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REsTRICTEDFEEDING, SPERMATOGENE$[S ANDOaowm[NAecnc CHARR,Salvelinusa/pi nus:THEIoENTlflCATIONOFTwoPOSS[BI..E

GAMETOGENICCONTROLPO[NTS

By

Corina D. Rice

A thesis submittedtotheScboolof GraduateStudiesinpartial fulfillment oftherequirem ents fortheDegree ofMasterof Science .

Departmentof Biology MemorialUniversity of Newfoundland

July 1999

St.JOM 'S Newfoundland

ABSTRAcr

Experimentswere conductedto determineiffood restrictionduring certain critical critica lseasonscouldreduce the incidenceof earlymaturity in Arctic charr(Salvelin us a/pinus L.)andto describe the stages ofspermatogenesis associated with those critical periods.

A seven-stagedescriptionof thespermatogenicprocess is presented along witha time seriesof thepro gres sion through the variousstages.Sperma togenesisin Arctic charr is initia tedin autumn under decreasingphotoperiods and temperatures,approximatelyone year before spawning. The onsetis characterizedbymitoticproliferationofspennatogoniabefore changes in GSI aredetectable.Thespermatoge niccycle is highly asynchronouswithin the testisof asingle fish, and between individualsof a populatio n.Two criticalpoints inthe spermatogenic cycle thatmaybesusceptibletonutritional controlwereidentified.Thefirst occursinautumn, affectingtheadvancementoftestes fromthe pre-spermatogonialstageto the immature stages; the second in spring,whenspermatogonialcysts form anda switch fromsperma togonialproliferation tosperma tocyteformation ensues.Reducedfeedin g temporarilyinterruptsmitotic divisionof germ cellsduring theearlystages of gonadal developme nt.

The Arctic charr,aged 1+inautumn,were subjectedto two different restricted feeding regimes. Expe riment I,cons isting ofalternating twoweek periodsof food deprivationwith excess feeding (2:2,14week duration),at any time from NovembertoJune

did not reduce the proportions of male fishshowing definite signsof maturity byJuly compared to acontrolgroup.InExperiment II.fishsubject ed to6-wee k periodsof continuous starvation (6;0)from Septemberto Aprilexhibitedtemporaryreductionsin gonadal activity. ByJuly.final maturation proportions of starvedfish were depressed relativetothecontro lgroup. Restrictedfeedinghad noeffect on thedegre eof gonadal investmentintermsof gonadosomatic index (GS£). No femal esmatured duringeither experiment.

Temporalchan gesinfish growth were monitoredoverthe course ofboth studies by retrospective examin ation oftaggedIndividuals. Afterperiods of food deprivation, resump tionofregularfeeding resultedinfish displayinghyperphagicfeedingactivityand compensatory growth.Growth differentialswere most pronouncedinfishstarved during periods of highertemperature, especially duringthe time of rapidtemperature increas e in May/Jun e.InExpe rim ent I,maturing males continued to grow andincrease bodyconditio n throughoutthewintermonths. while growthof theirimmature counterpartsremain ed low untilspring. Growthratesof maturing fish wereconsistentlyhigher thanimmature fish.In ExperimentII,growth patterns of maturingand immaturemales weresimilar,however.

conditionfactors ofmatwing maleswere slightly abovethose of immaturefish.

This study lend s support to the theory that there aretwo criticalperiodsinthe matura tioncycleofsalmonids.Food restrictio n alone,however, isnotaneffec tivemethod ofsuppressing early maturityinthis speciesowing tothelongand flexibleduration of the ide ntifiedcritical decisionperiods.

ACKNOWLEDGMENTS

The successfulcomp letio nofthisresearch would nothave beenpossiblewithoutthe immensehelp of so many people and organizations. Most especially,Iamgrateful to my supervi sor,Dr.MargaretBurton,who providedme with the opportunityto conductthis study and for allofherinval ua blehelp, guidance andinsight alongthe way.

The financial assistance oftheCanadianCentre for Fisheries Innovation, and the Department of BiologyandSchoolofGraduate Studies of MemorialUniversityis kindly acknowledged.

Thanksto Dr.David Schne iderforhis assistance with the statistical analysis of my data.

Thank you10Roy Fickenfor his patientphot ogra ph ic assistance. Thanks to Grant Dwyer for all his invaluab lecomputing assistance from rescuing mylost files to solvingallmy formattingandprint ingpro blem s. Thank you to thetec hni cal andcustod ialstaff atthe OceanSciences Centre foralloftheir helpincaring for my'babies ' .Ialsowishto acknowledge Dr.LaurenceCrim, Dr. SIeveGoddard andDr.GoverdinaFahraeus-Van Ree, for acting on my supervisorycommittee.

Iameternally gratefulto my parents without whosehelpIcouldnever havecompleted this thesis.They notonlyprovided me with financialand emotionalsecurity but pro ve d to be inv a lua ble fishmeas urin g assistants.Theirconstantsupport of my education and career choice is unwavering, eventho ughtheyanxiouslyawait my 'job'.

Avery specialthankstoallofmy familyandfriends, and most especially for the support of Grant,Karenand Matthew whohavebeenmy sources of strength andescapeduring my researchand writing.

iii

TABLEOF CONTENTS

Page ABSTRACT••• • ••••••••••• ••• ••••••• •• • • •• ••• •••••••• ••••• ••• •••••• •••••I

ACKN OWL EDGMENTS••••••• •••••• ••• •• •••• • •••••.•• •••••••• ••••• ••••.••III

LIST OFTABLES •••••• • • ••••••••••••••• •••• ••• • ••• ••• • • •••••••• •••••••••VI

LISTOFFIGURES .•••• •••••••• ••• •••• ••• •••••• ••••••• ••• • •• ••• ••• ••••••VJ1

LISTOF APPENDIXTABLES.• .•• • .•• ••••• •••• ••• •• •••• •••••••.•• •••• • •• •.•x

LIST OF ABBREVIATIONS•••••••• •••• •• ••• • ••••••••••••• •• •••.•• •• ••.•••.XII

1.0INT RODUCTI ON••••• •• ••••• •••••••••••••• •• ••• • •••••• • •••••• ••••••••• I

1.1Nutrition andReproduction I

1.2 CriticalPeriod . 5

1.3Spermatogenes is 8

1.4 Arcticcharr . .•...•9

1.5Objectives . II

2.0l\1ATERIALS ANDMET HO DS••••• ••• • ••••••••• •• •• •• ••• ••••••••••• • • •••12

2.1Fishand HoldingConditions. . 12

2.2ExperimentalDesign . .14

2.2.1ExperimentalSet-up. . 14

2.2.2Feedingand SamplingProtocols . .. .. ... .. .. . .. .. . 16

2.3Histology. . ..20

2.4Data Analysisand Statistics .. . ..20

2.4.1.Final MaturationProportions . . 20

2.4.2Growth andPerformance ... . ..22

2.4.2.1 Final Sampling Data .... . . ... . . ...22

2.4.2.2Tagged Fish. . 22

3.0RESULTS••••• ••••• • ••••• •••• ••• •• •••• • •• •• •••• • • ••••• •• • ••••• ••• • •24

3.1Testicular Morph ology 24

3.2Stages ofSpermatoge nesis . 25

3.3Effects of Starvation on Spermatogenesisand Maturation. .40

3.3.1FinalNumbersofFishandSurvival. ..40

3.3.2Starvation Effects on Spermatogenesis. 41 3.3.3Critical Poin ts oftheSpermatogenicCycle 45

3.3.4 Final Maturati on Proportions. . .. 48

3.4GonadalTissue Investme nt .. 53

3.5Effectsof Food RestrictiononGrowth 55

3.5.1Final GrowthResul ts . 55

3.5.2 Individuall y TaggedFish ...59

3.5.2. 1Effect ofMaturation on Growth. . .59 3.5.2.2EffectofRestricted FeedingRegimeonGrowth . ... 64

