Physiological Basis of Stomach Oil Formation in Leach's Storm-Petrel (Oceanodroma Leucorhoa)

PHYSIOLOGICAL BASIS OF STOMACH OIL FORMATION IN LEACH'S STORM-PETREL (OCEANODROMA LEUCORHOA)

ALLEN R. PLACE,• NINA C. STOYAN,2 ROBERTE. RICKLEFS,2 AND RONALD G. BUTLER 3 •Centerof MarineBiotechnology, University of Maryland,Baltimore, Maryland 21202 USA, 2Departmentof Biology,University of Pennsylvania,Philadelphia, Pennsylvania 19104 USA, and •Departmentof Sciencesand Mathematics, University of Maine,Farmington, Maine 04938 USA

ABSTRACT.--Weexamined the compositionof stomachoils and the gastrointestinaladap- tations responsiblefor their formation in chicks of Leach'sStorm-Petrel, Oceanodromaleu- corhoa.We used tritium-labeledglycerol triether (a nonabsorbablelipid-phase marker) and carbon-14labeled polyethylene glycol (a nonabsorbableaqueous-phase marker) to follow the gastrointestinaltransit of a homogenizedmeal fed to chicks.Dietary lipids remained in the gastrointestinaltract significantly longer (126 h mean retention time) than aqueouscomponents (12 h) due largely to different rates of gastricemptying. Mean gastricretention times were 0.35 h for aqueoussolutions and 70 h for neutral lipids. Stomachoils from Leach's Storm-Petrelchicks and adults consistlargely (>90%) of neutral lipids (triglycerides,wax esters,and glycerol ethers). The compositionis dynamic,and componentsare partitioned betweenan oil phaseand aqueousphase based largely on water-lipidsolubilities. The paucity of fatty acidsand phospholipidsin stomachoils derivesfrom limited solubility in neutral lipidsand rapidgastric emptying of polarlipid constituents.Proventricular lipolysis of neutral lipids is low (<3%) and we found no evidenceof proventricularlipid secretion.During the week or two before fledging, Leach'sStorm-Petrel chicks regulate the amount of stomachoil by restricting the rate of gastric lipid emptying. Received20 January1989, accepted10 June 1989.

THE ORDERProcellariiformes is entirely pe- of the gastrointestinal tract in petrels slowed lagic. Most speciesfeed on lipid-rich prey, and the drainageof oil into the intestine.The denser adults may commute long distancesbetween food and aqueousmaterials tend to separateas breeding sitesand feeding areas.With the ex- a bottom layer that blocks the accessof oils to ception of diving petrels (Pelecanoididae), all the pyloric opening. Those speciesthat regur- Procellariiformespossess stomach oils (Warham gitate stomachcontents as a defense (e.g. ful- 1977, Jacob 1982). These oils are found in chicks mars) eject the lighter oil first, and they eject and adults, breeders and nonbreeders, in birds solid or partly digestedmaterial only after the captured at sea or at breeding colonies (Jacob oil supply is exhausted.Recent studies on gas- 1982). The ability to concentratedietary items tric motility in Leach's Storm-Petrel chicks into high-energy mealsthat occupysmaller vol- (Duke et al. 1989) and on gastrointestinalpas- umes and have lower osmoticloads may pro- sageof dietary lipids and aqueouscomponents vide an advantage for both adults and chicks. in Gentoo Penguins (Pygoscelispapua) and Stomachoils are important to the breeding ecol- southern Giant Petrels (Macronectesgiganteus) ogy of these pelagic seabirds(Ashmole 1971). (Roby et al. 1989) establishedthat aqueousdi- Nearly 80% of the energy delivered to chicks etary componentsempty rapidly from the pro- of Wilson's Storm-Petrel (Oceanitesoceanicus) re- ventriculusof procellariiformswhereas dietary sidesin stomachoils (Obst 1986) and 30% of the neutral lipids are retained.Moreover, mean re- energy metabolized by incubating Leach's tention times in petrels for lipid phase (20 h) Storm-Petrels (Oceanodromaleucorhoa) resides in and for aqueous phase (10.7 h) were signifi- stomach oils (Ricklefs et al. 1986). cantly longer than in penguins(8.9 h for lipid Cheah and Hansen (1970b) thought that oils phase,and 7.6 h for aqueousphase) (Roby et al. accumulatein the stomachbecause lipids are 1989). digested more slowly than proteins. Warham We describe the mechanisms and kinetics of (1977)suggested that the physicalarrangement stomachoil formation in a representativepro-

