Variability in fatty acid composition of bottlenosedolphin (Tursiops truncatus) blubber as a functionof body site, season, and reproductive state

Asha M. Samuel and Graham A.J. Worthy

Abstract: Odontocete blubber has been shown to be variable in composition and can be separated into strata visually,histologically, and biochemically. The purpose of this study was to examine fatty acid composition of bottlenose dol-phin (Tursiops truncatus (Montagu, 1821)) blubber, and determine if differences exist between body sites, reproductivestates, and (or) seasons. The influence of these variables on blubber composition could aid in the creation of a modelthat would use fatty acid signature analysis to evaluate diet in free-ranging populations. Blubber samples were obtainedfrom freshly dead animals along the Texas and Louisiana coastlines. Samples from nine body sites were analyzed to in-vestigate site variability, and from one site to evaluate differences due to season, reproductive state, and blubber layer.All body sites of animals sampled in the winter were statistically indistinguishable, indicating that biopsy samplescould be obtained from any location on the animal for fatty acid analysis during this season; however, three distinctblubber layers were identifiable, and reproductive states were significantly different in terms of fatty acid composition.Seasonal differences in fatty acid composition were also highly significant for all one-site inner blubber layer samples.Ultimately, the differences in fatty acid composition could have resulted from dietary or physiological factors and needto be examined further.

Résumé : Le lard des odontocètes est connu pour avoir une composition variable et il peut être séparé en strates soit àl’oeil, soit par histologie et analyse biochimique. Le but de notre étude est d’analyser la composition en acides gras dulard du grand dauphin (Tursiops truncatus (Montagu, 1821)) et de déterminer s’il existe des différences entre le lardprovenant des différentes régions du corps ou récolté à différents stades de la reproduction et (ou) à différentes saisons.La détermination de l’influence de ces facteurs sur la composition du lard pourrait servir à créer un modèle pour éva-luer le régime alimentaire en nature à partir de l’analyse des signatures des acides gras. Nous avons obtenu des échan-tillons de lard provenant d’animaux morts, mais encore frais, sur les côtes du Texas et de la Louisiane. Nous avonsanalysé les prélèvements provenant de neuf sites corporels afin de déterminer la variabilité en fonction de la répartitionsur le corps, ainsi que des prélèvements à un seul point pour analyser les différences reliées à la saison, à l’état de re-production et à la couche particulière de lard. Les échantillons pris dans les différentes régions du corps sont statisti-quement impossibles à distinguer en hiver, ce qui indique qu’à cette saison les prélèvements de biopsie peuvent êtrepris indifféremment sur n’importe quelle partie du corps pour l’analyse des acides gras. Cependant, il est possible dedistinguer trois couches distinctes de lard et la composition en acides gras varie aussi en fonction de l’état reproductif.Il y a finalement des différences saisonnières de composition en acides gras dans tous les échantillons de lard de lacouche interne prélevés à un même site. La cause fondamentale de ces différences de composition en acides gras peutêtre reliée à des facteurs alimentaires ou physiologiques et doit être examinée plus à fond.

[Traduit par la Rédaction] Samuel and Worthy 1942

Introduction

An understanding of the foraging ecology of marine mam-mals is critical to evaluating their role in marine ecosystems.This area of research has been studied in greater detail overthe last decade because of recent advances in both technol-ogy and analytical techniques. Stable isotopes have been

successfully used to determine dietary habits of many verte-brate species, including marine mammals (Gilmour et al.1995; Pond et al. 1995; Smith et al. 1996; Kurle and Worthy2001, 2002). Information derived using this methodology islimited to interpretation of trophic level or geographic originof prey (e.g., Gilmour et al. 1995). More recently, fatty acidsignature analysis (FASA) has been utilized to evaluate

Can. J. Zool. 82: 1933–1942 (2004) doi: 10.1139/Z05-001 © 2004 NRC Canada

1933

Received 5 March 2004. Accepted 12 January 2005. Published on the NRC Research Press Web site at http://cjz.nrc.ca on23 February 2005.