4.0DISCUSStON•••• • •• ••• •••• ••••••• •••••• ••• • •• •• • ••••• • ••• •• •• • • •• • • •73

4.1Spennatogenesisand Maturatio n. . 73

4.1.1Spermatogenic Cycl eofArcticcharr 73

4.1.2 Nutritional Inhibition of Maturatio n. .. . 80 4.1.3Critical PointsintheSpermatogenic Cycle 81

4.1.4 MaturationProportions.... .... . 84

4.1.5GonadalTissueInvestment ... .86

4.2Growth Responses. . 87

4.2.1 VariationBetweenExperiments. . 87

4.2.2Sexual Status andGrowth .88

4.2.3Restric tedFeedin gRegimes andCompensat oryGrowth 90

4.3Aqu aculture Implications 92

4.4FutureResearc hDirectionsandRecommenda tions 94 5.0CONCLUSIO NS.•••••••••••••• ••••••• • ••••••• ••••. •• ••••• • •• •• •• • ••••97 6.0REFERENCES•••• ••••• •• •••••.•• ••••• • • •• •••••• •• • • ••• •• •••• • •• ••• •100 7.0ApPENDIX•• • • •• •••• ••••••• ••••• •• ••••• ••••••••• •• •••••••• •• • •• ••••110

LIST OF TABLES

Pa ge Table 1: Numberof fishpergroup atthe beginningof eachexperim ent. Groups

weredividedinto two replicatetanks each. _. . 15 Table 2: ExperimentI.Restrictedfeedingandsamplingschedule..

Table3: ExperimentIT.Restrictedfeedingandsam plingschedule

. 17

...18 Table 4: Summary ofthemicroscopiccharacteristi csdistinguishingthe

spermatogenic stage s of Arcticcharr. ... .. ._..._... ..38 TableS: Timin gand durationof the spermatogenic stag es ofArcticcharr 39 Ta ble6: Numbersof fishatthetermina tion ofboth ExperimentsI and U. 41 Ta bl e 7: Histologicalcharacteristi csandstages ofdevelopm entdefinin gtesticular

deve lopmentasusedinclassifyin g samp led fishover the time period of

ExperimentII. . 42

Table8: Numbers and proportions of virgin male fish showing signsof active

spermatogene sisdurin gExperim ent II _ 43

Table9: Finalmaturationproportionsofthecontro land experimental groups of

fishinExperi ment L 49

Table10: Final maturationproportionsof thecontrolandexperim ental groups of fishinExperiment II.. ... . . . . ... . 52

vi

LIST OF FIGURES

Page Figure1: Watertempera ture profileforbothexperimen tal periods .. .13 Figure 2: Testisofa completelyimmaturefish at StageI.Pre-sp ermatogonial. ... 32 Figure3: Testis of a fish at StageIla, Immature I,of develop ment sho wing lobular

structure and primaryspermatog onia, presenteither singly or few per

lobule. . 32

Figure4: Testis of afish at StageUb,Immature II, ofdevelopment.Lobulescontain greater numbersofspermatogonia withboth primaryand secondarytypes present.Mitotic proliferationofspermatogoniaare observableand lumens

arevisiblewithin thelobules. .32

Figure5: Fish testisatStageIll,Transient.Lobules arelargeand containprimaril y secondaryspermatogoni a. Mitotic activity ofspermatogoni ais increased

andcellscan beseendividingas acyst.. .32

Figu re 6: Testisat theinitial stageofspermatocyteformation,Stage IVa., Sperma tocy teFormationI.and meioticdivision . . .34 Figure 7: Testisshowin gsecond stage ofspermatocyte formati on, StageIVb,

SpermatocyteFormat ionIl:Primary spennatocytes and secondary spennat ocytes are observab le within testicular lob ules 34 Figu re 8: Testis atSpe rmatid Formatio nstageofdevelopm eot,StageV. ...34 Figure 9: Testisadvancedto Spermatozo a stage of developm ent, StageVI.

Spermatozoaarerecognizedbythe darkly stainin gconcentr ationofsperm

headsand thepresence oftails. . 34

vii

Figure10:Fish at stage of FunctionalMaturity, StageVII. Testisconsists of spermatozoalyingfreein thelumens of lobules andprimary spermatogonia

liningthelobulewalls... ..36

Figure 11:Testis showing asynchrony oflobular progression through the spennatogenicstages.Cellswithin acystproceed through developmen t together but not allcystsare synchronous within alobule.. 36 Figu re12: Fishtestis (September )showingan aborted orincompletematuration.Only

a fewspermatozo aare presentinthelumens of somelobu les amongst

spermatogonia.. . 36

Figure13: Testisof previouslymature fish (June) showing resorpti onof residual spermatozoa remainingfrom theprevious spawning season. . 36 Fipre14:Percentages of malefish showingsigns oftesticulac developmentduring

Experiment II.... .44

Figure15 :Final (JUly)proporti ons of maturingmalefishin the controland feed restri cted groups for A)ExperimentI, and B) ExperimentII... .50 Figure16: Meangonadosomatic indices(July) of A) females, B) maturing males ,and

C)immature malesineachgroupfor ExperimentsI andII. . 54 Figu r e17:Meanfinalweights and conditionfactorsofaLIfish inExpe rim entI...56 Figure18:Mean final weights and condition factorsof all fishinExperimentII... .57 Figure 19:Comparison ofmean growthparameters (::I:s.e.]overtimebetween tagged

maturing and immature males of ExperimentI. 62

Figu re20: Comparisonof mean growth parameters (::I:s.e.)over timebetweentagg ed maturingand immaturemalcfishof ExperimentII. . 63 Figu re21: Mean weights (::I:s.c.)of maturing andimmaturetaggedmale fishfrom

ExperimentsIandII. .. . ... 70

Figure22:Meancondition factors (::I:s.e.) of maturing and immature taggedmalefish

fromExperim ents I andD. .71

viii

Figu re23:Mean specificgrowthrates(±s.e.) of maturingan-d immaturetaggedmale

fish from Experiments IandIT. . 72

ix

LISTOF APPENDIXTABLES

Page

TableA-I: Binomialcompari sons ofmale maturationproportions betweenthe

replicatetanksinExperiment I.. . II I

TableA-2: Binomi alcomparisons ofmale maturationpropo rtion s betweenreplicate

tanksin Experime nt U. .. 112

TableA-3 : Three-wayANDVAresults for the final growthparametersofall fishin

Experim entI.. .113

TableA-4: Three-wayANDVAresultsfor the finalgrowth parametersofall fishin

ExperimentII. . 114

TableA-5 : Final meas urements ofmaturingand immature male fishinExperiments I andU. .. ... ... . ... . . . ... . . .115 TableA-6: ANCOVA results forweight ofindividuallytaggedArcticcharr of sex-

re gre ssed on time. .116

TableA-7: ANCOVAresul tsforlength ofindividua lly taggedArctic charrof sexa regr essed on time. . .. .. ... . . . ... ... ...116 Table A-8: ANCOVA resultsforcondition factor of individual lytaggedArcticcharr

ofsex" regressedontime... .117

TableA-9: ANCOVA results forspecificgrowthrateof individuallytagged Arctic

charrof sex'regressedon time. .117

TableA-IO:ANCOVAresultsfor weightof individuallytagg edmaleArcticcharr of thedifferenttreatmentgroup sregressed ontime.. .118 TableA-II:ANCDVAresultsfor length of individuallytaggedmale Arctic charr of

thedifferent treatmentgroupsregre ssed ontime.. 119

TableA·12:ANCOVA results forcondition facto rs of individuallyraggedmaleArctic charroftbedifferenttreatment groupsregressedontime._. ••.120 TableA-13: ANCQVAresultsforspecific growth rate of individuallyraggedmale

Arcticcharr oftbc different treatmentgroupsregressedontime 121

xi

LISTOFABBREVIATIONS

CF conditionfactor

GSI gcnadoso matic index

L Leydig cells

Lo lobular boundariesIwall

Lu lobularlum en

Mi mitotically dividingcell

MiG groupmitoses

ScA primaryspermatocytes ScB secondaryspermatocytes SgA primary spermatogonium(ia ) SgB secondaryspermatogonium (ia) SGR specificgrowthrate

St spermati ds

Sz spermatozoa

xii

1.0INTRODUcnON

1.1 NUTRITION AND REPRODUCTION

Reproductiveprocessesareenergeticallyexpensiveandrelyheavilyonbothenergy reservesand successfulattainmentof exogenous resources (Shul'man 1974). The association between nutritionand reproductionhas led to extensiveinvestigatio ninto the linksbetween thesetwo systems.especiallyin mammals(reviewed byI'Ansonet al.1991).