687 The Auk 106: 687-699. October 1989 688 PLACE•r AL. [Auk,Vol. 106 cellariiform, Leach's Storm-Petrel. We relate the ony on nearby Little Duck Island. Depending on the mechanicsof stomachoil formation to the pro- experiment,chicks were either taken to the laboratory cessby which procellariiformsare able to use for feeding experimentsand then returned at the ex- the dietary lipids. We show how birds concen- periment'sconclusion, or they were fed labeledmeals trate food into a store,the compositionof which in the field and then replaced in their nest burrows (wherethey remained).All the chicksfrom Little Duck is determinedlargely by propertiesof the meals, Island were of known age. Other studieswere con- the lipids, and a specialized gastrointestinal ducted in 1986 at the Bowdoin Scientific Station on morphology. Kent Island, New Brunswick, just south of Grand Manan Islandat the mouth of the Bayof Fundy.Chicks MATERIALS AND METHODS were removed from burrows and kept in a laboratory on the island until the conclusionof the study. The Radiolabelsand fiuors.--Tri[1-•4C]olein(122 mCi/ agesof Kent Island chicks were estimatedfrom mea- mmol), L-[•4C(U)]proline (266.4 mCi/mmol), [1-•4C] surementsof wing cord (Ricklefs et al. 1985). L-c•-dioleoyl, [dioleoyl- 1-•4C]-phosphatidylcholine Diet of captivesubjects.--During laboratory experi- (114 mCi/mmol), and L-c•-l-palmitoyl-2-oleoyl- ments, chicks were fed freeze-dried krill (Euphausia [oleoyl-l-•4C]-phosphatidylcholine(52.6 mCi/mmol) superba)and freeze-dried copepods(predominantly from New England Nuclear (Boston,Massachusetts), Limnocalanusmacrufus, 87% of biomass).A 20% (w/v) [1-•*C] cetyl alcohol (24 mCi/mmol), D-[•*C(U)] glu- homogenateof either of these items was made with cose(4.28 mCi / mmol), n-[1-•*C]-hexadecane(61 mCi / water and supplementedwith either 35% (w/w) olive mmol) from Amersham(Arlington Heights, Illinois), oil (triglyceridesupplement) or 35%(w/w) cetyl oleate [1-•4C] stearic acid (52 mCi/mmol) from Research (wax supplement). Nightly meals of 5-8 cc (ca. 88- ProductsInternational (Mount Prospect,Illinois) were 141 kJ/meal) were delivered with a disposable5-ml found to have radiopurities > 98%by thin layer chro- syringe attachedto a 10-cm length of polyethylene matography. We used [1,2-3H] polyethylene glycol tubing insertedthrough the esophagusinto the stom- (M.W. 4000, 1.4 mCi/mmol) from New England Nu- ach. All chicksaccepted meals without regurgitation clear (Boston,Massachusetts) and [U-•4C]polyethyl- and exhibited normal development, based on daily ene glycol (M.W. 4000, 15 mCi/g) from Amersham measurementsof wing growth. (Arlington Heights, Illinois) without further purifi- Monitoringaqueous-lipid phase partitioning.--Our ba- cation. Scintillation fluors were ACS II (Amersham, sic strategy was to monitor the movement of carbon- Arlington Heights, Illinois) and BiosafeII (Research 14 labeled dietary constituentsin relation to tritium ProductsInternational, Mount Prospect,Illinois). Du- labeled aqueous-phaseand lipid-phase markers. In plicatesamples were countedtwice on a BeckmanLS such studies it is important that each marker have 3801 scintillation counter, and a quench correction similar propertiesto componentsof the phaseunder curve was derived for different extracts and tissue study(Wiggins and Dawson 1961;Carlson and Bayley types using an H number calibration. The counting 1972a,b). Polyethyleneglycol (M.W. 4000)is a widely efficiencyfor •4Cin the samplesvaried from 88.0%to acceptedaqueous-phase marker (Wingate et al. 1972) 75.4%, and that for 3H from 24.0% to 11.0%. The coef- and glycerol triether is a suitable neutral lipid-phase ficient of variation for replicatesamples averaged 3.0 marker (Morgan and Hofmann 1970; Carlson and + 1.3% (ñSD) for tritium and 1.9 + 0.9% for carbon- Bayley 1972a,b; Meyer et al. 1986; Roby et al. 1989). 14. All radioactivities are expressed in /•Ci (1.00 Thesecompounds are nonabsorbable,nontoxic, non- microCurie = 37.0 kilobequerals). degradable by digestive or bacterial enzymes, and Tracerand carrierwax estersynthesis.--Labeled wax they do not influence the normal absorptionof di- ester([1-•4C] cetyl oleate) and carrier cetyl oleate was etary aqueousnutrients or fat. synthesized(Place and Roby 1986).The radio purity To test the phasespecificity of eachmarker, several (2.2 mCi/mmol) was 98.7%,the overall yield ranged samplesof stomachoil and excretawere subjectedto from 45% to 75%. The chemical purity was >98%. Bligh and Dyer extraction(1959). Of the *H-GTE ra- Glycerol triether [1-(9 cis-octadecenyl)2,3 didodecyl dioactivity, 93 ñ 1.2% (n = 4) was recoveredin the glyceroltriether] wassynthesized as described (Mor- chloroformlayer while 96 ñ 2.5 % (n = 4) of •4C-PEG gan and Hofmann 1970) by BACHEM. The tritiated radioactivity appeared in the aqueousphase. Thin triether (3H-GTE) was prepared by reduction with layer chromatographyof both excretedmarkers in- platinum asa catalyst(New EnglandNuclear, Boston, dicatedthat neither had been substantiallymetabo- Massachusetts)and purified (Roby et al. 1989). Carrier lized (i.e. radiopurity of each was at least 95%of that triacylglycerolwas purified from olive oil (Placeand prior to ingestion). Roby 1986). Samplingproventriculus contents.--We obtained Study areas.--Laboratory experiments were con- stomachsamples with a 5-ml syringeused to gently ducted in 1986 and 1987 at the Mt. Desert Island Ma- suckcontents through a 10-cmlength of polyethylene rine BiologicalLaboratory on Mt. DesertIsland, Maine. tubing from the esophagusinto the proventriculus Storm-petrelchicks were obtainedfrom a nestingcol- (Grubb 1971). Stomach oils were separated from the October1989] StomachOil Formation 689