A.M. Samuel1,2 and G.A.J. Worthy.2 Physiological Ecology and Bioenergetics Laboratory, Texas A&M University, Suite 105,5001 Avenue U, Galveston, TX 77551, USA.

1Corresponding author (asamuel@mail.ucf.edu).2Present address: Department of Biology, University of Central Florida, 4000 Central Florida Boulevard, Orlando, FL 32816-2368,USA.

predator–prey feeding interactions in marine systems. Thisapproach has been used to examine the fatty acid composi-tion of individual potential prey and match it to that of pred-ator body tissues to determine dietary history (Fraser et al.1989; Iverson 1993; Iverson et al. 1995, 1997, 2004; Kirschet al. 2000).

Lipids in marine systems have been determined to becomplex and diverse, and many long-chain polyunsaturatedfatty acids (PUFAs) can be traced back to a specific organ-ism and geographic location in the food chain (Fraser et al.1989; Iverson et al. 1997). It is well documented that dietaryfatty acids are incorporated virtually unchanged into the bodytissues of monogastric carnivorous animals (e.g., Iverson1993; Gilmour et al. 1995; Pond et al. 1995; Kirsch et al.1998; Logan et al. 2000). By examining the fatty acid com-position of fat in carnivorous marine mammals, it has beenpossible to discern diet. Fatty acids have been used as dietindicators primarily with pinnipeds (Smith et al. 1996;Iverson et al. 1997, 2004; Käkelä and Hyvärinen 1998);however, few published studies exist that match dietary fattyacids to body-tissue fatty acids in cetaceans (e.g., Hooker etal. 2001; Budge et al. 2004).

Marine mammal blubber plays a role in a variety of func-tions, including thermoregulation, streamlining, buoyancy,and energy storage (Ryg et al. 1988). Previous research hasshown that the lipid content and fatty acid composition ofthis tissue vary among species and populations dependingupon the physiological and environmental challenges facingan animal not only in a particular geographic location, butalso during a particular time of year. The lipid content ofblubber is known to be affected by the nutritive condition ofan animal (Aguilar and Borrell 1990), environmental tem-perature changes (Ryg et al. 1988; Aguilar and Borrell 1990;Worthy and Edwards 1990), and seasonal changes in bothprey quality and availability (Barros and Odell 1990;Cockcroft and Ross 1990; Scott et al. 1990; Shane 1990;Kirsch et al. 1998; Bradshaw et al. 2003). Fatty acid compo-sition of blubber has also been shown to vary among ageclasses (Koopman et al. 1996, 2003; Iverson et al. 1997;Kirsch et al. 1998) and between genders (West et al. 1979a;Cockcroft and Ross 1990), and reproductive states (Ackmanet al. 1975b; Aguilar and Borrell 1990).

Cetacean blubber has been described as vertically strati-fied. Connective-tissue density has been shown to increasefrom the outermost to the innermost surfaces (Ackman et al.1965, 1971), and fatty acid composition has also been shownto vary among layers (Ackman et al. 1965, 1975a; Aguilarand Borrell 1991; Koopman et al. 1996). In some species, atleast two distinct blubber layers were identified, one locatedjust under the epidermis and one immediately above themuscle layer (Ackman et al. 1965; Koopman et al. 1996).Other cetaceans, such as bottlenose dolphins (Tursiopstruncatus (Montagu, 1821)), have been shown to exhibit threelayers within the blubber (Ackman et al. 1975a; Aguilar andBorrell 1990). It also has been suggested that each blubberlayer performs a different function, with the stable outerlayer used for structural support and the more variable innerlayer used for energy storage (Aguilar and Borrell 1990).

The first objective of the present study was to examineblubber fatty acids at several sites on the body of bottlenosedolphins to determine if blubber has a relatively constant

biochemical composition within an individual animal. Thesecond objective was to determine if differences in fatty acidcomposition exist between layers, reproductive states, andseasons at a single site on the body. Since most biopsy sam-ples from wild populations are taken at one standard site, onthe dorsal surface just behind the trailing edge of the dorsalfin (Aguilar and Borrell 1991), this site was chosen to evalu-ate possible differences in fatty acid composition. By exam-ining the fatty acid composition of bottlenose dolphinblubber in detail, it may then be possible in the future tostudy the foraging ecology of this species using FASA.