Possiblelinksidentified range from interactionsbetween bod y stateand reproduction.for example, weight and proximatecompositio n(Frisch and Revelle 1970).to theinfluenceof gut peptides actingon thecentralnervous system (Kennedy and Mitra 1963).Themost thoroughlyinvestigatedlinks are those associated with the rolesof adiposityand growth.

Theinitiationofpubertyinfemaleshas been correlated with the attainment of a critical body weightinhumans. sheep. rats.chipmunks.andinsheep tothe attaimnent of a minimumlean to fat ratio andpercentbody fat (I'Ansonet at.1991).Insows,nutritionalstrategies aimed atincreas ing body fat reserves havebeen showntoimp rovefertility (Odowd1997).

Nutritional influencesonreproductionhavealso beenidentifiedin amphibians andreptiles (WhittierandCrews 1987).Inthe alpinesmooth snake(Coronellaaustriaca),reproduction is associatedwith energy level sand condition.Snakesthatrecoveredconditionsoonerinthe yearafter reproductionwere able toreproduc e againafterabrieferdelay (Luiselli 199 6).

The roleof nutritionon reproductio nintelecstshas been studiedless extensively,but accumulatingevidence suggests a close relationship .

Rejectingopportunitiesforreproductionis an occurrencewhich has been reported for a widerange of iteroparousanimals (Bull and Shine1979) . Inte leo sts,inadequa te nutritio nbasbeenimp licated as a causativefactorfor such om issions. Fedorov(1971) reportedthatsexuallymature,female Greenlandhalibut(Reinhardtius hippo glossoides)of the BarentsSea didnot spawnannually.Asimilarsituation has been describedfor other wildpopulationsof marine fish includingthe orangeroughy,Hoplostethus atlanticus(Bell etal.1992),and in Newfoundlandpopulationsofwinter flounder , Pteuroneaes amencanus (Burtonand Idler1984). Failureof post-maturefemalesto developvitellogenic oocytes and spawn annually hasbeen investigatedexperimeutallyfor haddock,Melanogrammus aeglefinus (Hislopet al. 1978;Hislop1988),winterflounder(Burton and Idler 1981), and plaice,Pteurone cte splatessa(Horwoodet al.1989).Ineach case,these conditions were induced byreducingfood supplies,providing evidenceof a connection betweennutrition and reproduction.

Theoccurre nce of non-annual spawninghas also beenreported forfreshwaterfish, mostnotablyinrelation to the tendencyof north emiteroparous fish tospawn at intervalsof two ormoreyears .This phenomenonhasbeen documentedinArctic charr,Salvelinus alpinus, in France(Jamet 1995),in Norway(Jergensenet at.199 1)andinNauyukLake, Canada (Dutil1986).Dutil(1983,1986)suggestedthatgonadal developmentdrains reserves

to such anextent that theeharr arcenergeticallypreventedfrom maturingtwoyearsin succession. The samehasbeenreported forseveralspeciesofanadromouswhitefish, Coregonus artedii, C.c/upea[ormis,andProsoptumcylindraceum (Morin et al.1982;

Kennedy1949,1953) livingin northerly,low prod uctivity environments. Sturgeon, Acipenserfidvescens andA.transmo ntanus,represent anextreme exampleof non-annual spawning.Inthe wild, females ofeach species only spawn every four to 11years (Bulland Shine 1979).Even in captivityunder ideal feeding conditions,the spawning interval of sturgeonisdifficultto reduce below two years(Williotand Bnm1998). Itmay be that the energy drain of gonaddevelopmen tand spawning is so greatthatatleast two yearsare requiredbefore the fisb.are re-conditionedenoughtoreproduce again, similartowhat is seen with the northerly fish. Non-annualspawninghas also beennoted for more southerly iteroparoussalmonids, includingAtlantic salmon,Sa/mo salar,and Salvelinus malma(Bull and Shine1979).

Nutritionalso appearsto playa pivotal rolein determiningfecundityinteleosts.

Scott (1962)and Bagenal(1969) foundthatinrainbow trout(Oncorhynchusmykiss) ,and in brown trout (Salma frutta), respectively,reduced feedingresultedin decreasedfecundity.

Similarly,reducin grations are reportedto have affectedeggproduction and maturation proportionsin the three-spined stickleback, Gasterosteus aculeatus (Wootton1973), haddock, (Hislop 1988),winterflounder(Tylerand Dunn 1976), herring, Clupeaharengus (Maetal.1998),Atlantic cod, Gadus morhua(Kjesbuetof. 1991;Karlsenelal ,1995 ),

plaice (Horwoodet al, 1989), and Atlantic salmon (Bromage et 01. 1992). Precocious maturation of malesalmonids, especially under hatchery conditions, has led to extensive investigationinto the roles of enhanced feedingopportunity and growth on maturation. Rowe and Thorpe (19903) found that springgrowthwas important for determining the onset of maturation of male Atlantic salmon parr.Subsequenttothis, it was shown thatincreas ing fat stores or therate of acquisition of fatinspring may be the determiningfactor (Roweetat.1991). Condition factors oneyear prior to spawning (autumn) have also been implicated (Bohlinet 01.1994).

Empiricalstudies on a widerangeof animals clearlyillustrates that nutrition can play a regulatory roleon reproductive processes ,even from thelarval stage of thelifecycle.

However,most of this information isbased on correlations between some aspect of somatic state (e.g.,size,fatness,growthrate) and reproductionwith little consid erationas to how this physical infonnation istransmittedto thereproductive axis.Determining thephysiological mechanismsresponsible for thisinteraction has beenproposed as a challengeto animal physiologistsand representsone of thegreat frontiers of biology(I'Ansonet of. 1991).

Inrecent years,muchexcitementhas beengeneratedby the identification of the protein leptin and its possiblerolein theregulationof food intake and body mass in mammals and in the understandingof the relationshipbetweenreproductivestatus and the neuroendocrinesystem. Dietary restrictioninrats is observed to be associatedwithlow plasma leptin levelsandsexual immaturity.Inthese animals,centralinfusionofleptin was

able to inducesexual maturation (Aubert 1998).Inmice it is suggested that rising plasma levels of leptin represents a signal to the brain that the animal is metabolically ready for sexualmaturity,andthe onsetof pubertyensues (Gruazet al. 1998). Low levels of circulatory leptinhave alsobeen implicated in menstrualdysfunction in women (Tataranni 1997). It has not yet been demonstratedhow this protein can act as a signal of metabolic status, butit has been suggested thatit may function by affecting neuropeptide-Y neurons inthe hypothalamus andloritmay affect peripheral endocrine targets, such asthe pituitary, ovary,testes orpancreas (Aubert 1998).

Inteleests,no such metabolic messenger has yetbeen described, but it is possiblethat such a somatic-reproductivelink is conservedin vertebratesanddoes exist. However, the reproductive systems offisharemore diverseandless understood than mammals so before such a regulatory factor can be sought,it is important to understand the reproductive cycle ofthespeciesinquestion.Knowing when the reproductive cycle may be sensitiveto nutritionalstatus is essentialbefore one can attempt to identify metabolic signals that may be acting at that time.

1.2CRITICALPERIOD

Attemptsto decipher the link between metabolicstateand reproductioninfish has ledto the development oftheconcept of a maturational'critical period' (Thorpe 1986,1994;

Burton1994). This refers to the time when fish may be sensitiveto nutritional status and

use this informationinmaking the physiologicaldecision ofwhether ornot to mature for a given year.The determination ofnutritionall y sensitive critical periods in the life cycles of teleostsis a crucial steptowards the eventual identification of metabolicsignals relaying information aboutsomatic conditiontothe reproductivesystem.