aqueousand particulate phasesby centrifugation at fed 3 ml of a 1:1 (v/v) mixture of purified olive oil 10,000 x g for 10 min. and cetyl oleate,with glyceroltri [9,10 (n)-•H]oleate Gastrointestinaltransit times for lipidand aqueous com- (12/zCi/ml) added. Aliquots were taken after 15 rain, ponents.--Fourstorm-petrel chicks (age = 33-37 days, and the chicks were returned to their burrows with mass= 58 + 4.98g) were fed a radioactivelytagged, the entrancesblocked to prevent adultsfrom return- homogenizedmeal (2 g) of calanoidcopepods. We ing with food. After 24 h, the chicks were fed 3 ml addedthe nonabsorbablewater solublemarker [1-•4C] of 1:1 (v/v) purified olive oil and cetyl oleatewith polyethyleneglycol (M.W. 4000,PEG) and 50 mg/ml labeled glycerol tri [1-•4C]oleate(0.3/zCi/ml) added. of unlabeledPEG to markthe aqueousphase (1.3/zCi/ Sampleswere removedafter 15 min. We usedlabeled meal). To label the lipid phase,we added (3.7 /zCi/ triglyceridesto estimate lipid volume becauselittle meal) [3H]-labeledglycerol trierher (1-octadecyl-2,3- (<1.0%) lipolysisoccurs in the proventriculusfor up diodecyl glycerol, GTE). The ratio of [3H]/zCi/•4C/zCi to 36 h (Placeand Roby 1986). of the food was 2.83 ñ 0.08 (n = 4). The meals were We calculatedthe apparent volume of aqueousor delivered by tube and syringe. After feeding, each lipid componentsof the proventriculus,(V) at time t chick was placedon a polyethylenemesh platform from marker dilution by the expression(V•C,/C•) - suspendedin a 2-galpolyethylene container to collect V,,where V• is the analyzedsample volume, V, is the excreta.Containers were kept in the dark and main- volume of the marker fed, C• is disintegrations/rain tained at 14 ñ 3øC which simulated the nest environ- (DPM) in the samplevolume, and C, is the DPM in ment. Chickswere periodicallytransferred to clean the fed solution(Diamond et al. 1986).Gastric emp- containers, and the excreta was collected. Unlabeled tying was modeled using the exponentialfunction meals(5-8 g) were fed nightly to chicks.Accumulated (Stubbs 1977, Smith et al. 1984), excreta in each container were extracted with 50 ml of chloroform:methanol(2:1) and filtered on glass V• = V, x e-•'xb• (2) fiber filters to obtain a single phaseextract. Aliquots (5 ml) were placed in scintillation vials and the sol- where V• is the gastric volume at time t, V, is the vent was removedunder nitrogen evaporation.Fluor initial volume fed, and b is the exponentialrate of was added and the vials counted. Food retention time emptying. was estimatedusing the exponentialfunction: Gastriclipolysis of stomachoils.--Six chicksfrom Kent Island(age = 68.6 ñ 3.14days, mass = 73.5 ñ 12.5g) nt = bo - eI-blXl•-b2• (1) and 4 chicksfrom Little Duck Island (age = 58 + 3.5 days, mass= 64.3 ñ 2.5 g) were fed 2 g of lipid where n, is the proportion of marker excretedfrom containing 12.5 •Ci of [•H-GTE]-marker and 5/zCi of initial administrationto time t (Hove 1984).The pa- [•C]-lipid. The carbon-14labeled lipids were either rameterb0 is the asymptoticrecovery of marker,b• is glyceroltri [1-•C]oleatein olive oil or [1-•C]cetyl the rateof excretion(per hour), and b2 is the delayin oleatein cetyl oleate.After 15 rain, a sampleof stom- hours before marker is recovered. The total mean re- ach oil was taken and the excreta collected. An ad- tention time (TMRT) is the sumof b2and the recip- ditional stomachoil samplewas taken 24 h after feed- rocal of b•. Estimates and 95% confidence intervals for ing. Aliquots of stomach oils were counted for dual thethree parameters were obtained by nonlinearleast label radioactivityas describedabove. squaresregression weighted by the varianceof rep- As a more sensitivetest of differentiallipolysis of licates (Johnson et al. 1981). waxesand triglycerides, 6 chickson Little Duck Island Aqueousand lipid emptyingrates of the proventricu- (age = 42.3 ñ 1.37 days,mass = 66 + 9.25 g) were lus.--To calculatethe contributionof proventriculus fed 3 ml of 1:1(v/v) mixtureof purifiedolive oil and emptying time to transit time, we used a double-iso- cetyl oleate with glycerol tri [9,10 (n)-•H]oleate(7.7 tope dilution technique (Diamond et al. 1986). To /zCi/ml) and [1-•C]cetyl oleate (0.14/zCi/ml) added. measureaqueous emptying rates,8 chicks(age = 64.1 Sampleswere taken after 15 rain, and birds were re- ñ 5.0 days, mass= 71.3 ñ 10.0 g) were first fed 5 ml turned to their burrows. The chicks were removed at of an isotonic(300 toOsin/l, accordingto Schmidt- 4, 24, and 48 h after feedingfor further samplingof Nielsen [1960]) glucose/water solution labeled stomach oils. The chicks were allowed to receive meals with•4C-polyethyleneglycol (50 mg/ml PEG; 0.017 from their parents.Aliquots were analyzedfor radio- /zCi/ml). After 5 rain (t = 0), an aliquot was removed activity and chemicalcomposition. and dilution of label determined. After 1, 2, 4, or 8 Stomachoil composition.--Sampleswere taken from h, we fed 2 ml of an isotonicglucose/water solution 12 Kent Islandchicks (age = 66.2 + 3.52days, mass labeledwith a secondmarker, •H-polyethylene glycol = 62.0 ñ 5.75 g) that had been removed from their (50 mg/ml PEG; 0.071 /zCi/ml). Again, 5 rain after burrowsand keptin the laboratoryfor 24 h. We with- each feeding, an aliquot was removed and dilution drew stomachoil from thesebirds 5 rain after they of the label determined.To measurethe emptying were fed labeledglucose or proline,and then again rateof lipid componentsin the proventriculus,6 chicks after 24 h. We alsotook samplesfrom randomlyse- (age = 48.5 ñ 4.6 days, mass= 76.3 ñ 9.65 g) were lectedchicks (age = 50.3 _+1.42 days, mass = 80.3 ñ 690 PLACEET AL. [Auk,Vol. 106

