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. 660 Vol.67, •o. 4 CandlersTo Determine Incubation Stage [661 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 em- bryo 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 l! Vol.67, No. 4 CandlersTo Determine Incubation Stage [663 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 pho- tograph incubationstages in eggsin 1993 and photographedthe daily stagesof egg developmentin 1994. Photographswere made from known- age eggscollected in the wild and incubatedin incubatorsuntil hatching. Egg photographswere mainly of speciesof smallpasserines with incuba- tion 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 D ['] Vol.67, No. 4 CandlersTo Determine Incubation Stage [665 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.