Inwinterflounder, Burton (1994)showedthat a non-reproductivefish is likely to occur if feeding is restrictedprior to and immediatelysubsequent to the current spawning season (mid-April). Feedingmanipulationsduring this time havebeen successful in inducingthe non-reproductivestate in thesefish, a state that can be subsequentlyreversed the following yearwitb return to adequate nutrition (Burton 1991).Histological observations ofthe ovary during this critical time indicate thatthenon-reproductivestateoccurs as a result of fish failing toundergo exogenous vitellogenesis,indicating thatnutritional status actsas acontrol mechanismearlyinthe gametogenic process . The failureof fishwith high post- winter condition tobecome non-reproductive when starvedduring this critical period suggeststhat it is not feeding level,butrather somemeasure of current nutritional status that isactingasa reproductiveregulator(Burton 1994 ).

Critical periods forthe nutritional contro lofmaturation have also been proposedfor salmonids. Since salm onid maturation was thought to be initiated under increasin g photoperiods(Scottand Sumpter 1983),it has been proposed that a criticalperiod for Atlanticsalmonmaturation should occurinspring (Thorpe1986). Thistheory was developedinresponse totwoobservationsmade on salmonid life histories. The first was that

somethingoth er than photoperiodmust becontro llingthe onset of gametogenesisinfish;

otherwise,allfish would maturethe first timetheyexperiencedtheappropriate light conditions.The secondstemmedfromtheidea ofage orsize atmaturi ty. Thorpe(1986) reasonedthat size at ageis ana prioriargument and that fish.before initiating maturation, must in someway assess its physiologicalstate. One suggestionwasthatthis assessmentis basedon the rateof storage,or turnover of surplusenergy, exceedinga genetically determinedthresholdduringa criticalseason, definedbytherateof increasein day length.

He defined this timeas the'seasonal window' forinitiation of salmonparr maturation.

'Ibismodelhas nowbeenextensivelytested on Atlanticsalmon (Adamsand Thorpe 1989;Roweand Thorpe 1990a,b;Herbingerand Friars 1992;Simpson1992;Berglund1995;

Kadriet al.l996)and itwas foundthat feeding opportunityinlatewinter/spring can have an effect on thepropo rtionof fishmaturingina given year.However,the timing and extentof foodrestri cti on required to producethiseffect, andthe proportions of immature fish resulting. have not beenconsistentbetweenstudies so a setnutritionallysensitive'critical period'remains obscure. Since maturatio nmay bedepen den t on other factorssuchas temperature.photoperiod, and genetics,itis importantto knowwhich stage ofgametogen esis isbeing affected.Inthis way,a criticalperiod canbe identified for agiven populationby recognizingwhenthe fish are at a gametog enicstage suscep tible to feed deprivation .

The recent realization that the annual cycleof gonadalgrowthinsalmonidsbegins in autumnrather than under theincreasingday-lengthsof spring bas ledto arevision of

Thorpe's(1986) critical period modelin thatthe decision taken atthis timeis not whether or nottoinitiatematuration, but ifitshould bepermitted to continue (Thorpe1994). There are,to date,no published accounts providingevidence thata criticalperiod mayalso occur inautumn controllingthe ons et of early gonadalgrowth,butthepossible existenceoftwo primary annual switch points controllingreproductive function in salmonidsispostulated (Thorpeetai.1998).

Early maturityis anundesirable trait incultured fish.Suchfish sho w deteriorating flesh quality,lower growth rates, smaller size,aggressivebehavio ur and impaired smelting, allof which interruptproductionschedulesforfarmers(Aksne set al.1986).Theability to controlmaturationalproblemsthrough nutritional mechani smscouldreducerelianceon the need to useother morelabo ur intens iveprocedures such as triploidizationor hormonal manipulations.Therefore, interestinthe identification ofa critical periodinsajm onids and themechanism co ntro lling theonsetofmaturationremainshigh.

1.3 SPERMATOGENESIS

Theproces sofspermatogenesis inte tecsts isnot wellunderstood , owing tothe diversityof thegroup andthus, a widerangeofreprod uctivestrategies (Pudney1995) . Severalstudiesof spermat ogen esis infishhave been conducted onsalm o nids (Henderson 1962; Grier1981; Billard1992),however,descriptions ofthe processarestillconfus ing and inco nsistent, and controllingmechanismsstillundefined (Pudney1995).Most studiesof

spermatogenesis that do exist are concentratedon laterstages ofthe process when hormonal and steroidprofiles areapparent and the testes welldeveloped.Verylittle informationis availableon theearly stages ofgonadal development (Chiba et at. 1997). Miura et al.(1997) proposed thatinJapanese eel, Anguilla fapontca, entry intospermatogenesis is initiated by gonadotropinsstimulatingLeydigcells to producel l-ketotestestercuewhich in tum induces spermatogenesi sthroughtheactivation of Sertoli cells.However.thismodelisfar from complete. Itdoes not address many of the still remainingquestions about control mechanisms within thereproductivesystem.Confusion in theliterature stillremains as to the specific events occurringduring spermatogenesis . Forexam ple. how and when spermatogonial renewal takes place (Henderson 1962;Grier 1981). the timing ofgonadal recrudescence(Scott and Sumpter 1983; Thorpe1994; Thorpe et at. 1998)and thefactors regulating entry of germ cells into mitosisand meiosis (Miura et at, 1997). The identification of a nutritionalcritical period corresponding with certain stages ofthe spermatogenic cycle could help decipher wherecontrolmechanisms in the cycleact.

1.4 ARcncCHARR

Arctic charrare the mostnortherlydistributedfreshwater fish with a circumpolar range inthe Northern hemisphere(Scott and Crossman 1973).Unlesslandlock ed.most strains are anadromous, migrating downstream after spring thaw and enteringthe ocean for asumm erfeeding season.Juvenile fishusuallyremaininfreshwater for a number of years

before makingtheir first seaward migration.Inthe marine environment, Arctic cbarrare not highlymigratory ,remainingclo seto shore andtheirnativestreamsto whichtheyreturnin mid- to late-s ummer tooverwinter(Leim andScott 1966) . Anadro mousstrainsof Arctic cbarrfrom the Fraser River,Labrador,average 6.9yearsand 38.1cm at maturity (Delabbio 1995) .InArctic waters,cbarr spawninearlyautumn,usuallyin Septemberor October,in mo resouther lyregions, spawning extends later into autumn., occurrin g in November or Decemb er(S cottand Crossman 1973).Spawning is reportedto occur overgravelorrocky shoalswhenwatertemperatures reach 4°C (Scottand Crossman1973;Joblinget at.199 8).

The eggs develop over the wintermonths and fryhatchinearly spring (Joblingetal.1998).

Arctic cbarr are morpho logicallyandphysiologically simi lar to Atlantic salmonand rainbowtrout.Sincethe 1970s,they have been regardedas apromis ing candidatefor aquaculture., owing tohighergrowthratesincold waterthan eithersalmonor trout(Delabbio 1995 ). However,theexpansionof cbarr fanninghas notbeen as rapid as originally predi c ted.Inarccentreview,Joblinget al. (1998) outlined.the problemswith charrculture which have troubledthe industry.One of the mainprob lemsdescribed is early maturi ty.

Theprovision ofgoodgrowthconditionsallowcharrto reachin one yearthe sizeitwould nonn aUyrequireseveral yearsinthe wild to attain, an occurrencewhichhasbeenassociated withhigh percentagesofearlymaturin gmale fish.Becauseofthistendency forearly matura tion underacceleratedgrowthregimes,and the paucityof infonnationon the gametogenic andreproductivecycles ofArctic charr,it wasdec ided that thisspecies would

to

make a good modelfish onwhichto conductfeed restrictionand gonadal development studies.

