"enlightenment Through Science: Illuminating The Path Of Understanding"

VirtualUniversityofPakistanLaboratoryManual:DevelopmentalBiology(ZOO501P)1ContentsS.NoPracticalP.No.1Studyofthestructureofgametesinsomerepresentativecases,i.e.frog,fishandmammal22Hensegginternalandexternalstructuraldetails53Studyofcleavageandsubsequentdevelopmentfrompreparedslidesand/ormodelsinvariousanimalsi.e.,frog,mammalsandchicketc.114Studyoffertilization,earlydevelopmentoffrog/fishthroughinducedspawningunderlaboratoryconditions.165Studyofdevelopmentalstagesofnematodesthroughmicroscopicanalysisofanimaldung196Dactylographyanditsusesindevelopmentalbiology282PracticalNo:1Studyofthestructureofgametesinsomerepresentativecases,i.e.frog,fishandmammalMaleGametesThemalegamete(sperm)issmallandmotileandonlycontributesthemaleshaploidnucleustothezygote.SpermAtypicalhumanspermatozooncanbedividedintothreesections,1.Head,2.Mid-piece3.TailHeadregion:Theheadregioncontainsthreestructures,AhaploidnucleusAnacrosomecapPairedcentriolesThehaploidnucleuscontainsthepaternalDNA(thiswillcombinewithmaternalDNAiffertilizationissuccessful)TheacrosomecapcontainshydrolyticenzymeswhichhelpthespermtopenetratethejellycoatoftheeggThecentriolesareneededbyazygoteinordertodivide(eggcellsexpeltheircentrioleswithintheirpolarbodies)Mid-piece:Themid-piececontainshighnumbersofmitochondriawhichprovidetheenergy(ATP)neededforthetailtomoveTail:Thetail(flagellum)iscomposedofamicrotubulestructurecalledtheaxoneme,whichbendstofacilitatemovement.3Fig1.1:DetailofspermFemaleGametesAtypicaleggcellissurroundedbytwodistinctlayers1.Thezonepellucida(jellycoat)2.CoronaradiateZoneorZonapellucida:Thezonapellucidaisaglycoproteinmatrixwhichactsasabarriertospermentry.Coronaradiate:Thecoronaradiataisanexternallayeroffollicularcellswhichprovidesupportandnourishmenttotheeggcell.Withintheeggcellarenumerouscorticalgranules,whichreleasetheircontentsuponfertilizationtopreventpolyspermy.Eggcellscommonlyincludeahaploidnucleus.Thecytoplasmoftheeggiscalledooplasm.Itcontainsaverylittlemountofyolkinhumanfemaleandthereforeitisalecithal.Inanimalswherehugeamountofyolkispresent,thecytoplasmofeggconsistsoflipoproteins,pigmentgranules,waterandalongwithothercytoplasmicorganelles.4Fig1.2:Detailofhumanegg5PracticalNo:2HensegginternalandexternalstructuraldetailsChickeneggqualityhasbeendefinedbyKramer(1951)asthepropertiesofanygivenfoodthathaveaninfluenceontheacceptanceorrejectionofthisfoodbytheconsumer.Eggqualityisageneraltermwhichreferstoseveralstandardswhichdefinebothinternalandexternalquality.Externalqualityisfocusedonshellcleanliness,textureandshape,whereasinternalqualityreferstoeggwhite(albumen)cleanlinessandviscosity,sizeoftheaircell,yolkshapeandyolkstrength.Fig2.1:Detailofegg1.EggshellTheoutereggshellismadealmostentirelyofcalciumcarbonate(CaCO3)andiscoveredwithasmanyas17,000tinypores.Itisasemipermeablemembrane,whichallowsairandmoisturetopassthroughitspores.Theshellalsohasathinoutermostcoatingcalledthebloomorcuticlethathelpskeepoutbacteriaanddust.62.Outershellmembrane3.InnershellmembraneThesetwomembranes-outerandinner-arejustinsidetheshellsurroundingthealbumen(white).Thetwomembranesprovideanefficientdefenseagainstbacterialinvasionandaremadepartlyofkeratin.Theoutermembranestickstotheeggshellwhiletheinnermembranestickstothealbumen.Whenaneggisfirstlaid,itiswarm.Asitcools,thecontentscontractandtheinnershellmembraneseparatesfromtheoutershellmembranetoformtheaircell(see14below).4.ChalazaChalazaistwistedinoppositedirectionsandservestokeeptheyolkcentered.Themoreprominentthechalazae,thefreshertheegg.5.Exterioralbumen(outerthinalbumen)Theouterthinalbumenisanarrowfluidlayernexttotheshellmembrane.6.Middlealbumen(innerthickalbumen)Theinnerthickwhite(chalaziferouslayer)isadense,matted,fibrouscapsuleofalbumenaroundthevitellinemembraneoftheyolk.Themattedfibrouscapsuleterminatesoneachendinthechalazae,whicharetwistedinoppositedirectionsandservetokeeptheyolkcentered.Thispartoftheeggisaexcellentsourceofriboflavinandprotein.Inhigh-qualityeggs,theinnerthickalbumenstandshigherandspreadslessthanthinwhite.Inlow-qualityeggs,itappearsthinwhite.7.VitellinemembraneTheclearcasingthatenclosestheeggyolk.Whenaneggissaidtobemottled,theyolksurfaceiscoveredwithmanypalespotsorblotches.Thestrengthandintegrityofthevitellinemembraneareveryimportantinpreventingeggyolkmottling.8.NucleusofpanderNucleusofpanderaplugofwhitishyolk,withnoparticularsignificancefordevelopmentandwhosefunctionispurelyanutritiveone,liketherestoftheyolk.9.Germinaldisk(blastoderm)Asmall,circular,whitespot(2-3mmacross)onthesurfaceoftheyolk;itiswherethespermenterstheegg.Thenucleusoftheeggisintheblastodisc.Theembryodevelopsfromthisdisk,andgraduallysendsbloodvesselsintotheyolktouseitfornutritionastheembryodevelops.10.Yellowyolk7Amajorsourceofvitamins,minerals,almosthalfoftheprotein,andallofthefatandcholesterol.Theyolkcontainslesswaterandmoreproteinthanthewhite,somefat,andmostofthevitaminsandmineralsoftheegg.Theseincludeiron,vitaminA,vitaminD,phosphorus,calcium,thiamine,andriboflavin.Theyolkisalsoasourceoflecithin,aneffectiveemulsifier.Yolkcolorrangesfromjustahintofyellowtoamagnificentdeeporange,accordingtothefeedandbreedofthehen.11.WhiteyolkAlsoknownas,thelatebraisanareaofwhiteyolklocatedinthecenteroftheyolk.ItislowerinfatandthereforestandsoutasabrightwhiteareainmanyMagneticResonanceImages.Thespecificfunctionofthelatebraisuncertainbutitmayactasacentralstructurearoundwhichtheadditionallayersoftheyolkareformed.12.Internalalbumen(Chalaziferousalbumen)Theinnerthickwhite(chalaziferouslayer)isadense,matted,fibrouscapsuleofalbumenaroundthevitellinemembraneoftheyolk.Themattedfibrouscapsuleterminatesoneachendinthechalazae,whicharetwistedinoppositedirectionsandservetokeeptheyolkcentered.13.ChalazachalazaeItistwistedinoppositedirectionsandservestokeeptheyolkcentered.Themoreprominentthechalazae,thefreshertheegg.Chalazae,whicharetwistedinoppositedirectionsandservetokeeptheyolkcentered.14.AircellAnairspaceformswhenthecontentsoftheeggcoolandcontractaftertheeggislaid.Theaircellusuallyrestsbetweentheouterandinnermembranesattheeggslargerend.Astheeggages,moistureandcarbondioxideleavethroughtheporesoftheshell,airenterstoreplacethemandtheaircellbecomeslarger.15.CuticleorbloomTheshellisproducedbytheshellgland(uterus)oftheoviduct,andhasanoutercoating,thebloomorcuticle.Thecuticlesomewhatsealstheporesandisusefulinreducingmoisturelossesandinpreventingbacterialpenetrationoftheeggshell.Mostofcuticleisremovedfromtableeggswhentheyaremechanicallywashed.8PracticalNo:3Studyofcleavageandsubsequentdevelopmentfrompreparedslidesand/ormodelsinvariousanimalsi.e.,frog,mammalsandchickCleavageofChickThezygotenucleusundergoesaseriesofmitoses,withtheresultingdaughternucleibecomingpartitionedoff,bycytokinesis,inseparate,andever-smaller,cells.Thefirstcleavageoccursshortlyafterthezygotenucleusforms.Afurrowappearsthatrunslongitudinallythroughthepolesoftheegg,passingthroughthepointatwhichthespermenteredandbisectingthegraycrescent.Thisdividestheeggintotwohalvesformingthe2-cellstage.Thesecondcleavageformsthe4-cellstage.Thecleavagefurrowagainrunsthroughthepolesbutatrightanglestothefirstfurrow.Thefurrowinthethirdcleavagerunshorizontallybutinaplaneclosertotheanimalthantothevegetalpole.Itproducesthe8-cellstage.Fig4.1:DetailofzygotedevelopmentVariousstagesofcleavageinafrogzygoteThenextfewcleavagesalsoproceedinsynchrony,producinga16-cellandthena32-cellembryo.However,ascleavagecontinues,thecellsintheanimalpolebegindividingmorerapidlythanthoseinthevegetalpoleandthusbecomesmallerandmorenumerous.Bythenextday,continuedcleavagehasproducedahollowballofthousandsofcellscalledtheblastula.Afluid-filledcavity,theblastocoel,formswithinit.9Fig4.2:StagesofblastulaFrogBastulaDuringthisentireprocesstherehasbeennogrowthoftheembryo.Infact,becausethecellsoftheblastulaaresosmall,theblastulalooksjustliketheoriginaleggtotheunaidedeye.Notuntiltheblastulacontainssome4,000cellsisthereanytranscriptionofzygotegenes.Alloftheactivitiesuptonowhavebeenrunbygeneproducts(mRNAandproteins)depositedbythemotherwhensheformedtheegg.CleavageofAmphibianCleavageinmostfrogandsalamanderembryosisradiallysymmetricalandholoblastic,justlikeechinodermcleavage.Theamphibianegg,however,containsmuchmoreyolk.Thisyolk,whichisconcentratedinthevegetalhemisphere,isanimpedimenttocleavage.Thus,thefirstdivisionbeginsattheanimalpoleandslowlyextendsdownintothevegetalregionasshowninFig.Intheaxolotlsalamander,thecleavagefurrowextendsthroughtheanimalhemisphereataratecloseto1mmperminute.Thecleavagefurrowbisectsthegraycrescentandthenslowsdowntoamere0.02-0.03mmperminuteasitapproachesthevegetalpole.10Fig4.3:CleavageofafrogeggCleavagefurrows,designatedbyRomannumerals,arenumberedinorderofappearance.(A,B)Becausethevegetalyolkimpedescleavage,theseconddivisionbeginsintheanimalregionoftheeggbeforethefirstdivisionhasdividedthevegetalcytoplasm.(C)Thethirddivisionisdisplacedtowardtheanimalpole.(D-H)Thevegetalhemisphereultimatelycontainslargerandfewerblastomeresthantheanimalhalf.Hrepresentsacrosssectionthroughamidgastrulastageembryo.CleavageinMammalsItisnotsurprisingthatmammaliancleavagehasbeenthemostdifficulttostudy.Mammalianeggsareamongthesmallestintheanimalkingdom,makingthemhardtomanipulateexperimentally.Thehumanzygote,forinstance,isonly100mindiameterbarelyvisibletotheeyeandlessthanone-thousandththevolumeofaXenopusegg.Also,mammalianzygotesarenotproducedinnumberscomparabletoseaurchinorfrogzygotes,soitisdifficulttoobtainenoughmaterialforbiochemicalstudies.Usually,fewerthanteneggsareovulatedbyafemaleatagiventime.Asafinalhurdle,thedevelopmentofmammalianembryosisaccomplishedwithinanotherorganism,ratherthanintheexternalenvironment.Onlyrecentlyhasitbeenpossibletoduplicatesomeoftheseinternalconditionsandobservedevelopmentinvitro.Withallthesedifficulties,knowledgeofmammaliancleavagewasworthwaitingfor,asmammaliancleavageturnedouttobestrikinglydifferentfrommostotherpatternsofembryoniccelldivision.Themammalianoocyteisreleasedfromtheovaryandsweptbythefimbriaeinto11theoviduct(Fig).Fertilizationoccursintheampullaoftheoviduct,aregionclosetotheovary.Meiosisiscompletedatthistime,andfirstcleavagebeginsaboutadaylater.Cleavagesinmammalianeggsareamongtheslowestintheanimalkingdomabout12-24hoursapart.Meanwhile,theciliaintheoviductpushtheembryotowardtheuterus;thefirstcleavagesoccuralongthisjourney.Fig4.4:DevelopmentofahumanembryofromfertilizationtoimplantationCompactionofthehumanembryooccursonday4whenitisatthe10-cellstage.Theembryohatchesfromthezonapellucidauponreachingtheuterus.Duringitsmigrationtotheuterus,thezonapreventstheembryofromprematurelyadheringtotheoviductratherthantravelingtotheuterus.Inadditiontotheslownessofcelldivision,thereareseveralotherfeaturesofmammaliancleavagethatdistinguishitfromothercleavagetypes.Thesecondofthesedifferencesistheuniqueorientationofmammalianblastomereswithrelationtooneanother.Thefirstcleavageisanormalmeridionaldivision;however,inthesecondcleavage,oneofthetwoblastomeresdividesmeridionallyandtheotherdividesequatorially.Thistypeofcleavageiscalledrotationalcleavage.12Fig4.5:ComparisonofearlycleavageA)echinoderms,B)mammalsComparisonofearlycleavagein(A)Echinodermsandamphibians(radialcleavage)and(B)Mammals(rotationalcleavage).Nematodesalsohavearotationalformofcleavage,buttheydonotformtheblastocyststructurecharacteristicofmammals.Thethirdmajordifferencebetweenmammaliancleavageandthatofmostotherembryosisthemarkedasynchronyofearlycelldivision.Mammalianblastomeresdonotalldivideatthesametime.Thus,mammalianembryosdonotincreaseexponentiallyfrom2-to4-to8-cellstages,butfrequentlycontainoddnumbersofcells.Fourth,unlikealmostallotheranimalgenomes,themammaliangenomeisactivatedduringearlycleavage,andproducestheproteinsnecessaryforcleavagetooccur.Inthemouseandgoat,theswitchfrommaternaltozygoticcontroloccursatthe2-cellstage.13PracticalNo:4Studyoffertilization,earlydevelopmentoffrog/fishthroughinducedspawningunderlaboratoryconditionsInducedspawningisoneofthecommonmethodstostimulateovulationofthefishinthehatchery.Severalnaturalandartificialhormoneshavebeenusedtoinducebreedingoffish,whileOvaprimisoneofthemostpopularandeffectivesolutionstostimulatethematurationofmaleandfemalebroodfish.HormoneadministrationThreedosagesofOvaprimweretestedinthisstudy:forfemales,0.3mlkg-1bodyweight(BW),0.5mlkg-1BW,and0.7mlkg-1BW;formales,0.15mlkg-1BW.Thebroodfishincontrolgroupswereinjectedwithphysiologicalsolution(0.9%NaCL)atthedosageof0.25mlkg-1BW.SpermandeggcollectionThefemalebroodfishwereinjectedwiththeirrespectivedosagetwotimes;thefirstinjectionwithhalfofthetesteddosage,andthesecondinjectionwasconducted6hoursafterthefirstinjection,withtheremainderofthetesteddosage.Thefemalebroodfishwasovulated6hoursafterthesecondinjection.Theeggswerecollectedbygentlefingerpressuretotheabdomen.Thecollectedeggswereputinaplasticjarandkeptinaniceboxat4C.Themaleswereinjectedwithasingledoseof0.25mlkg-1BW.ThemalesibanfishwassacrificedwithMS-222(Merck),preparedbydissolving4gofMS-222in5Ltapwater.Thetesteswereremovedandwashedwithphysiologicalsolutionandthenperforatedandchoppedwithscissors.Thesemenwasgentlysqueezedoutandputinatube,andkeptinanicebox(4C).Thesemenwasmixedwithphysiologicalsolution(0.9%NaCl)atdilutionratio1:100(v/v).FertilizationandincubationAtotalof1mleggsand1mlofdilutedspermweremixedhomogenouslyinaplasticbasin(Calista,Volume500mL)andwithapproximately1mloffertilizationsolution(contains4gureaand3gNaClL-1).Theresultingmixturewasleftincontactfor5minutes.Theincubationbasinwasinstalledwith24-hourportableaeratorandLEDlamp.14Atotalof100eggsweretakenrandomlyandthenincubatedinaplasticbasin(Calista,volume:2L),withthreereplicatesatawatertemperatureof25-27Cinawaterheater.Successfulfertilizationwasobserved8hoursafterincubation.Unfertilizedeggswereidentifiedbytheiropacity;theunfertilizedeggswereremovedfromthejar,whilehatchingratewasmonitoredattwo-hourintervals.ThelarvaewerefedonTubifexsp.adlibitumonday5afterbeinghatchedandrearedinthesamejarfor40days.MeasuredparametersanddataanalysisThelatencyperiod,therelativenumberofovulatedeggs,eggdiameter,fertilizationrate,hatchingrate,andsurvivalrateoflarvaeonday40afterhatchingweremeasured.Thelatencyperiodwasdeterminedbycalculatingthetimebetweenthesecondinjectionandovulation.TherelativenumberofovulatedeggswascalculatedbydividingthetotalnumberofreleasedeggsafterOvapriminjectionbythetotalbodyweightofthefemalebroodfish.Fertilization,hatching,andsurvivalrateswerecalculated.ResultsTable5.1:Latencyperiod,fertilization,hatching,andsurvivalrateofsibanfishCyclocheilichthysapogonbasedonOvaprimdosagesNoParameterOvaprimdosage(mlkg-1bodyweight)03571Latencyperiod(hour)10.010.01d7.270.06c7.180.04b7.070.04a2Therelativenumberofovulatedeggs(eggsg-1ofBW)5.50.71a10.676.03b11.000.00b19.0011.53c3Fertilizationrate(%)40.001.41a60.008.19b60.007.55b76.679.07c4Hatchingrate(%)25.552.12a62.942.05b56.955.31b76.002.92c5Survivalrateonday40afterhatched(%)40.002.83a61.951.80b58.601.08b79.999.13c15Ingeneral,therelativenumberofovulatedeggs,fertilization,hatching,andsurvivalrateswereincreasedwithincreasingOvaprimdosage.However,therewerenosignificantdifferencesbetweenthevaluesseenat3mlkg-1BWand5mlkg-1BWdosage.16PracticalNo:5StudyofdevelopmentalstagesofnematodesthroughmicroscopicanalysisofanimaldungMaterialrequiredZincsulphateCentrifugetubeFecalsamplesCentrifugationmachineMicroscopeCoverslipSlidesProcedure1.Fecalsampleswerecollectedfromtherectum,andexaminedbystandardparasitologicalmethodofzincsulphateflotation.Forthismethod,approximately1goffecalmaterialwasdilutedwithtapwaterandpassedthroughasieve(No150)intoacentrifugetube.2.Thetubewascentrifugedat200gfor3min,thesupernatantfluidwasdischargeddowntoapproximately1cmabovethesedimentandzincsulphate(ZnSO47H2O)solution33.2%(w/v)wasaddedtothesediment.3.Afterthoroughdilutionofthesediment,zincsulphatesolutionwasaddedtojustoverthetopofthetubeandacoverslipwasplacedonthetopofthetube.4.Aftercentrifugationat150gfor1min,thecoverslipwascarefullyremovedandplacedonamicroscopeslide.5.Allthepreparationswereexaminedundertheopticalmicro-scopeat100,400and1000magnification.Observation1.ThemostprevalentnematodewasTrichostrongylusretortaeformis(50%)(Fig