Materials and methods

Sample collectionThe Texas Marine Mammal Stranding Network collected

blubber samples from dead-stranded or incidentally caughtbottlenose dolphins along the Texas and Louisiana coastlinesbetween 1990 and 2000. Only adult animals (standard lengthgreater than 200 cm; Turner and Worthy 2003) were used forthis study. Blubber was collected from animals that had diedwithin the previous 12 h and showed no signs of skin peel-ing or bloating (National Marine Fisheries Service code 2;Geraci and Lounsbury 1993) to eliminate decomposition as apotential variable. Samples were frozen at –20 °C immedi-ately after collection. Gender, reproductive state, and bodycondition were determined at necropsy. It was possible todefine two sampling seasons based on changes in coastalwater temperatures throughout the year. “Summer” animalswere sampled between April and October (water temperature26 ± 0.49 °C (mean ± SE)), and “winter” animals were sam-pled between November and March (mean water tempera-ture 16 ± 0.52 °C). In the first phase of the study, blubberwas analyzed from five animals that were sampled fromeach of the dorsal, lateral, and ventral body surfaces at threegirth rings (axilla, trailing edge of the dorsal fin, and anus)for a total of nine sites (Fig. 1). These five animals consistedof three lactating and two nonlactating dead-stranded fe-males from the winter season. Only winter samples wereused to minimize the effects of season as a variable. For thesecond half of the study, samples were analyzed from onebody site (trailing edge of the dorsal fin, dorsal surface) for22 animals (19 dead-stranded, 3 incidentally caught), whichincluded the 5 animals from the first phase of the study. Ofthese 22 animals, 11 were male and 11 were female (5 lac-tating and 6 nonlactating). Within each gender there werefive summer and six winter animals.

Sample preparationAfter the samples were collected, skin and muscle were

removed and blubber was separated into three layers (outer,middle, and inner), based on visually discernible differencesin structure and color (Fig. 2). The transition zone betweenlayers was removed to ensure that each layer was sampledwith no cross-contamination. Two subsamples, each weigh-ing approximately 0.5 g, were removed from each layer.Lipids were then independently extracted from each of theblubber samples with a solution of 2:1 chloroform–methanolusing a modified version of Folch et al.’s (1957) method (seeIverson 1993; Iverson et al. 1997) as described in Samuel(2000).

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Esterification was performed by adding 8% boron trifluor-ide in methanol to the extracted lipids followed by a seriesof hexane extractions to isolate and purify the resultant fattyacid methyl esters (FAMEs) (see Iverson et al. 1997; Samuel2000). FAMEs were stored at –20 °C until further analysis.FAMEs were also produced using acid-catalyzed methyl-ation (see Iverson 1988) to cross-validate the esterificationmethod described above. Esters were created by adding1.5 mL of dichloromethane and 3.0 mL of 0.25 mol sulfuricacid/L in methanol to the extracted lipids, and the resultantsolution was placed in the dark for 72–96 h. After this timeperiod, FAMEs were extracted using hexane and distilledwater as previously described.

Gas–liquid chromatography was performed on FAMEs us-ing a Perkin–Elmer Autosystem XL gas chromatograph fit-ted with a 30 m × 0.25 mm i.d. column coated with a0.25 µm thick 50% cyanopropyl polysiloxane film (DB-23,J&W/Agilent, Folsom, California). The gas chromatographwas connected to a computerized integration system usingthe software package Turbochrome Workstation® (versions 4and 6.1.2.0.1; Perkin–Elmer Instruments LLC 2000). Heliumwas used as the carrier gas. The injector temperature washeld at 250 °C and the detector temperature remained at270 °C. The initial oven temperature, 153 °C, was main-tained for 2 min, then ramped at 2.3 °C/min to 174 °C, heldfor 0.2 min, then ramped at 2.5 °C/min to a final tempera-ture of 220 °C and held for 3 min (S.J. Iverson, personalcommunication). Total program duration was 32.73 min.Fatty acids in the sample were identified from known stan-

