al.,1989;Carragher&Sumpter,1990 ; Pottin ger&Pickering,1990 ;Foo&Lam,1993)
62
Resorptioninfemal es is another problemreg arding reproductive perfonnance and has been encounteredin captivenubot..ScophJhaJmusmarimus.andsole.Soleo soIea (Bromleyet al.,1986;Howell&Scott,1989).The impact of atretic processesonthe poten tialfecundityofcod.Gadusmorhua,wasexaminedinKjesbuetaJ.(1991)_Erosion ofpotentialfewndity duringthespawningseasonwaslinkedtomaternalnu tritional status.Femalesspa wned between20%to80% of
mar
potentialfecunditydepending on theirsomaticconditio n.Atresia wasnot observedinprespawnin g ovaries excep tforfishin very poorconditio n, butinc rease dinspawningfish as theseason progres sed.The energy made availableby atresi a mayfuel reproduction when somat ic condition ispoo r (Kjesbuetal.,1991 ).Whether femaleyello wtail which resorbed wereinpoorer condition thanfemales whichcompleted their spawning season could notbedetermi ned Weightand length data didno t indicateany dist incti o n forthese females.Aconnectio n mayexistbetween irregul arovulatoryactivityandresorption.Allfemaleswhichreso rbed displayedirregular ovulat oryactivity,yet notaUfemaleswithirregular intervalsexhibitedresorption.
IrregularovuIar.ory activityextendsthe spawningduration of an individualwhichmaybe delet eri o us forsomatic condition.particularly inthe current experiment wher eyellowtail wereno t fed duringspawning.Reso rp tionmaybemo re likelyundersuchcircumstancesif anutrit ionallink with resorp tion exists.Regarding thedecreasebetw een years in resorb ing females,thismayhavebeenduetoimpro vementsinnutritio nalcompo sition or