7.20 g) on Little Duck Island for stomachoil com- ach oils and excretaby thin layer chromatography position analysis. (TLC). Aliquots containing equivalent counts were Field observationsindicated that chicksretain sig- spottedon the preabsorbentarea of channeledsilica nificant(5-8 ml) quantitiesof stomachoil during pre- G plates(Uniplates, Analtech), developed with hex- fledgingweight loss.An experimentwas performed ane: diethyl ether: aceticacid (80:20:1),and scanned with 6 chicks(age = 62 _+0.95 days,mass = 66 + 2.44 with a BioScan 100 radiometric scanner. This solvent g, wing cord = 158 ___1.7 mm) to determine whether systemresolves wax esters,triacylglycerols, fatty acids, stomachoil compositionand volume might be reg- fatty alcohols,1,3-diacylglycerols, 1,2-diacylglycer- ulated in older chicks. The 6 chicks were fed a meal ols, monoacylglycerols,and complexlipids, in order of 20%(w/w) homogenizedkrill containing35% (w/ of decreasingrelative mobility (Rf). To separatecho- w) of a 1:1mixture of cetyl oleateand olive oil. [•H]- lesterolesters from wax esters,double development GTE (0.48 •Ci/g) and tri [1-•4C]oleate(0.16 •Ci/g). with hexane: diethyl ether (98:2) was necessary Stomachoil was sampled 15 min later. Chicks were (Christie1982). The carriergas used in the radiometric placed in polyethylene containersto collect excreta. scanningwas P-10 (90%argon, 10% methane) at a flow On the subsequent6 nights, the chickswere fed 3 g rate0.5-1.01/min. The spatialresolution for eachscan of unlabeledhomogenized krill containing35% (w/ wasset at 4 mm. The distributionof label amonglipid w) olive oil. Beforefeeding, we took stomachoil sam- classeswas estimated by integration of the counts ples for lipid and radioactivityanalysis, weighed the under each peak after subtractionfor background. chicks,and measuredwing cords.An additional ex- Theoverall counting efficiency for •4Caveraged 10.5% periment was conductedwith 7 chicks that would whereasthe countingefficiency for 3H averaged0.5% have fledgedif not in captivity. acrossthe plate.Labeled cetyl oleate, cholesterol oleate, Polar and nonpolarlipid repartitioning.--Labeled triolein, oleic acid, and cetyl alcohol were used as phospholipids, L-oe-12-palmitoyl-2-oleoyl-[oleoyl-1-standards to determinethe R! of thesemajor lipid •4C]phosphatidylcholine and L-oe-dioleoyl,[dioleoyl- classes. 1-•4C]phosphatidylcholine, were usedin two separate Quantificationof lipidsusing TLC-fiame ionization de- experimentson phospholipidgastric emptying. We tection.--Operatingconditions for the IatroscanTH- added5 •Ci of labeled phospholipidand 50 •Ci 3H- 10 analyzer (Iatron Laboratories,Tokyo) were as de- GTE to 49.5 g of homogenizedcopepods. We used 6 scribedby Rigler et al. (1983). Sampleswere spotted birds (age = 42.2 ___1.47 days, mass = 76.7 ___14.18 g) on type S-II chromarods(Iatron Laboratories)which for eachexperiment; and we fed eachbird a 5-ccmeal. were activatedby scanningthrough a hydrogenflame We withdrew samples5 min after the meal was ad- twice. Integration was performed by a Hewlett-Pack- ministered, and 1, 3, 5, 24, and 48 h thereafter. The ard 3390-A integrator (Avondale, Pennsylvania). 5-min sampleprovided an initial ratio of the labels Chromarodswere developedin equilibratedfilter pa- for comparisonwith subsequentratios. Becauseen- per-lined glass tanks containing 75 ml of solvent dogenousphospholipase activity was high in the ho- (Harvey and Patton 1981,Kaitarana and Ackman 1981, mogenized copepod meal, we repeated the experi- Parrishand Ackman1983). Samples were prefocused ment using the same labels added to pure olive oil. with acetone.Samples of stomachoil were filtered, In these experiments,the ratio of labeled phospha- centrifuged,and 10 •1 was added to 500 •1 of chlo- tidylcholine to glycerol triether was 5:1. Eachchick roform. Total lipid content was determined by de- (age = 45 ___3.95 days,mass = 80.2 ___12.3 g) was fed veloping the rodsin chloroform:methanol (1:1, v/v) 3 ml of labeledlipid, and proventricularsamples were along with a lipid standard.The solvent front was taken at 1, 2, and 3 h. run to 2 cm, and quantificationwas performed by Six chicks(age = 45.5 + 2.5 days,mass = 77.2 _+ TLC-FID. Lipid quantitieswere determinedby com- 4.9 g) were fed 5 ml of a homogenizedcopepod meal parison with a standard curve for concentrationsof containing[1-•4C] stearic acid (0.31 •Ci/ml) and 3H- olive oil between 1 and 20 •g. For determination of GTE (1.85 •Ci/ml) to determinefatty acid emptying stomachoil lipid compositions,rods were developed rates.Stomach oil sampleswere taken at 5 min, 2 h, in one of severalsolvent systems. The first was 85 ml 5 h, and 24 h. To test whether nonpolar lipids (i.e. hexane: 15 ml diethyl ether: 0.1 ml formic acid for alkanes)accumulate in the proventriculus,we fed 6 50 min. Phospholipidcomposition was determined chicks(age = 41.3 ___1.03 days, mass = 70.5 _+8.12 g) with double sequentialdevelopment. Rodswere first 0.5 ml of a copepodmeal labeledwith n-[1-•4C]hexa- developedfor 50 min in the abovesolvent, dried in decane (1.16 •Ci/ml) and 3H-GTE (1.74 •Ci/ml), and the oven, and burned halfway. Theserods were then we withdrew aliquotsat 5 min, 24 h, and 48 h. developeda secondtime with 75 ml CHC1•:35 ml Meal and stomachoil sampleswere centrifugedfor MeOH: 3.5 ml H20. Finally, sampleswere analyzed 10 min to separateaqueous and lipid phasesbefore for glycerolether componentsusing a doubledevel- we removed aliquotsfor scintillationcounting and opment system.Samples were prefocusedtwice in lipid analysis. acetone,and air-dried between each prefocusing. Rods Tracerrecovery and distribution.--Wedetermined la- were then developedin hexane:diethyl ether:for- bel distribution among various lipid classesin stom- mic acid (99:1:0.05) for 25 min, air-dried for 5 min, October1989] StomachOil Formation 691 and then redeveloped for 20 min. After drying at 100' 1000C for 5 rain, the rods were burned to a point behind the wax esters.After this partial burning, rods were dried and then developed for 40 rain in hexane: diethyl ether: formic acid (80:20:0.1);they were then burned the entire length of the rod. A standardcurve wasgenerated for eachmajor class of lipids observed (wax esters,dialkyl glycerolethers, triglycerides, fatty acids, fatty alcohols,mono- and diglycerides,and ; 40 phospholipids). Enzymaticdetermination of lipid components.--Cho- •. 2o lesterol concentrations were determined with cholesterol oxidase (Sigma Diagnostics,Procedure No. 351). Triglyceride concentrations were determined using glycerol phosphate oxidase after lipase pro- 0 2'0 4'0 6'0 8'0 1•0 120 ductionof free glycerol(Sigma Diagnostics, Triglyc- HoursPost Ingestion eride [GPO-Trinder] Procedure No. 338). Fig. 1. Time coursesof gastrointestinalemptying Statistics.--Resultsare expressedas means + stan- of aqueous(I) and lipid (0) phasesin 4 young (34- dard error of the mean (œ+ SEM); n representsthe day-old) Leach'sStorm-Petrel chicks. The lines rep- number of measurements.Comparisons that involve resentthe fitted exponentialfunction (Eq. 1) that best percentageswere performed on arcsin transformed describedthe rate of gastrointestinalemptying. data. Differenceswere consideredsignificant when P -< 0.05, except for comparisonsinvolving ratios of disintegrations/minute (DPM) of two markers;in this When 65-day-oldchicks were fed an identical case, P -< 0.001 was chosen. In no case were fewer meal, 86.0 + 0.34% (n = 12) of the aqueous than 1,000 DPM/sample observedfor either isotope. markerwas recoveredafter 24 h while only 1.8 Lines were calculated by a nonlinear least squares + 0.2% (n = 12) of the lipid marker was re- iterative procedure(modified Gauss-Newtonmethod) covered.The unexcretedlipid markerwas found (Johnsonet al. 1981). Parameterestimates are given in stomach oils. Older chicks with substantial with their 95% confidence limits in brackets. Other stomach oil (i.e. 9.1 + 1.5 ml, n = 6) excrete statisticalprocedures used are identified in the text. significantly more lipid marker (31.4 + 7.2%,n RESULTS = 6). This higher excretion rate wes accom- panied by a low lipid assimilation efficiency Gastrointestinaltransit times for lipidand aqueous (46.1 + 1.88%, n = 6) as comparedwith 94 + components.--Theexcretion curves differed for 0.35%, n = 12) (Place and Roby 1986, Place et lipid and aqueousmarkers (Fig. 1). The aqueous al. 1986, Roby and Place 1986). The unassimi- marker was excretedsignificantly faster than lated lipids are excretedlargely unhydrolyzed the lipid marker. By 24 h, 83.7 + 1.8% (n = 4) in older chicks. of the aqueousmarker was recoveredcompared Aqueous-and lipid-phaseemptying rates of the with only 11 ñ 1.1% (n = 4) of lipid marker. proventriculus.--Initialaqueous proventricular After 4.5 days,nearly all of the aqueousmarker volumesfor eight 65-day-oldchicks varied from was recovered (94.3 ñ 0.9%, n = 4) while only 0.9-5.1 ml. After 1 h, the gastricaqueous volume 44 + 2.1% (n = 4) of the lipid marker was re- decreased to 0.65 + 0.19 ml. We assume this covered.A major portion (38 + 2.4%, n = 4) of indicated a proventriculusempty of aqueous the unrecoveredlipid marker residedin stom- components.The proventriculusstill contained ach oils aspiratedfrom the proventriculus. 3.42 + 2.09 ml of stomach oil. The average The fitted lines (Fig. 1) best describethe rate aqueousvolume emptied was 6.6 + 1.2 ml, and of gastrointestinalemptying accordingto Equa- the mean emptying time was 0.35 h. When the tion 1. The timefor firstappearance (b•) of either volume of aqueousmaterial in the proventric- marker was similar: 1.4 h [0.4, 2.5] for lipids and ulus exceeded8 ml, emptying was not complete 0.9 h [0.7, 1.1] for aqueouscomponents. The rate by 1 hour. of passage(b•) for aqueouscomponents was 0.09. Gastricemptying of lipid was significantly ßh -• [0.08, 0.10] while that for lipids was 0.008' slower. The initial stomach oil volume was 1.9 h -• [0.007, 0.009]. The total mean retention time + 0.5 ml (n = 6). After 24 h, the volume was (TMRT) was 12.0h for aqueouscomponents and reduced to 1.5 + 0.2 ml (n = 6) with a mean 126 h for lipid components. emptying time of nearly 70 hours. 692 PLACEET AL. [Auk, Vol. 106

TABLIt1. The [3H]-GTE to •4C-labeledlipid ratio in TABLE2. Stomach oil composition (as weight per- stomach oils from Leach's Storm-Petrel chicks 24 h centages)of Leach'sStorm-Petrel chicksfrom Kent after feeding. Island and Little Duck Island.