dard mixtures (68B, 68D, 87, 463, Nu-Chek Prep, Inc., Ely-sian, Minnesota) and secondary reference mixtures. Fatty ac-ids were named using International Union of Pure andApplied Chemistry nomenclature, number of carbons : num-ber of double bonds, where n–x indicates the position of thefirst double bond in relation to the terminal methyl end ofthe fatty acid, and were converted to percent amount of thetotal sample. All unknown peaks within the sample werequantified but not used for further analysis, since it was notpossible to determine their origin.

Statistical analysisAfter gas-chromatographic analysis, duplicate subsamples

were averaged to produce one value for each fatty acid. Fattyacid percentages were divided by the total amount of identi-fied fatty acids in the sample to standardize the data. Onlyfatty acids with chain lengths ranging from 12 to 24 carbonsand amounts greater than or equal to 0.5% that were foundin all samples were used for further analysis. The amountsof saturated fatty acids (SFAs), monounsaturated fatty acids(MUFAs), and PUFAs were calculated for each sample,along with the total amounts of n–3 and n–6 fatty acids andthe ratio of n–3 to n–6 fatty acids. Classification and regres-sion tree (CART) analysis was used for statistical compari-sons (SYSTAT® version 7.0.1 (SPSS Inc. 1997) and S-PLUS® version 6.1 (Insightful Corporation 2002)). This pro-cedure was selected because it performs multiple simulta-neous comparisons of many variables, which in this caseconsisted of 16 fatty acids. The algorithm makes binary

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Fig. 2. Transverse section through the blubber of a bottlenose dolphin sampled at the trailing edge of the dorsal fin on the dorsal sur-face. Three blubber layers are visible along with the skin and underlying muscle tissue.

Fig. 1. Blubber-sampling locations (×) on the dorsal, lateral, and ventral surfaces of the body of bottlenose dolphins (Tursiopstruncatus). Samples were collected from three girth rings at the axilla, trailing edge of the dorsal fin (TED), and anus on each surface.

splits, dividing the data into homogeneous groups based onmaximum deviance between nodes, until a terminal node isreached in which no further differences can be detected be-tween samples. Misclassification ratios (MRs) were calcu-lated as the number of misclassified samples in a node overthe total number of samples in a node. The MRs were con-sidered to be similar to a “p value”, whereby a misclassifi-cation ratio less than or equal to 5% was deemed statisticallysignificant. Terminal nodes that were completely distinctfrom each other had a MR of zero.

Results

No differences were observed between dead-stranded andincidentally caught animals, since they did not separate fromeach other during CART analyses. Similarly, CART analysesshowed no differences between the two methods of trans-esterification. Sixteen fatty acids were employed in allCART comparisons because they were found in amountsgreater than or equal to 0.5% of total fatty acids: 14:0,14:1n–5, 16:0, 16:1n–7, 16:1n–9, 16:3n–4, 18:0, 18:1n–7,18:1n–9, 18:2n–6, 20:4n–6, 20:5n–3, 22:4n–6, 22:5n–3,22:5n–6, 22:6n–3.

To examine potential differences in fatty acid compositionat different body sites, samples were analyzed from five ani-mals at nine body sites (N = 135 samples). Body sites werestatistically indistinguishable from one another using CARTanalyses (MR = 51/135; p = 0.38). Blubber layers hadstatistically significant differences in fatty acid composition(MR = 5/135; p = 0.04). The middle layer grouped with theouter layer on the same side of the tree 90% of the time. Theouter and inner layers separated from one another at the firstCART split, with only one misclassification (1/90; p = 0.01).