1.SOSIECIlVES

The primary purpose ofthis study was toexamine the effect of foodrestrictio non reproductivedevelopmentinArctic cbarr. The specific objectives were:

1. To definethe critical period(s)inthe gametogenic cycle ofcharr;

2. To identify the stage(s) of the spermatogeniccycle affected by food restriction;

J. To describe the annual cycle of spermatogenesisinArctic charr;

4. Toexamin e the effects ofrestricted feeding on growth and performance.

11

2.0MATERIALSANDMETHODS

2.1FISH ANDHOLOING CONDmONS

Hatchery raised Arctic charr from Daniel'sHarbour Arctic ChaIT Hatchery, Newfoundland, were used in this study. The strain originatedfrom the Fraser River, Labrador (56°39'N,63°1O'W).The fish used in Experiment I were fertilized in thefallof 1994 at Hidden Valley Charr Farms,PEl, and shipped to Daniel's Harbo ur wherethey hatchedin winter1995.They were obtained from the hatchery in October 1996at age 1+_

Fish for Experiment ITwere fertilizedinDaniel' sHarbourinFal11995andhatchedinwinter 1996.InSeptember1997 they were obtained from the hatchery at age1+.In bothyears, the average initial weight of thefishwas 18g and represented the mid-sizedrangeoffish from theindividualcohorts.That is,the largestand smallest fisb of eachindi vidu al yearclass were not usedinthis study.

Experiments were conductedat the Ocean Sciences Centreof MemorialUniversity ofNewfoundland,Logy Bay,NF (47"30'N,52°10'W)under a simulatednatural photoperiod maintained bya photocell controlled timing system. Thefishwerehe ld in 1mx1m(270l) fiberglasstankssuppliedwithflow-throughfreshwaterat rates of appro ximately8['min-]_

The watertemperature profile forboth years isshowninFigureI.The fishwereheldunder ambient water tempera turesullntil May of each yearwhen aheat exchang erwas installed

12

16 Experiment1 Experiment2

14 ^{199 6-1997 1997-199 8}

E

12

~

¹⁰

... ~

~

e

~

$

0NDJ FMAM J J ASOND J F M A M J J Months

Figul"e1: Meanwater temperances for10-dayintervals throughoutbothexperimental periods.Vertical line represents timeof division between ExperimentsIand ll;asterisks indicatetime ofheatexchangerinstallation.

13

to cool thewater.Thissystem worked effectivelyuntil salt-water temperaturesincreased beyond 1O"CinlateJuly.Low dissolvedoxygenconcentrations(50"10saturation) associated withelevated water temperatures (max.16°C)inlate summerof 1997necessitatedthe installationofanoxyge n injection syste m,whichbrought oxygen lev elsbackto90%.

Itwas necessaryto keep watertemperatures below12°Cto prevent the onsetof proliferativekidneydisease,a disease caused bythe parasitic organismPKX,which is endemic tothe watersupply atthe Ocean Sciences Centre (Brownetal.1991).Regardl ess ofefforts madetocircumvent theonsetofthis disease,mortalities occurringinlate sunun er (September 1997)in ExperimentIwere attributed to this caus e.

2.2EXPERIMENTALDESIGN

Thisstudyconsisted oftwo experim ents. ExperimentIwas conductedinthe period of November 4,1996,toOctober2,1997; ExperimentIIran fromSeptember27,1997 , to July10, 1998.

2.2.1ExperimentalSet-up

Fish werestocked into12 tanks(5 treatmentsplus a control,induplica te)according tothe distributio noutlined in Table1.InExperiment I waterdepthwasadjustedtoensure stocking densitieswere comparablebetween tanks(initiall y- 24kg/m3). Treatment consistedof restricted feedingof the fishinthefive pairs ofexperimentaltanks during

14

different time periods. Contro lfish were fedtoexces s throughout the experime n t, with excess feeding defined as feeding to the point wherefishleft foodrem ainin g on the'bo ttom oftbetank,Observationsindica tedthatfish were capableof. and did feed fromthe "bottom of the tanks. Extra food was removed fromthe tanks daily by siphoning.Througho-utboth experiments fish were fed a commerciallyprepared trout diet (CoreyVigorTrc u.t Feed.

Corey Feed Mills.NewBrunswick) containin g-40% crude protein and-18%lipid.

Table1: Number offish per group at thebeginning ofeach experiment.Grou pswere divided into two replicatetanks each.

GfOoUpS Treatmenttanks

Group2 Group3 Group 4 Groupl

~O=l

_

Experiment

II 624 306

280 306

260 306

240 306

220 306

21)0 31)6

Twen ty fish from eachtankwere individuallytaggedusingFloy@FingerLiJng tags stitched through the dorsalmusc ulature immediatelyanteriorto the dorsal fin.Fo r tagging andperiodicmeasurements.fish wereanaesthetized using2-phenoxyetbanoJata concentrationof 0.5ml-z'.

15

2.2.2 Feeding and SamplingProtocols

Tables 2 and 3 outline the feeding,histologicalsam p lingand tagged fish measurementschedules for ExperimentsI andII, respective ly.InExperimen t I, fish were subjected to a14- weekrestricted feeding regimeinwhich a twoweekstarvati on period was followedby a two week period of excess feeding(2:2regime).The firstrestrictedfeeding periodcommencedin Novemberwith Group I andended in Junewith Group5.For Experiment IIa stricter feeding regimewas implementedwhere fish were subjectedto six weekstarvationperiods (6:0regime) withno intermittentfeeding.Starvationperiodsinthis experiment commencedearlier,runningfromSeptember(GroupI) throughApri l(Group 5).

Fishweresamp ledthroughoutboth experiments to followthe normalprocess of gonadal development andto assesstheeffects of restrictedfeed ingongrowth.body conditionand gonadal development.At the beginning of each experimentsamplesoffish were killed to establisha baseline measurementof gonadaldevelopment.InExperiment I, this sample consistedof3 6 fishinjured during transport.InExperimentII,a representative sampleof 40 fisb was selectedand used for this measurement. Samplesize atother measurement times,with theexceptio nof the final samples,wastenfisb per tank. All sampledfisb were killedby overdose of anaesthetic,eitherMS222 or2*pbcnoxyethanol, lengtbcd totheneare st 0.1em,weighed tothenear est gram,anddissected10remove the gonads.The gonads were macroscopicallyexamin ed andclassified for sexand maturity, weighed to the nearestgram"andthen immediatelyfixedinBollin'ssolutio nforhistological

16

ExperimentI.Restrictedfeedingandsampling schedule.•,sampletaken forhistologyanalysis;shadedblocks, starvationweeks;T, terminationof

S#ta edlish ttim

Tab le2:

group; gg m = =en <S.

!'otoath ^WHO Taoed

,.u

bcgia.niag

.

me.,.nmeDU^SO eo_tro l

_·

1 1 3

•

⁵

N~ II 18

2S

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²

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Doc ²³16

· ·

3.

6 SI

·

Jm _2.¹³

·

27³

·

Feb

I.

¹⁷

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I.

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Mu 17

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31⁷

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AI"

_" I

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53

· · · · .

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J= 16

· .

23 54

·

3.⁷

· · ·

101 ²¹_za

I '

₅₅

·

·

^{T T T T T}

Au

I ' ·

Sop ²⁹

, ·

^T

17

ExperimentIl.Restrictedfeedingandsamplingschedule.-,sampletaken forhistologyanalysis;shadedblocks,starvationweeks;T,termination of

S#ta ed fish easurem. ttimes Table3:

group; gg m on

Mouth WHk _lied roo

begUllIoillog_IS Me1lS.rcmeat_SO Control 1 2 J

•

^S

·

Sop zz

29

·

Oa ¹³

20

27³ 51

· · · · ^· ·

Ncv 10

13 2.

I

•

⁵²

· · ·

Dec IS

29 3

1~ 12

19 53

· · · ·

26 2

F'b

,

16 23

2

.. _{· ·}

,

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23 30

•

Ap< IJ²⁰ 53

· · · · ·

27

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M.y 11

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2S8^I 56

· · ^· · ·

1= IS

22 29

101

•

^{57 T T T T T T}

18

analysis. Conditionfactor (CF) and gonadosomatic index: (GSl) werecalcula ted by EquationsIand 2asfollows:

Con d iti on Factor .. ~x:100 (I)

Length

GODa d oso mati c IDdell:= ~x 100 (2)

Weight

Tagged fish were monitoredforlength and weight periodically throughouteach experiment. Condition factorswerecalculatedas described above and as well,specific growthrates (SGR), basedon changesin weightover time, werecalculatedbetween sampling periods according to Equation3:

Sp ec ifi cGrowth Rate= ~j x 100 (3)

~-tl

whereinwt,andin J.¥tare the natural logaritluns of the weightsattimesIand 2, respectively, andt1-t1isthe number of days between sampling times.