Kent Island Little Duck Island Little Duck (n = 4) (n = 6) Kent Island Island Initial ratio Final ratio Initial ratio Final ratio Lipid (n = 18) (n = 12) Wax ester 5.8 ñ 0.6 28.6 + 3.5 2.8 + 0.03' 2.9 + 0.006 2.5 ñ 0.02 2.5 ñ 0.07 3.1 + 0.02 • 3.2 _+ 0.114 Triglyceride 65.6 + 2.0 35.9 + 3.3 Glycerol ether 0.8 + 0.2 28.7 + i0.6 Wax ester supplemented diet. Fatty acid 6.3 ñ 0.3 1.8 ñ 0.18 Triglyceridesupplemented diet. Cholesterol 9.5 + 0.9 2.0 + 0.2 Polar lipid a 12 + 0.6 2.9 ñ 0.43 Predominantlymonoglycerides. Gastriclipolysis of stomachoils.--Because the tritium label resided in a nonmetabolizable, determined enzymatically (0.89 ñ 0.01 M, n = nonabsorbablelipid marker while the carbon- 10). We believe the differenceis due to glyceryl 14 label residedin neutral lipid (triglyceride or ether diesters which co-migrate with triglyc- wax ester),lipolysis would lead to an increased erides in our solvent systemand which are not [3H]/[14C]ratio as neutral lipid was hydrolyzed assayedenzymatically. The organic phosphate and carbon-14 label emptied from the proven- content of oil indicated a phospholipid level of triculus.There was no significantchange in the 0.29 ñ 0.26%(n = t0). The relative proportions ratio after 24 h on either Kent Island or Little of eachlipid componentin stomachoils did not Duck Island (Table 1). changesignificantly after 24 h (i.e. in a repeated Similar results were recorded when chicks measures ANOVA, the amount (gg) of each received mealsfrom their parents.The volume component in the 24-h sample and the initial of stomach oil after we fed chicks 3 ml of a t:t samplewas not significantlydifferent). (w/w) mixture of olive oil and cetyl oleate was The compositionof stomachoil samplesfrom 5.9 ñ 0.4 ml (n = 6). On the subsequent two chicks on Little Duck in 1986 and 1987 was nights, chicks received meals from their par- different from that of samplestaken from Kent ents. The ratio of triglyceride to wax ester ra- Island chicks(Fig. 2). Wax esterand triglyceride dioactivity remained constantfor the first 24 h content were nearly equivalent. A component despite a nearly threefold dilution of both with chromatographicproperties similar to di- markers(addition of unlabeledlipid from meals acyl glyceryl ether was detected. Estimated fed by their parentsdiluted the markers).At 48 phospholipid content was 0.21 ñ 0.26% (n = h post-ingestion,wax esters apparently were 12). As with the Kent Island chicks, the relative retained in stomach oils at higher levels than proportions of each lipid component did not triglycerides.However, < 0.4%of either labeled changesignificantly after 24 h in unfed chicks. lipid was present, and the significanceof this Thus, the composition of stomach oil is stable finding is questionable.Significantly faster gas- for at least 24 h provided no dietary lipids are tric emptying was observed in chicks fed by eaten. We believe that lipid secretion by the their parentsthan in thosefed artificially. We proventriculus, if present, is extremely low. attribute this to larger and fresher natural meals. The compositionof stomachoil changedwhen Stomachoil composition.--Instomach oil sam- chickswere fed nightly meals(Fig. 3: A and B). piestaken from randomlychosen chicks on Kent After an initial meal of homogenized krill sup- Island in 1986 (Table 2), the oil contained most- plemented with a t:l mixture of triglyceride ly triglycerides, with only a small proportion and wax ester (and containing labeled triglyc- of wax esters (Fig. 2). Cholesterol concentra- eride and triether), we fed subsequentmeals of tions,determined by TLC-FID (0.12 ñ 0.008 mM, homogenizedkrill supplementedwith triglyc- n = 14) and enzymatically (0.14 ñ 0.01 mM, n eride. Each meal (53 kJ) contained ca. 1.0 g of = 14),were statisticallyindistinguishable (paired lipid. With each meal, triglyceride content of t-test,t = 0.575,df = 13, P -- 0.575).Triglyceride stomachoil increased (3.68 ñ 0.93%/day, R2 = concentrationsdetermined by TLC-FID (0.94 ñ 0.246) while wax ester content decreased(8.36 0.03 M, n = t0) were significantlyhigher (paired ñ 2.52%/day, R2 = 0.319). The free fatty acid t-test, t = 3.96, df = t0, P = 0.0033) than those content remained constant while the cholester- October1989] StomachOil Formation 693

8O ß Kent Island LittleDuck

40'

20'

Fig. 2. Stomachoil compositionfrom Leach'sStorm-Petrel chicks on Little Duck Island (1986 and 1987) and Kent Island (1986). ol content increased (6.1 _+ 1.69%/day, R2 = in the field for Leach's Storm-Petrelsprior to 0.267).These changes in lipid compositionare fledging (Ricklefs et al. 1980a, b, 1985). consistentwith replacementof the volume sam- Additional evidencefor volume regulation of pled (0.364 _+0.014 ml, n = 30) by dietary tri- stomachoil was observedin daily weight gains glycerJde(assuming constant volume). The ini- exhibited by 7 chicks(70 daysold) after removal tial volume of stomach oil was 7.45 _+ 0.459 ml of all stomachoil. Eachchick was weighed prior (n = 6). Sampling removed 4.9% of the stomach to being fed nightly meals of krill supple- oil per day. From fecal collections on the first mented with triglylceride (2.2 g/meal). Aver- two days, 2.3 + 0.53% (n = 12) of [3H]-GTE was agebody massincreased linearly for 7 days(1.12 excretedwith chicksassimilating 94.2 _+0.58% + 0.06g/day, R2 = 0.97)until eachchick gained (n = 12) of labeled triglyceride. The daily de- on average 8 g. Subsequentweighings indicat- crease(7.35 + 0.85%/day,R 2 = 0.705) in specific ed no additional changeJn mass.Aspiration of activity of [3H]-GTE Jsconsistent with addition the proventriculus on day 9 provided 7.75 _+ of dietary lipid equivalent to the volume re- 1.46g of lipjd, and no net changein body mass moved Jn sampling plus the volume emptied from the initial weighing at the start of the from the proventriculusfor assimilationby the experiment. chick (Fig. 3C). This storageof gastriclJpid oc- Fateof polarlipids and hydrocarbonsin stomach cursas body weight decreases(Fig. 3D). Except oils.--Phospholipidsrapidly repartJtionedout for one individual, all chicks had completed of stomachoils (Fig. 4: A and B). In contrast, growth.The dailyweight loss(1.81 + 0.21g/day, fatty acidsand alkanesslowly repartitJonedout R 2 = 0.93) observed is similar to that observed of stomachoils (Fig. 4: C and D). 694 PLACEET AL. [Auk,Vol. 106