The strongest classifier of all nine-site samples wasreproductive state, with 97% of the samples from non-lactating females separating from the lactating females at thefirst CART split. Within each reproductive state, layers haddifferent fatty acid compositions, but the differences werenot statistically significant for either nonlactating (MR =3/54; p = 0.06) or lactating (MR = 5/81; p = 0.06) females.

A larger set of animals were compared at a single bodysite to examine potential differences in fatty acid com-position between seasons, reproductive states, and blubberlayers. Using CART analysis, 22 animals were examined.Samples that had not produced a clear, accurate chroma-togram were excluded from further analysis (two winter fe-male middle layers, one winter female inner layer, and twowinter male inner layers), yielding a total of 61 samples inthe analysis. CART analysis indicated differences in fattyacid composition between summer and winter animals(MR = 9/61; p = 0.15) and for gender/reproductive states(MR = 9/16; p = 0.15), but the differences were not statisti-cally significant. Outer and inner layers were separate afterthe initial split, while middle layers separated on both sidesof the tree, thereby increasing the p value (MR = 12/61; p =0.20) (Fig. 3).

Differences were found between all layers and seasons forSFAs, MUFAs, PUFAs, and total n–3 and n–6 fatty acids.The proportion of both SFAs and PUFAs increased from theouter layer inwards to the inner layer in both summer andwinter samples (Fig. 4). The proportion of MUFAs, however,decreased from outer to inner layers in both summer andwinter samples (Fig. 4). Summer samples were relativelysimilar to winter samples in terms of SFAs, but had largeramounts of MUFAs and smaller amounts of PUFAs thanwinter samples (Fig. 4). The amounts of n–3 and n–6 fatty

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Fig. 3. Classification and regression tree analysis for all samples (N = 61) from 22 animals at the trailing edge of the dorsal fin, dorsalsurface, separated according to blubber layer (outer, middle, and inner). Classification of each node is based on the majority of sam-ples within that node. Ratios in each node represent the number of outer:middle:inner samples within each node. Fatty acids used toseparate groups are placed between the lines, with splitting values (percent amount of fatty acid) on either side.

acids generally increased from outer to inner layers, with n–6 fatty acids found in similar concentrations between sea-sons, and n–3 fatty acids found in larger amounts in the win-ter than in the summer.

The inner blubber layer (N = 19 animals) was examined ingreater detail using CART analyses because it is thought tobe the most energetically active layer and therefore the mostappropriate to study dietary history. The inner blubber layerhad greater amounts of 16:0, 18:0, 22:5n–3, 22:5n–6, 22:4n–6 and 22:6n–3 and smaller amounts of 14:1n–5, 16:1n–7,16:1n–9, and 16:3n–4 than the middle and outer layers. Allthree layers had comparable amounts of 14:0, 18:1n–7,18:1n–9, 18:2n–6, 20:5n–3, and 20:4n–6.

Differences in fatty acid composition between reproduc-tive states and seasons were evaluated within the inner blub-ber layer (Fig. 5). Males, nonlactating females, and lactatingfemales were statistically indistinguishable using CARTanalyses (MR = 6/19; p = 0.32); however, males and lactat-ing females grouped on the same side of the tree. Males hadlarger amounts of 18:1n–9 and 22:4n–6 than all females(Fig. 5). Nonlactating females had greater amounts of 20:5n–3than males (Fig. 5). No trends were seen between reproduc-tive states for all other fatty acids examined. Summer andwinter animals also had significantly different fatty acidcompositions in their inner blubber layers (MR = 1/19; p =0.05). Samples from animals in the summer season had

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Fig. 4. Error plots of changes in amounts of saturated (A), monounsaturated (B), and polyunsaturated (C) fatty acids between blubberlayers (outer, middle, and inner) and seasons (summer: April to October; winter: November to March) in 22 animals sampled at thetrailing edge of the dorsal fin, dorsal surface. Values plotted are means ± standard error.

larger amounts of 14:1n–5, 16:1n–7, 16:1n–9, and 18:1n–9(Fig. 5). Samples taken from animals in the winter had more16:0, 22:5n–3, and 22:6n–3 (Fig. 5). The amounts of allother fatty acids analyzed were similar in the two seasons.

