Using Candlers to Determine the Incubation Stage Of

J. Field Ornithol., 67(4):660-668

USING CANDIdERS TO DETERMINE THE INCUBATION STAGE OF PASSERINE EGGS

JOHN T. LOKEMOENAND ROLFR. KOFORD• Northern Prairie Science Center National BiologicalService 8 711 3 7th Street SE Jamestown,North Dakota 58401 USA Abstract.--Determiningthe incubation stage of birdeggs can provide important information to investigatorsconducting nesting studies. We describethe use of candlersin the field to determinethe incubationstage in eggsof Lark Buntings(Calamospiza melanocorys) and othersmall birds with an incubationperiod of 11-13 d. Candlingwas accomplished easily usingsimple tools and did not involvethe destructionof eggsor lengthydisturbance of nests.Candling is often preferableto other methodsthat rely on egg mass,mass-growth curves,or immersionof eggsin water.

UTILIZACION DE ALUZADORES PARA DETERMINAR LA ETAPA DE INCUBACION DE HUEVOS DE PASERINOS Sinopsis.--Eldeterminer la etapadel periodo de incubaci6npuede proveerie al investigador informaci6nsumamente importante. Describimos el usode aluzadores,en el campo,parc determinerla etapade incubaci6nde huevosde Calamospizamelanocorys y otros paserinos con periodo de incubaci6nentre 11-13 dias.El aluzadose 11ev6a cabofJcilmente utilizando herramientassencillas que no conllevanla destrucci6ndel huevoo disturbiosprolongados. E1 aluzamientomuchas veces es preferiblea otrosmttodos que dependende la mesadel huevo,curves de incrementoen mesao la inmersi6ndel huevoen ague. In nesting studies, it is often desirable to determine the incubation stage,in days,of an egg or clutch of eggs.By knowingthe incubation stage,investigators can estimatethe dateson which eggswere laid and incubationwas initiated, as well as predict the date of hatchingand fledging.This informationhelps investigatorscalculate the optimum date to determine nest fate and the bestdate to band or mark young (Bart 1977). In previousstudies of waterfowl,we (Lokemoen et el. 1984, Koford et el. 1992) usedfield candlers(Weller 1956) to age embryosin eggsbe- causecandlers are a rapid, simple,and accuratemethod. Eggscan be canalledin the field usinga simplecanalling device, ambient light, and a chart illustrating incubation stages.A candler can be used to view the embryosin mosteggs, but not within shellsthat are denseor too heavily pigmentedto transmitlight. In comparison,other embryo-agingmethods are more difficultto usein the field or requiresacrificing embryos. The flotation method of aging eggshas drawbacksdue to errors in aging larger eggsor later-laideggs (Westerskov 1950). In addition,warm water mustbe carriedin the field. Determiningembryo age from eggdensity requiresaccurate field measurementsof eggmass, length, and width, and developmentof a linear regressionof egg sizeto eggmass from eggsof

• Currentaddress: National Biological Service, Iowa Cooperative Fish and WildlifeResearch Unit, Iowa State University,Ames, Iowa 50011 USA.

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Air ½'ell

Yolk

Shell

Albumen

Inner Membranes

Blood Vessels

Embryo

Heart

FIGURE1. Major componentsof a Lark Buntingegg early in development.The heart is distinguishableas the darkenedarea within the embryo. knownage for each speciesof interest(Green 1984) Examiningthe embryo directly to determine age is a problem becauseone or more eggs in eachclutch must be destroyed(Caldwell and Snart 1974). During nestingstudies in 1991-1993,we adaptedand developeda can- dlingmethod to ageembryos in eggsof passerinesand othersmall birds. 662] j. T. Lokemoenand R. R. Koford J. Field Omithol. Autumn 1996 Lark Bunting