DISCUSSION

In Leach's Storm-Petrel, we found that stomach oils originate in the diet, but their composition need not match that of the diet pre- cisely.We determinedthat extensivedifferential lipolysisof wax estersvs. triglyceridesin the 2 proventriculus was not responsible for the [] prevalenceof wax estersin the stomachoils of some species,and that the proventriculus se- creteslittle, if any, neutral lipid. The lipid com- Days positionof stomachoils reflectsnot only the lipid composition of prior meals but also the relative solubility eachclass of lipids hasin the stomachoils previously accumulated.Finally, 1.0 duringthe week or two prior to fledging,chicks S o regulatethe amount of stomachoil by restrict- 0.8 ing the rate of gastricemptying. 0.6 Physiologicalbasis of stomachoil formation.-- Gastricemptying in animals is a highly regu- 0.4 lated and coordinatedprocess. Gastric empty- ' ing is slow when the stomachcontains nutrient- 0.2- rich food (suchas lipids) and is more rapid when the contentsare less energy rich. In humans, 0 O.C the half-time for gastric emptying of a non- Days nutrient saline solution is only 7.9 + 1 min U. while that for a homogenousmeal is on the order of 60 min (Smith et al. 1984). In other mammals,gastric emptying of lipids is nearly half that for aqueous components (Jian et al. ß c 1982, Meyer et al. 1986). Comparablediffer- 0.18 encesexist between passagerates of lipids and aqueouscomponents in seabirds (Roby et al. 1989). In Leach's Storm-Petrel chicks, the half- time for gastricemptying of neutral lipids is 70 h while that for aqueoussolutions is only 0.35 h. A unique gastrointestinalanatomy and cor- 0.10 respondinggastrointestinal motility are impor- ,•_ 0.08 o tant determinants of this slower gastric emp- Days tying of dietary lipid (Duke et al. 1989,Roby et al. 1989).We have describedpreviously the gastrointestinalanatomy and motility pattern fol- lowing a meal in Leach'sStorm-Petrels (Duke

80- et al. 1989). Low gastric motility, slow gastric D emptying, and the positionof the pylorus relative to the proventriculus all contribute to stomach oil accumulation.

Fig.3. Changesin the contentof stomachoils from 62-day-oldchicks fed nightly: (A) triglyceride(.) and wax ester ([•); (B) fatty acid (¸) and cholesterol(0). (C) The glycerol triether marker (ß) was diluted by dietarylipids. (D) Prefledgingweight lossoccurs even Days with nightly feedings. October1989] StomachOil Formation 695

,0.8 The mammalian stomach is the site of differ- ential emptying of lipids and aqueouscompo- -0.6 .•, nents, and it also plays an important role in neutral lipid emulsification.Ingested fats must be emulsifiedor solubilizedin the stomachprior '0.4 0 *• to the formation of micelies in the aqueouscon- tents of the duodenum. Potential emulsifiers - 0.2 i•) that can function in the acid milieu of the stomach include peptic digestsof dietary protein, 0.0 4 complexpolysaccharides, and dietaryphospholipids (Carey et al. 1983). Some enzymatic hy- Time(hours) drolysisof triglyceridesis known to occurwhich resultsin digestionof up to 30%of dietary fats (Careyet al. 1983).The monoglyceridesformed during this gastriclipolysis aid in the emulsification process. In seabirds,we found very little gastric lipolysis(Roby et al. 1986,Place and Roby 1986). '0.5 A possibleexplanation of the efficient assimilation of nonpolarlipids is the unique character of the "enterogastricreflex." In fowl, asin mam- -0.3 mals,gastric motility is inhibited by intraduodenal injections of 0.1 N HCL, 1600 mOsm solutions of NaC1, corn oil, amino acids, or by 0.• 0 I 2 3 intraduodenal balloon inflation (Duke and Evanson 1972, Duke et al. 1973, Sklan et al. 1978). Time(hours) An aspectof this feedbackregulatory mecha-

7.0 nismpeculiar to birds,including seabirds, is the occurrence of one or more intestinal refluxes ß 0.5 • during the period of gastricmotility inhibition 6.5' 0.4 • (Duke et al. 1973, 1989). Intestinal refluxes occur approximatelythree timesmore often in Leach's .,_•0•6.0' C10.6 Storm-Petrels than in fowl (Duke et al. 1989) and involve the movement of intestinal con- 0.2 • • 5.5' tents back to the proventriculus. Thus, gastric o.1 O) emptying is tied closelyto the receptivenessof

5.0 0.0 the duodenum for additional digesta, and the 0 1'0 2'0 30 reflux returns the digesta(both gastricand duo- Time (hours) denal) for further processingin the gizzard. In the process,duodenal products like monoglyc- eridesand fatty acidsare refluxedto the gizzard 2.5 '1,0 alongwith biliary (bile salts,phospholipids, and

.o.8 Fig. 4. Repartitioningof dietaryphospholipids (A) '0.6 L-a-12-palmitoyl-2-oleoyl-[oleoyl-l-14C]phosphati- dylcholineand (B) L-a-dioleoyl,[dioleoyl-l-"C] phos- '.0.4 phatidylcholine, fatty acids (C) ([1-•4C]stearic acid) and hydrocarbons(D) (n-[I-"C] hexadecane)from '0.2 stomachoils. All comparisonsare made relative to [sH]-GTE.The ratio of [3HI #Ci/"C #Ci in stomachoils