.6.1),followedbyTrichurisleporisfound18(21.42%)(Fig6.2)animalsandPassalurusambiguusin4(4.76%).172.Mixedinfections(Fig.3)weresignificantlymorecommonthansingleinfectionsFig6.1:PosteriorendofmaleTrichostrongylusretortaeformisFig6.2:PosteriorendofmalePassalurusambiguus18Fig6.3:NematodeeggsTrichostrongylusspecies1.Trichostrongylusspeciesarenematodes(roundworms),whichareubiquitousamongherbivoresworldwide,includingcattle,sheep,donkeys,goats,deer,andrabbits.2.Atleast10Trichostrongylusspecieshavebeenassociatedwithhumaninfections.3.Infectionsoccurviaingestionofinfectivelarvaefromcontaminatedvegetablesorwater.4.HumaninfectionsaremostprevalentintheMiddleEastandAsia,withaworldwideestimatedprevalenceof5.5millionpeople.Lifecycle1.Trichostrongylusspp.areinfectiveasathirdlarvalstageandareingestedalongwithplantmaterialoninfectedpastures.2.Fromthisstagetheydevelopthroughanotherstagetoanadultstage.Theadultparasiteshedseggsintotheintestineofthehostwhicharepassedintotheenvironmentthroughthefeces.3.Theeggsarestrongyletypeandtheyhatchreleasingafree-livinglarvalstageoftheparasite.4.Thetrichostrongylelarvaedevelopthroughasecondlarvalstagetothethirdlarvalstagewhichistheinfectivestage.19Fig6.4:TrichostrongylusspeciesFig6.5:Trichostrongylusspecies-lifecycleReferences1.https://www.researchgate.net/publication/267761569_Endoparasites_found_in_European_brown_hares_Lepus_europaeus_hunted_in_Macedonia_Greece2.https://en.wikipedia.org/wiki/Trichostrongylus20PracticalNo:6DactylographyanditsusesindevelopmentalbiologyDactylographyreferstothescientificstudyoffingerprintsasamethodofidentification.Expertsindactylographyrecognizethatpalmprintshavethecharacteristicofuniquenessandthattheycontainreferencepointsthatenableaccurateandconclusivecomparisonsjustasdofingerprints.Fingerprintscanbeusedinallsortsofways:Providingbiometricsecurity(forexample,tocontrolaccesstosecureareasorsystems)Identifyingamnesiavictimsandunknowndeceased(suchasvictimsofmajordisasters,iftheirfingerprintsareonfile)Conductingbackgroundchecks(includingapplicationsforgovernmentemployment,defensesecurityclearance,concealedweaponpermits,etc.).Fig8.1:DactylographyFingerprintsareespeciallyimportantinthecriminaljusticerealm.Investigatorsandanalystscancompareunknownprintscollectedfromacrimescenetotheknownprintsofvictims,witnessesandpotentialsuspectstoassistincriminalcases.Forexample:AkillermayleavetheirfingerprintsonthesuspectedmurderweaponAbankrobbersfingerprintsmaybefoundonarobberynoteInanassaultcase,theperpetratormayhaveleftfingerprintsonthevictimsskinAburglarmayleavefingerprintsonabrokenwindowpane21AthiefsfingerprintsmaybefoundonasafeInaddition,fingerprintscanlinkaperpetratortootherunsolvedcrimesifinvestigatorshavereasontocomparethem,orifprintsfromanunsolvedcrimeturnupasamatchduringadatabasesearch.Sometimestheseunknownprintslinkingmultiplecrimescanhelpinvestigatorspiecetogetherenoughinformationtozeroinontheculprit.IntheabsenceofDNA,fingerprintsareusedbythecriminaljusticesystemtoverifyaconvictedoffendersidentityandtracktheirpreviousarrestsandconvictions,criminaltendencies,knownassociatesandotherusefulinformation.Officersofthecourtcanalsousetheserecordstohelpmakedecisionsregardingacriminalssentence,probation,paroleorpardon.