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METHODS Studyprofile.--In 1991, we estimated the age of embryosin passerine eggsin the field usinginformation providedfor waterfowleggs by Weller (1956) and for Mourning Dove (Zenaida macroura)eggs by Hanson and Kossack(1963). The successof these efforts prompted us to rate the ability of our field assistantsto age embryosin 1992. We compared the estimatesof embryo age by six field assistantswith the actual date of hatchingfor 40 passerinenests. The sampleincluded Lark Bunting (Cal- amospizamelanocorys), Horned Lark (Eremophilaalpestris), and Vesper Sparrow (Pooecetesgramineus) clutches that were found during the egg- layingstage and that ultimatelyhatched. Although we had no picturesof passerineegg development in 1992, we describedthe technique to the field assistantsand providedpictures of waterfowlegg development(Wel- ler 1956) scaledfor passerineeggs. The 1992 testswere encouragingso we developeda techniqueto photograph incubationstages in eggsin 1993 and photographedthe daily stagesof egg developmentin 1994. Photographswere made from knownage eggscollected in the wild and incubatedin incubatorsuntil hatching. Egg photographswere mainly of speciesof smallpasserines with incubation periodsof about 11-13 d. Field candlingtechnique.--For candling eggsin the field we used only ambientlight and portablecandling tubes. The tubeswere madeof 15-cm lengthsof foam pipe insulation.Foam insulationwith insidediameters of 1.3 cm and 1.6 cm is suitablefor candlingmost small bird eggs.The foam is easilyshaped around the egg to screenout light from the side when viewing. Eggsare held at the distalend of the candlingtube but are not placed entirelyinside the tube. The egg is held vertically,with the blunt end or air cell of the egg upward and the pointed end of the egg downward. Generally,eggs incubated fewer than 3 d are best viewedwhile looking toward the ground, and eggsincubated 3 d or more are best viewed againsta bright sky.Embryonic developmentis not symmetricalso the egg must be turned on its long axis to view the air cell, embryo, and other detailsfrom variouspositions. In the field, we candied at least 3 eggsin each clutch, becausesome eggsmay be dead or infertile. The eggsof all species,especially passerines, require careful handling because they are quite fragile. Photographingembryonic development.--Most of our photographicwork

FIGURE2. The incubationstages of a Lark Bunting egg.The top two rowsof picturesshow developmentalstages from day 0 through day 5 with the yolk or embryo in the center of the egg. The third row of picturesshows the samedevelopmental stage as row two, but the eggis turned 90ø and the embryois at the sideof the egg.The fourth row shows egg developmentstages of day 7, day 9, and day 11 with the embryohaving grown so that it fills most of the egg interior. 664] j. T. Lokemoenand P,. P,. Koford J. Field Ornithol. Autumn 1996

Robin Dove Blackbird

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on embryonic developmentwas done with Lark Bunting eggs (n = 6), which photographedwell becausethe egg shellsare translucentand un- marked. For comparativepurposes, we alsoexamined and photographed the incubation stages of eggs from Mourning Doves (n = 3), House Wrens (Troglodytesaedon, n = 2), Eastern Bluebirds (Sialia sialis, n = 1), American Robins (Turdus migratorius,n -- 2), Red-winged Blackbirds (Agelaiusphoeniceus, n = 5), Brown-headedCowbirds (Molothrusat• n = 2), and Common Grackles (Quiscalusquiscula, n = 1). Eggswere placed in an incubator that automaticallycontrolled tem- perature (38 C), relativehumidity (50-55%), and turned the eggseach hour. Incubation periods of eggs in the incubator were compared with those reported by Harrison (1979) to ensure that we were quantifying natural embryonic development. Eggswere removed from the incubator each day to photograph and document embryo development.Each egg wasplaced horizontallyinto a cradle that held the egg firmly on its side, but allowed artificial light to be projected from below. The cradle was lined with plasticineto make a light-proofseal. A C-700 Super Chromegaenlarger colorheadwas used as the sourceof light. We found that green or cyan light projected from the enlarger gave the most natural portrayal of blood vesselsand embryo within the yellowish-hued egg interior. A single-lens reflex camera, equipped with a 50-mm macro lens, was held above the egg on a copy stand, with the lens pointed vertically at the side of the egg. We used shutter speedsof 0.5-12 s and F-stopsof 4.0 to 16.0 and three typesof black-and-white film: Kodak Plus-X Pan (125 ASA), T-Max (100 ASA), and Tri-X Pan (400 ASA). Use of brand names does not constitute endorse- ment by the U.S. Government.

RESULTS Candlingproved to be a usefultechnique for determiningembryo ages in eggsof Lark Bunting and specieswith similar incubation periods. Most passetineshave egg shellsthat are sufficientlytranslucent to be viewed easilywith field candlers. Some speciesthat have heavy shell markings, such as the Red-winged Blackbird, can be aged by candling, but other species,such as the House Wren, are more difficult to view.Also, in de- veloping our technique, we found that it was not possibleto age eggsof most shorebirds,such as Killdeer (Charadriusvociferus), Marbled Godwit (Limosafedoa), and Willet (Catoptrophorussemipalmatus), because the dense shell blocks light penetration.