At thetermin ation of bothexperiments all fish were killed,measuredand dissected.

However,inExperiment I, only80 fishfrom each contro ltankwerekilledalongwiththe rest

19

ofthe groups.Theremainingfishwerekept to followgonadal developmentuntilautumn.

Samplingof thesefishcontinuedas scheduled until termin ation ofthe experimenton 2 October1997, necessitated by theonset ofPKD.

2.3 HISTOLOGY

Gonadsremoved from sampled fish were fixed andpreservedinBouin's solution(15 saturated picricacid:5 fonnalin:1 glacial aceticacid)until the time of histological examin ation. Whole testes of immaturefish were fixedandusedforexamination; in maturing fish, only the anteriorportion (-1 13)ofonetestis waskept forprocessing. Tissues selectedfor microscopic examination weredehydratedin ethano l,cleared in xyleneand embedded inPara pl ast PlUS®paraffin. Blocksweretrimmed andcut transversely at5J,lm on a rotary microtome.Sectionswerefloatedonslidessmeared with Mayer's glycerine- albuminfor adhesion anddriedfor24 hours at 400C ona slidewarmer.Sections were stained with Ehrlich'shematoxylin, counterstained with eosin Y andmounted with Permountt».

2.4 DATAANALY SISANDSTATISTICS

2.4.1.Final Maturation Proportions

Differences inthe proportions of fish maturing between treatment replicatesand betweenthe controlandeach experimentalgroup were comparedusinga Generalized Linear

20

Model for the analysis ofbino mialfrequencies.The formatof the model usedwas :

where f isthe observ ed frequency,el'Nisthe expected frequencyandeistheresidualsof thefitof the modelto thedata.The statistic usedwas the G-tes t.,calc ulatedusing Log- Likelihood Ratios (Sakal andRohlf1995;Equations 17.1 and 17.3,p.689-690) . Toleran ce forTypeI errorwas setat5%, and theobservedp-valuefound using the Chi-square freq uen cydistribution in theMinita bll!>statisticalpackage(Minita b®Release9.2for Windo ws ).

Differencesbetween replica tetanksandbetween groups were conduc tedby binomi al compariso n of tbeproportio ns offishidenti fied as maturing(Sokaland Rohlf1995; Chapter 17). Additionall y,a binomial analysisfitting thetreatedgroupsdirectlyto the control was designedas a more sensitivetest for treatmen t effects.Inthis analys is, the control fishwere takenas a fixed effect,ratherthan arandomsamplefrom a largerpopulation. That is,the expec tedfrequencyto which the experimentalgroups werecomparedwastakentobethe observed frequency ofmaturingmalesin the contro lgroup, ratherthan comparinghowthe groups and controldeviatefrom anexpected meanvalue.Altho ugh thisapproachisnot ideal, since it basesthemodel on anexpected frequen cywith inherent error,it isjustifiedin

21

that the internalcontro lgrouparetheonlyfishthatthe treatmentgroups can be legitimately comparedwithfor thedetectionoftreatm ent effects .

2.4.2 GrowthandPerforma nce

2.4.2.1FinalSampling Data

Finalgrowthparameters ofweight,length.condition factor and gonadosomati cindex werebasedon individual measurem entsofallfish ofa given sexualstatus(maturin gmale, immaturemaleor female).Analysis ofVari ance (ANOYA)wasusedtotestforgrowth differences betweenreplicates and treatment groups.,and for differences attributableto sexual statewithin groups.Tukey ' spairwise comparisonswitha famil yerror rateof0.05wereused toisolatedifferences between groups.Fo ralltests.toleranceforTypeIerror.lX,wassetat 0.05. Residual softhe fitof thedatatothemodelswere tested for normalityusing histogram sandnorm alprobabilityplots.Datawhich did not meet theassum ptionsof normalitywerelog-transformedand whennecessary .randomized5000 times togen erate newp-valu es.

2.4.2.2TaggedFish

Analysi sof datafortagged fish was conducted using individualfish measurem e nts.

adjustingretrospectivelyfrom thefinalsam pling forsexualstatus.Analysi s of Covariance (ANCOYA).withtimeascovariate.wasused tocomp areslopesofgrowth parameters

22

between treatments for maturing and inunature male fish.All data were logtransformed to improvethe fit of the data to a linear model. When theoverallANCa VAmodel revealed significant interactions between time and the other explanatoryvariable (either sex or treatment group). the model was discarded. Differences between groups werethen assessed at each discrete measurement timeby one-wayANaVAto determine when differences between treatmentgroupsoccurred .Tukey's pairwise comparisons wereemployed when it wasof interestto know which groupsdeviated significantly from the others.

23

3.0RESULTS

3.1 TEsTICULAR MORPHOLOGY

InArctic cbarr the testes arepaired elongatestructures situated dorsal tothe gutand extending the fulllength of the coelomiccavity.They arephysic allyattached to.and supportedby,theswimbladder alongtheir lengthbymesorchiaarisin g fromthe peritoneum covering the swimbladder. Posteriorly.gonoducts leaving eachtestisuniteto form a commonductwhichopensinto the urogenital papilla.The testes are ofthe unrestrictedtype (Grier 1981 ; Billard 1986;Pudney(995)wher e the tissues are org anizedas a systemof lobules in which spermatogonia are distributedalongtheentirelength . In thenon- reproductivestate.the lobul es are composedof primary spermatogonia, secondary spermatogonia,and Sertoli cells.Extra-lobularmaterialis madeupofLeydig cellsand connective-typetissues. With the onset of spermatogenesis,spe rmatogonia rapidly proliferate,lobular hrmens,into which spermatozoa are eventuallyreleased. formandcysts of germcellsdevelo p.

24

3.2STAGESOFSPERMATOOENESTS

Spermatogenic stageswere classifi ed accordingto thecriteria describedbelow.This classification, although derived specificallyfrom observations made in this study, is comparable topreviousdescriptionsof teleosttesticular developmentdescribedby Henderson(1962) andGrier (1981).

StageI.Pre-sp ermatogonial

Macroscopically,testesat thisstageofdevelopm entarebarely identifiableasdistinct organs.Theyare thin, transparent,thread-likestructuresrunningparallelalongthe swimbladder.Typically,theyare associated with agonadosomaticindex (GSn oflessthan 0.05.Microscopicall y,neither testicularlobulesnor spermatogoniaare distinguishable.The tissueconsistssimply ofamass of diffuse connective-typ e tissue, andsome interstiti alcells that staindarklyandappear tobeLeydig cells(Figur e 2).

Sta ge fia .Imm atureI

Testesoffish classifiedasimmature are more advancedthanpre-sperm ato gonia l individualsin that,macroscopical ly,thetestesdispla yan obvious increase in width.They rema in transparent at this stage,buthave characterist ic pinkish huesnotdetectable atthe earlier stag e, a possible consequence ofincreasedvascularization. GS I valuesare approximately0.05. Undertight microsco py,thelobularstructureof thetestesis

25

distinguishable,.butatthis stageindividuallobules are smallandper section., usuallycontain only one to a few uA" type, or primary spermatogonia(Figure3). This typeof spermatogonium is recognized by its large size, distinctcytoplasmand central, roundnucleus containing usuallya single nucleolusand several chromatin patcheswhichstain darklywith hematoxylin.The tissue is verycompact aslobular lumens havenot yetformed. Mitotically dividing spermatogonia can be identified but theiroccurrence is rare indicating that thetissue isin aslow phase of spermatogonialproliferation .