0.0 is given on the left ordinatewhile the specificactivity 10 20 30 40 50 (#Ci/ml) of "C is given on the right ordinate.The arrow indicatesthe initial valuesat the time of inges- Time (hours) tion. 696 PL^CEET ^L. [Auk, Vol. 106 triglycerides)and pancreaticproducts (lipases). low their melting temperature,the solubilityof Gastric production of lipid emulsifiers is re- thesecompounds can be estimatedby the equa- placedin birds by productsof normal intestinal tion: lipolysiswhich are refluxedto a highly efficient Log(molefraction solubility) emulsificationmill (the gizzard).We believethat = --ASt(Tm-- T) intestinal reflux is unique to birds and permits 2.303RT' (3) high assimilationefficiencies for suchnonpolar where ASfis the entropyof fusion,and Tm is lipids as wax esters(Roby et al. 1986,Place and the melting point (Patton et al. 1984). For ex- Roby 1986). We also believe that intestinal re- ample, palmitic acid (melting point 61.8øC)has flux is responsiblefor gastricemptying of polar a 0.76% (w/w) solubility at 14øCwhile at 37øC lipids dissolvedin stomachoils. the solubility is 4.72% (w/w) in trioleoylglyc- Effectsof solubilityon stomachoil composition.- erol. Lewis (1966) found glyceryl ether diestersto Recently,the presenceof fossil fuel hydro- dominate stomach oils from Leach's Storm-Pe- carbons in stomach oils has been used to mon- trels. Lewis (1966) felt the presenceof glyceryl itor water quality in the marine environment ether diesterssupported a secretoryorigin, be- (Boersma1986). Our findings with n-hexadec- cause these compounds were not common in ane indicate that ingestedhydrocarbons persist zooplankton and other marine organisms.Our in stomach oils of Leach's Storm-Petrels at least results establishthat the composition of stom- as long as the nonmetabolizablelipid marker ach oils in Leach's Storm-Petrels is determined (TMRT > 70 h). Becauseresidue from a single by dietary lipid and the composition can be contaminatedmeal may persistin stomachoils highly variable(e.g. Fig. 2). Stomachoil com- for extended periods, care must be taken in as- positionis further influencedby the solubility sessingfrequency of contamination. of each dietary lipid classin previously accu- Physiologicaladvantages of stomachoil forma- mulated stomachoils. When major dietary lip- tion.--An energeticadvantage can be realized ids are changed,the compositionof the resident by metabolizingstomach oils in preferenceto stomachoil changesbut not in a manner nec- fat from adiposetissue during fasts(Roby et al. essarilyreflecting the lipid compositionof a 1989). Such gastricstorage of lipids precludes meal(e.g. Fig. 3). The absenceor low abundance the energy costsof synthesizingtriglycerides of polar lipids (such as fatty acids and phos- from assimilatedfatty acids, transporting the pholipidsin stomachoils) is due to limited sol- triglyceridesto the fat depots,and of later mo- ubility in stomachoils and rapid gastricemp- bilizing those energy reserves.These energy tying of these polar lipids with the aqueous costs amount to ca. 25-30% of the assimilated phase(Fig. 4). energy (Ricklefs 1974,Spady et al. 1976). Lipid analysisof mealsdelivered to chickson A correlateof increasingthe energy density Kent Island recorded nearly 21.7 ___10.5% SD of a meal through concentrationof dietary lip- phospholipid (Place and Roby 1986) yet the ids is a lower dietary osmotic(i.e. salt) load for phospholipidcontent of stomachoil was neg- the chick. Marine prey items (particularly in- ligible (<1%). We can explain this from the vertebrates)impose large salt loads. Excessdi- physicalproperties of phospholipids.Phospho- etarysalts are excretedfrom the nasalsalt glands lipids are insoluble in neutral lipids (Sander- through energy dependentmechanisms which mann 1979)and partition into the aqueousphase might consumereserves otherwise available for either as monomersor as micelies.Partitioning maintenanceand growth. Leach'sStorm-Petrels enriches neutral lipids as the aqueousphase of produce highly concentratednasal secretions a meal emptiesrapidly from the stomach.The (Schmidt-Nielsen1960). Through inclusion of solubilitiesof other polar and nonpolar lipids stomach oils in chick meals, procellariiform in stomachoils (suchas long chain fatty acids, chicksmay experiencelower costsof digesting alcohols,alkanes, triglycerides, and chlorinated marine prey items. aromatic and aliphatic hydrocarbons)are de- Salt load reduction also lowers a meal's cal- terminedlargely by their meltingpoints. Above cium load, which for marine prey items can be their melting temperature(i.e. asliquids), all of high. In fat absorption,high dietary calcium thesecompounds are theoreticallymiscible with levels frequently result in calcium soap for- liquid trioleoylglycerol(Patton et al. 1984).Be- mation of fatty acids and subsequentpoor ab- October1989] StomachOil Formation 697 sorptionof fats(Carey et al. 1983).Calcium soaps A secretoryorigin for stomachoils was gen- are poorly solubilizedby bile salts(Graham and erally accepteduntil analysesof mesopelagic Sakman1983) and up to 50%of fecalfat in ste- andbathypelagic marine organisms were found atorrhea has been shown to be calcium soaps to contain high concentrations of wax esters (Bliss et al. 1972). (Nevenzel et al. 1965, Lewis 1967) and Imber Gastric lipid storage could also affect the (1973)found primarily mesopelagicand bathy- digestive efficiency of nonlipid dietary com- pelagicorganisms of freshpetrel meals.In sup- ponents.Isocaloric substitution of caloriesfrom port of a dietary origin was the wide variation fat for nonlipid calories in the diet improves in lipid compositionfound in additionalstudies feed efficiency, increasesweight gain, or both (Lewis 1969; Cheah and Hanson 1970a, b; Watts in chickens (Fuller and Rendon 1977). In hens, and Warham 1976; Clarke and Prince 1976). For a clear positive correlationexists between the example, Cheah and Hansen (1970a) showed percent lipid in a meal and first appearanceof that stomachoil of Fulmarisglaciaiis contained a nonmetabolizable marker (Mateos and Sell mostly triglycerides,whereas stomachoils of 1981,Mateos et al. 1982). This slowing of gas- Puffinustenirostris showed mostly wax esters. trointestinal transit in responseto added di- Watts and Warham (1976) found various mix- etaryfat mayexplain improved feed utilization tures of triglycerides, wax esters, and di- in chickensthat receive lipid supplements.By acylglycerylether in 16 speciesof petrel. This retaining neutral lipids in the proventriculus, extremevariability strongly supported a dietary procellariiforms may slow gastrointestinal origin asproventricular secretions would show transit uniformly and thereby increase assimi- much more uniformity in lipid composition. lation efficiencyof nonlipid dietary compo- Current opinion now holds that these oils are nents uniformly. the product of food breakdownin the acidic Regulationof stomachoil volume.--An unex- and glandular proventriculus (Warham 1977, pectedobservation was the apparentregulation Jacob,1982). We clearly establisha dietary or- of stomachoil volume by Leach'sStorm-Petrel igin for stomachoils in chicksof Leach'sStorm- chicks nearing fledging. While experiencing Petrel. prefledgingweight loss,chicks fed nightly meals retainedsignificant dietary lipid in the proven- ACKNOWLEDGMENTS triculus (ca. 8 ml). In 7 chicks which had completed growth and attained adult body mass We are grateful to S. W. Kress and the National (38.2 + 1.60 g), a previously emptied proven- AudubonSociety for accessto Little Duck Island Sanc- triculus could be refilled with stomach oil with- tuary. We are grateful to C. E. Huntington and the Bowdoin Scientific Station for the use of facilities on in 8 days if fed nightly. The ecologicalsignif- Kent Island. The authors also wish to acknowledge icanceof departing the nest with nearly 7-days the laboratoryassistance of Tong Ma and the gener- energy needs (if indeed the fledglings retain ous gift of the tritiated glycerol triether from James the oil rather than dumping the extra ballast Meyer, Veteran's Administration, Sepulveda, Cali- before flight) remains to be ascertained. fornia. Helpful commentson improving earlier ver- Prior work on stomachoil formation.--Early sionsof the manuscriptwere provided by D. D. Roby studies (Smith 1911; Carter 1921, 1928; Carter and A. H. Brush. This study was supported by the and Malcolm 1927; Rosenheim and Webster National Science Foundation (USA) DCB87-09340, a 1927) described the major lipid component in Lucille Markey Fellowship,and a Departmentof Navy stomach oils as wax esters. Such early work, Contract (N00014-86-K-0696) to Place and by the En- supplementedby other studieswhich failed to dangeredand NongameSpecies Program of the Maine Departmentof Inland Fisheriesand Wildlife (Butler). find wax esters in littoral and pelagic food This is contribution No. 116 from the Center of Ma- sources (Imber 1976, Lewis 1966), led to the rine Biotechnology,University of Maryland. speculationthat stomachoils resultedfrom proventricular secretions or were the result of in- LITERATURE CITED gestedsecretions from the preen gland (Carter and Malcolm 1927). Matthews (1949) provided ASHMOLE,N. P. 1971. Seabirdecology and the ma- morphological and histological evidence that rine environment. Pp. 223-287 in Avian biology, supported a secretory origin of stomach oils. vol 1 (D. S. Farner, J. R. King, and K. C. Parkes, Hagerup(1926) argued for a dietary origin. Eds.). New York, Academic Press. 698 PLACEET AL. [Auk,Vol. 106

