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 aqueouscompo- nents (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