StagelIb.Im m atur eII

This stage ofspermatogenesis isdistinguished from the previousby a furtherincrease inwidth ofatransparenttestis and GSI values typically approaching 0.10. Atthis stage,the testisdoesnot increase uniformly in width alongits length, resultingina convo luting, irregular shaped structure. Under lightmicroscop y,the development of thetissueis obvious bythe increase in thenumber of spermatogoniaat various stages ofmitotic division.This proliferationof spermatogoniaresultsin therebeing an increasednumberof germina lcells perlobularsection.Mostspermatogoniaare stilluA" type, butnumerous"B"typecan be discrim inated. "B"type spermatogoniaare sligh tlysmaller than primary cellsas their cytoplasmic and nuclear content is reduced.Theirnuclearstn.teture alsodiffers in thatthere areusuallytwo orthree nucleolipresentand thechromatin patches dispersegiving the

26

nuc leusadarker andmore uniformappearance.Lumensbegin to open within the lobules decreasingthedensi ty of the tissue(Figure 4).

Stage II].Trllnsien t

Fishatthis stage of spermatogenesis showdrastic increases in theleve l of mitotic activi ty of the sperma togonia.Spermatogoniacan now be identified dividin g as a group, making cysts of cellsreadilydistin gui shable(Figure5). Most spermatogonia atthis stage are "B"type,and evenfurther reduced in sizefromhaving undergonerepeateddivisions.

Primaryce llsare stillidenti fiable,but are fewer in nwnber andseem to berestric tedtothe boundari es of the lobules. Lobu larlumenswidenandbeco me more distinct. Further irr egul ar increasesintesticularwidth are noticeable macroscopically,however,the testis remains translucentandlittl e changein GSl value (ca. 0.10)is observedfromthe previous stage.

Stage IVa.Spermatocyte FormlltionI

This stage is distinguished by the appearanceofprimary spermatocytes which are initially recognizablebythe aggregation ofthechromatin into onepole within a nucleus (Figure 6). The transformation from spennatogonia "8" into primary spermatocytes occurs synchronouslyin all celts within a cyst, andis not associatedwith any changeincell size.

The differencesbetweenthese cellsare restrictedto nuclearstructure and appearance.In

27

primary spennatocytes, the nucleolidisappear and the cellsundergo thefirstmaturation division of meiosis.Atthispointthe testesundergorapidincreases in size,GSI valuesrise quickly beyond0.10, usuallyto values above1.0. Thetestesare00longer transparent,they become increasinglyopaque, but continue to retain their pinkish coloration.This stagecan onlybeidentifiedduringthe period of timeextendingfrom March until June.

StageIVb.Spermatocyte FormationII

This stage ischaracterizedbythe appearanceofsecondaryspennat ocyt es,theend productsof thefirst meioti cdivision (Figure7).These cells resemble theirprec ursors in appearan ce,but arenoticeabl y smaller.Lobular lumens are no longerverydistin ct as the expanding cysts crowd intothe space.Macroscop ically,the testis is simi larto theprevious stageexceptthatsizeandGSI aresrill increasing.

Stage V. Spermatid Formation

This stageismarkedby the forma tion ofspennatids, theproductsof thesecond meioticdivision.Sperm atidsare distinguis hedfrom secondaryspermatocytesby a further reduction in sizeand the distinctly roundshapeof thedarkly stainingnuclei (Figure8).The testes continuetoincreas einsize andaSIvalue, and becomeincreasingly opaque.

28

StageVI.Spermato'loa

1his stage is characterized macroscopically by distinctchangesinthe coloration of thetestes.As thisstage progresses, bands of white coloration form along the testes correspondingto thetransformationof spermatids into spermatozoa This transformation is recognizedbythe concentrationof thenuclear materialinto onepoleof Utenucleusand the addition of a flagellumtoeach of thecells.As they are produced,spermatozoa are released from their cystsintothe lumen of thetesticular lobuleand are seen as dark concentrations ofsperm heads(Figure9).Primaryspermatogonia can still beobserved along theboundaries of the lobule,supposedlyacting as a reservoir of germinal cellsfor subsequentreproductive cycles .GSI and testicularsizecontinueto increase until theentire testis is composed of spermatozoalyingfreewithin thelobules.

Stage VII.Funct ion al Matu rity

Asspermatozoa formation continues, thetestes become progressively composed of spermatozoalyin g freeinthe lumensoftbelobules(Figure 10). At this stage,the testes are milkywhiteandsperm readilydischarges when pressure is appliedtothe testes.

Theclassification system as described above is summarizedin Table 4. It is important to note that each of thedescribed stages does not occur synchronouslyin allfish ofa population.Although the length and prevalence of thedifferent stagesvariesgreatly

29

withindividuals,anattemptis made herein todescr ibethebasic timeframefor the various stages ordevelopment(fable5).Inaddition, although cells ofan individual cyst develop synchro no us ly, thetestesoranindividualfishusuallycontain cystswithcharac teri sticsor more than one stage (Figure 11). Therefore.for thisstudy,individualfishwere classified basedonthe stageof developmentwhich was most prevalentinthehistol o gical sections.

Severalfishinthis studydisplayedpartial developmenr:of thetestis.completetothe fonn ationofspermatozoainwhich it appeared thatonlyafew cystsproceeded through the maturation cyclewhile the rest ofthe testisremaineduoe-reproducdve (Figure12).Asthis state ofdevelopmentwasobservedinSeptember samples.itisunlikely that these were fisb whichwerejust commencing develop me nt. Theabsenceof anyofthe intermediate, spermatocyt eor spermatid,stagessupports this. Thesefish wereconsidered non- reproductiveas it isunlik ely that sucharedu ced sta teofdevelop ment couldever constitu te a functionally mature male.Inaddition,thes e fish did not display signifi can tly increased GS I values, whichlendssupport to the idea that such developmentwas minor.It is possible that these fishreprese ntindivid ualsin whic hgonada l devel opm entwhich hadbegunwas subs eq uen tly arrested,andthosecystswhichhadproceededtodevelop beyondacertain criticalpoint (StageIV)were compelledto produce spermatozoa.

A smallpercentage (approximately 3%)ofthe fish were already reproductiveatthe beginning of both experiments.Amongstthese fish,thosethat underwenta subseq uent

30

maturationprogressed throughsimilarstages ofspennatogenic activity as those observed for virgin fish.Gonadalrecrudescencebegan by proliferation of the primary spermatogonia in thelob ules and continuedto progress through the stages of developmentwhile concurrently, residual spermatozoa from the previous cyclewere phagocytosed.Itwas observed that the comp leteresorption of spermatozoaremaining from a previous cyclecan take up to six months or lo nger as residual sperm could be identifiedintestis up untilJune of the following year(Figure 13). Fish undergoing a repetitivematuration cycleare easilydistinguished fromvirginfishin that the testes are muchenlargedandhave a whiteto grey colordepending on thedegree of resid ualsperm resorption.Microscopically,pre viously reproductivefish are distinguished from thosefish showing incomplete maturation by the enlarged testicular lumensinthe former{SeeFigures12 and 13).

31

Figure2: Testis ofacompletelyimmature fishatStage I,Pre-spe rmatogonial.No spermatog oniaorlobular structureis visible.L,Leydigcells;C,connective tissue.Bar=50~.

Figure3: Testisofa fish atStage ITa,ImmatureI,of development. Lobu lar structure is definedand primaryspermatogonia, recognizableby thelarg e central nucleolus, arepresent eithersingly orfew per lobule.Lo,lobule wall or bound ary;SgAprimary spenn atogo niwn.Bar=50Jim .

Figure4: Testis of a fish atStageIlb,Immatu reII of developme nt. Lobul es contain greaternumbers of spermatogoniawithboth primary and secondarytypes present. Mitotic proliferationof spermatogonia isobservable and lumens open within thelobules.SgA.primary spermatogo nium;SgB, secondary spermatogo nia;Mi.spermatogo ni umundergoingmitoticdivision; Lu,Iwn en..

Bar=50 urn.

Figu re5: Fishtestis atStageill,Developing.Lobules are large and contain primarily secondaryspermatogonia. Drasti cincreasesinthenumber of mitotically dividing cells are seen andcellsbeginto divide as a cyst.SgA, primary spermatogonium;Mi,mitosisoccurring singly in a spennatogonium; MiG, group mitosesoccurring simultaneouslyincellswithin a cyst. Bar=50 urn.

Figure6: Testis at initial stageof spermatocyte formation and meiotic division, Stage IVa,Spermatocyte FormationI.SgB.secondary spermatogonium;SeA, primary spermatoeytes ;MiG, group mitoses.Bar=50 urn.