BLIGH,E.G., & W. J. DYER. 1959. A rapid method of lated intestinalenvironment. Digestive Diseases total lipid extractionand purification.Can. J.Bio- and Sciences 28: 733-736. chem. Physiol. 37: 911-917. GRUBB,T. C. 1971. Stomach oil in Procellariiformes: BLISS,C. M., D. M. SMALL, & R. M. DONALDSON. 1972. an extraction technique. Ibis 113: 529. The extractionof calciumand magnesiumfatty HAGERUP,O. 1926. Communities of birds in the North acid soapsin steatorrhea.Gastroenterology 62: Atlantic Ocean. Vidensk. Medd. dansk naturhist. 724. Foren. 82: 127-156. BOERSMA,D. 1986. Ingestion of petroleum by sea- HARVEY,H. R., & J. S. PATTON. 1981. Solvent focus- birds can serve as monitor of water quality. Sci- ing for rapid and sensitivequantification of total ence 231: 373-376. lipids on chromarods. Anal. Biochem. 116: 312- CAREY,M. C., D. M. SMALL,& C. M. BLISS.1983. Lipid 316. digestionand absorption.Annu. Rev. Physiol.45: HOVE,K. 1984. Intestinal radiocalciumabsorption 651-677. in the goat: measurementby a double-isotope CARLSON,W. E., & H. S. BAYLEY.1972a. Preparation technique.Brit. J. Nutr. 51: 145-156. and useof glyceryl triether asan indicatorof fat IM•ER, M. J. 1973. The food of Grey-faced Petrels absorption. Brit. J. Nutr. 28: 295-305. (Pterodrornarnacroptera gouldii (Hutton)) with spe- ., & ---. 1972b. Digestionof fat by young cialreference to diurnalvertical migration of their pigs:a study of the amountsof fatty acid in the prey. J. Anim. Ecol. 42: 645-662. digestivetract using a fat-solubleindicator of ab- 1976. The origin of petrel stomachoils: a sorption. Brit. J. Nutr. 28: 339-346. review. Condor 78: 366-369. CARTER,C.L. 1921. A chemicalinvestigation of mut- JACOb,J. 1982. Stomachoils. Pp. 325-340 in Avian ton-bird oil. J. Soc. Chem. Ind. London 40: 220. biology,vol. 6 (D. S. Farner,J. R. King, and K. C. 1928. A chemical investigation of mutton- Parkes, Eds.). New York, Academic Press. bird oil. Part II. Comparisonof stomachoil and JIAN, R., N. VIGNERON,Y. NAJEAN,& J. J. BERNIER. body fat. J. Soc.Chem. Ind. London 47: 26. 1982. Gastricemptying and intragastricdistri- CARTER,J. L., & J. MALCOLM. 1927. Observations on bution of lipids in man. A new scintigraphic the biochemistryof "mutton bird" oil. Blochem. methodof study.Dig. Dis. Sci. 27: 705-711. J. 21: 484-493. JOHNSON,g. L., J. J. CORREIA,D. A. YPHANTIS,& H. CHEAH, C. C., & I. A. HANSEN. 1970a. Wax esters in R. HALVORSON.1981. Analysisof data from the stomachoil of petrels. Int. J. Biochem. 1: 198-202. analyticalultracentrifuge by non-linear squares --, & . 1970b. Stomachoil and tissuelip- techniques.Biophysical J. 36: 575-588. ids of the petrelsPuffinus pacificus and Pterodroma KAITARANA,J.K., & R. G. ACKMAN.1981. Total lipids rnacroptera.Int. J. Blochem. 1: 203-208. andlipid classof fishroe. Comp. Blochem. Phys- CHRISTIE,W.W. 1982. Lipid analysis,2nd ed. New iol. 69B: 725-729. York, Pergamon Press. LEWIS,R.W. 1966. Studiesof glyceryl ethersof the CLARKE,A., & P. A. PRINCE.1976. The origin of stom- stomachoil of Leach's petrel Oceanodrornaleuco- ach oil in marine birds: analysesof the stomach rhoa(Viellot). Comp. Blochem.Physiol. 19: 363- oil from six speciesof sub-Antarcticprocellari- 377. form birds. J. Exp. Mar. Biol. Ecol. 23: 15-30. 1967. Fatty acid compositionof somemarine DIAMOND, J. M., W. H. KARASOV,D. PHAN, & F. L. animals from various depths. J. Fish. Res. Board CARPENTER.1986. Hummingbird digestive Canada 24: 1101-1115. physiology,a determinantof foragingbout fre- 1969. Studies on the stomach oils of marine quency.Nature 320: 62-63. animals: II. Oils of some procellariiform birds. DUKE, G. E., & O. A. EVANSON. 1972. Inhibition of Comp. Blochem.Physiol. 31: 725-731. gastricmotility by duodenal contentsin turkeys. MATEOS,G. G., & J. L. SELL. 1981. Influence of fat Poult. Sci. 51: 1625-1636. and carbohydratesource on rate of food passage --, J. G. CIGNANEK,J. F. 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