FIGURE3. The incubation stagesof the American Robin, Mourning Dove, and Red-winged Blackbird. The top two rows of pictures show development during the third day of incubation for the three species.The embryo is in the center of the egg in row one, but the egg is turned 90ø in row two and the embryois at the sideof the egg.The third and fourth rows of pictures show egg incubation during the sixth and ninth daysof developmentwhen the embryo beginsto fill the center of the egg. 666] J. T. Lokemoenand R. R. Koford J.Field Ornithol. Autumn 1996

Egg development.--Developmentof eggsin the incubator paralleled that we observedin the field. For the focal speciesin this study,the period of egg incubation that we observedin the incubatorwas similar to that listed by Harrison (1979). For eggsartificially incubated in 1994, the two Lark Bunting eggs hatched in 12 d, the American Robin egg in 12 d, the Mourning Dove egg in 14 d, and one Red-wingedBlackbird egg in 11 d and the other in 12 d. Major egg featuresare depicted in Figure 1. The general stageof incubation is immediatelyapparent as soon as the egg is viewed through the candler.Eggs that are relativelyclear are either infertile, unincubated, or in the first 4 d of incubation. Eggs that show dark patternsmainly in the centerof the egg are in mid-incubation,about 5-8 d, and eggs that are quite dark are in late incubation, about 9-11 d for passerinesor 10--13 d for Mourning Doves.Development of embryos in Lark Bunting eggs is shown in Figure 2 and the comparativedevel- opment of embryosin Mourning Dove,American Robin, and Red-winged Blackbird eggsis shownin Figure 3. A descriptionof the daily stagesof embryonic developmentis presentedin Table 1. For the 40 nests in our test of field assistants, the mean error rate between the hatch date estimated when the nest was located and the actualdate of hatch was1.27 d (SD = 1.037,range = 0-4 d). Given the inherent variabilityinvolved in egg incubation,it is probablynot possible to estimatehatching with much greater accuracy.

DISCUSSION Basedon our results,trained field assistantscan routinely age embryos in eggs of Lark Buntings and similar speciesto within a day or two by candling. However, estimatesof hatching date can vary by two or more daysfor a varietyof reasons,including: (1) environment-inducedchanges in the length of the incubation period, (2) inherent variation among individual embryos, (3) investigatorerror in age estimates,and (4) embryo developmentcontinues throughout the day. The progression through developmental stagesappeared to be the same for many speciesof bird eggs that we examined in the field. By scalingfor longer incubation periods, we were able to obtain reasonably accurate ages for eggs of Mourning Doves and Western Meadowlarks (Sturnella neglecta).Some featuresare recognizableacross a broad range of taxonomic groups.For example, the distinctline seenseparating blood vesselsfrom clear albumen on days4-6 for passerinesis similar to that seen on days8-12 in Mallard (Anas platyrhynchos)eggs (24-d incubation, Weller 1956). Some features seem to be speciesor genus specific.For example, the air cell tips well down the side of the egg in the Mourning Dove and to a lesserextent in waterfowl, but tips little in passerineswe examined. Welty (1982:356) noted that most passerinespecies lay eggsat 1-d in- tervalsuntil the clutch is completed. The day of clutch initiation can be estimatedby counting back one day for each day of incubation and one day for each egg present in the clutch, assumingthat incubation began Vol.67, No. 4 CandlersTo Determine Incubation Stage [667

TABLE1. Descriptionsof the daily incubationstages of AmericanRobin, Lark Bunting,and Red-wingedBlackbird eggs with comparativeinformation for Mourning Dove eggs.a

Day Yolk or embryo Air cell Blood vessels Interior is uniformlylight and Smallor possiblynot visi- None clear;yolk is indistinctlocated ble if egg is stillwarm. equidistantfrom sidesof egg. 1 Yolkbecomes more clearlyde- Identifiable,but remains None fined and moreorange in col- small. or. Smallembryo visible, perhaps Remainssmall. A few narrow vessels at- late in day.Embryo appears as tachedto embryo. smalldark spot (red) in center of egg.Yolk rotates freely and floatstoward upper side with embryoon top. Embryois nowdiscernible as a Quite apparentin all Extendfrom embryoto curveddark expansein the species. form a networkacross centerof the egg.With good one sideof the eggbe- conditionsthe beatingheart low air cell. can be seen,but embryo moves little. Embryoexpands in size;in good Similarto day 3. Dark vesseledareas sepa- light embryocan be seen rated from clear areas movingquite rapidly. by line whichusually extendsvertically mid- egg. Egginterior is largelyobscured Increase in size for all Vesseled areas near air by vessels,but embryocan be species. cell expandand clear seenand it movesregularly. areas decrease. Embryoindistinct often appear- Largerand easilyseen. Completelyencircle the ing as two dark lobes. egg interior. Embryoseen as a dark shapebe- May tip slightlyto sidein Continueto expand low air cell. Embryostill shows passefines. withinthe shell. some movement 8 Developmentsimilar to day 7; Similarto day 7. Similarto day 7. embryosof passerinesshow little movement. 9 Due to embryogrowth, details Prominentfor all species. Vesselscan be seenat become difficult to observe. eachend of egg. 10-11 Egg is almosttotally opaque; At greatestextent. Mainlyobscured. onlylight at air cell.Embryo doesnot moveuntil just before hatching,when bill breaks inner shell membranes. Passerineembryos chip through eggshell and hatch.