Figu re 7: Testis showing secondstage of spermatocyte formation, StageIVb, Spermatocyte Formation II. Primary spermatocytes and secondary spermatccytesare observablewithin testicularlob ules.SgB, secondary spermatogonia; ScA., primary spermarocytes;ScB.secondary spermatocytes;

Lo,lobularwall.Bar=50 um.

Figu r e8: Testis atSpermatid Formationstage ofdevelopment,Stage V. SgA., primary spermatogonium; SgB, secondary spermatogonia; ScA, primary spermatocytes;ScB, secondary spermatocytes;St,spermarids.Note:

magnificationis lower,Bar=100 pm.

Fig u re9: Testis advanced to Spermatozoa stage of development, Stage VI.

Spermatozoa are recognized by the darklystainingspermheadsand the presence of tails.SgA., primary spermatogonium; SgB.sec ondary spermatogonia; SeA, primary spermatocytes; St, spermatids Sz, spermatozoa;

T, sperm tails. Bar"50 um.

FilUr"e10: Fish atstageof Functi ona lMaJurity,Stage Vll.Testi s consists of darkly stainingspermatozoa lying freeinthelumens of lobules.Primary spermatogonialinethelobulewalls.SgA.,primary spcnnatogonium; Lo, lobular wall.Bar-50um,

Flgul'"e11: Testisshowingasynchronyofl obular progression of spermatogenicstages.

Cellswithinacyst proceedthrough developmenttogetherbutnotallcysts are synchro nouswithin thelobule. SgA, primarysperm atogonium;SgB, secondarysperm atogoni a;ScA,primal}'spenn atoc ytes ;ScB,secondary spermatocytes; St,spermatids;Sz,spermatozo a.Note:magnificationislower.

Bar

=

100um.

Figure11: Fishtestis(Septem ber sample)showing an aborted or incompletematuration.

Onlyafewspermatozoa are presentinthe lumensof some lobules amongst spermato goni a. SgA, primary spermatogonium; SgB. secondary spermatogoni aSz, spermatozoa;Lo,lobularwall.Bar-50 J.UO.

Figure13: Testis (June sample) ofpreviously maturefishsbowing resorptionof residual spermatozo aremainingfrom theprevious spawning season.Primary spermatogonia linethe lobule walls.Rz,residualspermatozo aundergoing phagocytosis.SgA,primaryspermalogonjum;MiSgA.,mitoticallydividing spermatogonium.Lo,lobular wall,Lu, lumen.Bar'"SOJim.

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3.3 EFfECTS OFSTARVATION ON SPERMATOGENESISAND MATURAnON 3.3.1 FinalNumbersofFishand Survival

The numbers offishinthe finalsamples,separated accordingto sexualstatus,are showninTable6. Survivalof fish durin gbothexperimentswas veryhigh. Durin g ExperimentI, an accidentalloss of 38 fish from one controltankoccurre das a result of a standp ipe probl em. No natural mortaliti es occurredduringany ofthestarvationperiodsin either the firstor secondexperiment, indicatingthat the restricti on periods were not overly severe. When tem peratures increasedinthe spring , there was an insignificantloss of approximatelyone fishperday.These losses occurredinfish that we reinpoor condition.

Although the fish werediagnosed withPKD,durin g theactual experimentalperiods,no losses were attributab le to thisparas itic infestation.Thepre sence of this diseasewasonly recognize dinthose fish whichwere held beyond the terminati onof ExperimentI.Thesefish werenot usedin any quan titativeanalyses.

40

Table6: Numbersof fishatthetermination ofbotb ExperimentsI and II.

Sox Espert meneI Expe ri m eot lI

N % N %

Maturingmales 354 E~ 39.7

Ed'

291 E~ 28.8 E~

Immaturemales 158 512 17.8 57.5 308 599 30.5 59.3

Females 379 42.5 412 40.7

Total 891 100 lOll 100

3.3.2 Starvation Effects on Spermatogenesis

The staggered sampling schedule used throughoutExperiment I did not permit direct comparisons of the effects of starvation on the spermatogeniccycletobe madebetween the groups during theexperiment.However,sampling throughout theexperimental periodin Experiment II,which wasallconductedon the same day,made such comparisonsfeasible.

The chara cteristics thatwereused at the different sampling timesto assign fish into categorieswheretesticulardevelopmentwas considered to beongoing are described in Table 7.Table 8 andFigure14show the resultsof this sampling .

Ateachsampl ing timeduringtheperiods ofstarvati on (September to April) ,the group whichexhibited thelowest proportion offi shwithsigns oftesticulardevelopmentwas alwaysthat group whichwasstarved inthesix weekspreviousto sampling.Theprobability that the starved group,out ofapossible6 groups, always endedupinthis position by chance

41

Table7: Histologicalcharacteristicsandstagesof developmentdefiningtesticulardevelopmentasusedinclassifying sampledfishoverthetimeperiod of ExperimentII.

Time Stage(s) Characteristics or testesshowingtesticulardevelopment S<p15 11,111 SpermalogonilA,andlor B obviOlls,Ind in lobulci

Ocl27 11, 111 SpcnTlltogoniaA, andlorBobvious,lndin lobulcl

Dec 1I,IlI Spemutoioni.A,andlor D obvioUJ,end inlobu.J«,mllosilclcarly cvidcnt

Janl9 III SpermalogoniaA,andBobvious,incrcasednumbercells/Iobule, lumensopening, mitosisclearlycvident Mar3 1lI, IV SpermatogoniaA, andB obvioos,increlxdnumber cells/lobule,l umensopening,milosis,orcynformllion

Wilhspennalocytes A or B

Ap<2J III,IV SpcrmalogoniaA,llIdBobviOlls,increlScdnumbercells/IobuIe,lumensopening,mitosis,or,Spermatocyte, A,lndB orlnyfw1heradvancedltlte

MayS ^Ill.VII Spermatogonil A,and Bobvious,incltlScd number celli/lobule, lumensopening,mitosis,or,Spermatocytci A,llIdDol lnyfurtheradvanccd sllle

Jul3 IV. VII AtlelSllts pennatocytc stagc, ora nya dvallcedslagc tospcrrnalOlOI Stagc l,Prt-s~'nl(JIOfOnllJl, lilhisnotconsidcrcdtObeshowing tcsticu llTdcvclopmc nt

42

TableS: Numbersandproportions ofvirginmale fish showingsigns of activespermatogenesis during Expcrimentll. D- numberofvirgin male fish exhibiting testiculardevelopment,T«tctel numberofvirgin malefishinsample,P- percentage exhibitingspermatogenesis andtesticular development. Numbersin boldrepresentthegroupshowing thelowest proportionof fishshowing testiculardevelopmentfor the samplingperiod.

SepIIS Oc:121 Dtt. Jan19 Mar2 Apr il May2 S Jul 6

D T P D T P D T P D T P D T P D T P D T P D T P

Inililll

,

21 23.8

Control 2 7 28,6 S 9 SS.S 3 9 33.3 S 7 71.4 S 8 62.S 8 IS 53.3 51 90 56.1

GroupI 2 14 14.3 31121.3 S IS 33.3 , 10 SO , 12 SO 4 10 40 42 974].3

Group 2 4 12 33.3 3 13 23.1 4 II 36.4 1 12 S8.3 S 9 SS.S 817 41.1 S1 112 50.9

Group) 5 12 41.1 8 1457.1 1 911.1 3 9 33.3 5 9 SS.S s 10 SO 49 102

..

Oroup4 5 12 41.7 .10 40 41233.3 ) 10 JO 5 7 71.4 7 14 SO 4796 49

Group S 5 1338.5 6 II 545 4 1330.8 5 12 41.7 6 14 41.9 2

•

^{as 45 10144.6}

43

Sep Oct Dec Jan Mlr Apr May Jul

Figue14: Percentages of malefish showingsignsoftesticulardevelopmentduring ExperimentU.Testiculardevelopm ent ateachsampling time wasasdefined inTable7.Dashedvertical lineindicatestheend of the starvationperiods.

44