aEgg development for all threepasserine species is similarthroughout incubation period and for MourningDoves through day 7. However,air cellsof MourningDoves tilt downthe sideof shell, whereaspasserine air cellsextend across the top of shell.After about 7 days,embryo development in MourningDoves lags behind embryo development in passerinesbecause Mourning Doves require two additionaldays to hatch.As a result,passerine embryos stop movement on day 9, whereas MourningDove embryos stop movement on day 11. Also,it is possibleto viewdetails in passerine eggsthrough day 9, but eggsbecome mostly opaque on days10 and 11, whereasit is possibleto viewdetails in MourningDove eggs through day 11, but eggsbecome mostly opaque on days12 and 13. 668] J. T. Lokemoenand R. R. Koford J.Field Ornithol. Autumn 1996 on the day that the last egg was laid. If incubation in a given species typically begins before the last egg is laid, appropriate adjustmentscan be made. Candling eggscan increaseaccuracy of nesting data collected in field studiesof passerinesbecause the age of the embryo is known. Candling also increasesthe efficiency of field work because fewer nest visits are required to determine egg hatching success.Reducing the number of visits at a nest can reduce bias such as increased nest abandonment and increasedpredation associatedwith nesting studies(Bart 1977, West- moreland and Best 1985). Knowing the age of the embryos would be doubly important for researcherswanting to mark or study young at hatching. The candlerswe employed were easyto use and easyto trans- port. Young (1988) describedanother type of portable egg candler made from a plasticjug, but this styleis bulkier and more difficult to carry.

ACKNOWLEDGMENTS

We thank Ray Thielman for photographic advice and fine work in the darkroom. We appreciatethe helpful commentson the manuscriptby reviewersJane Austin,Larry Igl, Pam Pietz, and Carol Vleck.

LITERATURE CITED

BART,J. 1977. Impact of humanvisitations on arian nestingsuccess. Living Bird 16:187-192. CALD•%LL,P.J., ANDA. E. SN•mT.1974. A photographicindex for agingmallard embryos. J. Wildl. Manage. 38:298-301. GVmEN,R. 1984. Nomogramsfor estimatingthe stage of incubation of wader eggsin the field. Wader StudyGroup Bull. 42:36-39. HANSON,H. C., ANDC. W. KOSSACK.1963. The mourning dove in Illinois. Illinois Nat. Hist. Surv. Tech. Bull. No. 2. 133 pp. HARmSON,H. H. 1979. A field guide to westernbirds' nests. Houghton Mifflin Co., Boston. 279 pp. KOFORD,R. R., J. R. S^UER,D. H. JOHNSON,J. D. NICHOLS,AND M.D. SAMUEL.1992. A stochasticpopulation model of mid-continental mallards. Pages 170-181, in D. R. McCulloughand R. H. Barrett,eds. Wildlife 2001:populations. Elsevier Applied Science, London, United Kingdom. 1163 pp. LOKEMO•N,J. T., H. E DUEBBERT,AND D. E. SHARP.1984. Nest spacing,habitat selection, and behavior of waterfowl on Miller Lake Island, North Dakota.J. Wildl. Manage. 48: 309-321. WELLER,M. W. 1956. A simple field candler for waterfowl eggs.J. Wildl. Manage. 20:111- 113. WELTY,J. c. 1982. The life of birds. 3rd ed. SaundersPublishing, Philadelphia, Pennsylvania. 754 pp. WESTERSKOV,K• 1950. Methodsfor determining the age of game bird eggs.J. Wildl. Manage. 14:56-67. WESTMOVmLAND,D., A.'•DL. B. BEST. 1985. The effect of disturbance on mourning dove nestingsuccess. Auk 102:774-780. YOUNG,A.D. 1988. A portable candler for birds' eggs.J. Field Ornithol. 59:266-268. Received3 Oct. 1995; accepted3 Feb. 1996.