Discussion

Overall fatty acid compositionBoth the total array of fatty acids measured as well as

those found in large proportions (greater than 5% of total oc-currence; see Samuel 2000) in the present study were consis-tent with those found in other cetaceans (Ackman et al.1965; Williams et al. 1987; Koopman et al. 1996; Guitart et

al. 1999; Smith 2002), and represented both dietary and bio-synthesized fatty acids. However, it is important to note thatdifferent lipid extraction and analysis techniques were used,different arrays of fatty acids were examined, and fatty acidcomposition was not always expressed as relative percentamount of total fatty acids identified.

Sampling-site comparisonsBased on fatty acid composition, body sites were indistin-

guishable in winter female animals. These results are consis-tent with those from studies on other marine animals, includingcetaceans (Koopman et al. 1996), pinnipeds (Iverson 1988),and sea turtles (Holland et al. 1990). Koopman et al. (1996)

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Fig. 5. Differences in fatty acid composition between the inner blubber layers of adult bottlenose dolphins (N = 19 animals) for differ-ent reproductive states (nonlactating females, lactating females, males) (A) and seasons (summer: April to October; winter: Novemberto March) (B). Bars represent the mean ± standard error.

did note that blubber composition at the caudal pedunclewas different from that at the other sites, but suggested thatthese changes were due to the locomotory requirements atthat site.

Studies on cetaceans by Ackman et al. (1975a, 1975b)noted higher concentrations of PUFAs in the ventral blubber,while Koopman et al. (1996) did not find any differences be-tween surfaces in terms of fatty acid composition. The re-sults of the present study suggest that it is highly unlikelythat dietary fatty acids are preferentially stored in one partic-ular area of the body of bottlenose dolphins. Minimal varia-tion in fatty acid composition with respect to body sitesuggests that it should be feasible to use any location on thebody for biopsy sampling to evaluate the fatty acid composi-tion of winter animals.

Layering effectsBiochemical stratification occurred in the blubber, with all

three layers (outer, middle, and inner) exhibiting differentfatty acid compositions (Figs. 3, 4), especially in the five an-imals examined at nine body sites. Fatty acids that variedamong layers were of both dietary and endogenous origin.These results are consistent with those from previous studieson cetacean blubber showing that it is heterogeneous and di-vided into layers (e.g., Ackman et al. 1975a; Lockyer et al.1984; Koopman et al. 1996, 2003) that can be differentiatednot only visually and histologically but also biochemically.

Differences in fatty acid composition could result fromvariable fatty acid turnover rates within blubber layers (Aguilarand Borrell 1991; Koopman et al. 2003). Aguilar and Borrell(1990) suggested that the outermost blubber layer is the moststable and plays only a minor role in energy storage, whilethe middle layer represents a transitional zone between theouter and inner strata. The innermost blubber layer has beenshown to be the most variable in composition, and likely ex-periences active lipid deposition and mobilization (Ackmanet al. 1975b; Lockyer et al. 1984; Aguilar and Borrell 1990).Fatty acid turnover rates have not been studied in cetaceans,and with the suggestion of different functional roles for eachblubber layer, it is quite likely that different turnover ratescould affect the fatty acid composition of each layer.

Diet could also play a significant role in determining fattyacid composition. Long-chain PUFAs are prevalent in ma-rine systems and are usually of dietary origin (Fraser et al.1989; Iverson et al. 1997). When fatty acid classes (SFAs,MUFAs, and PUFAs) were considered, differences in fattyacid composition between layers became more evident. Theouter layer had a higher MUFA concentration, while theinner layer had significantly larger amounts of SFAs andPUFAs, consistent with other marine mammal species.Ackman et al. (1975b) suggested that PUFAs are depositedfirst in the innermost blubber layer and later in the outermostlayer. In fin whales (Balaenoptera physalus (L., 1758)),long-chain PUFAs tended to occur in the innermost blubberlayer (Ackman et al. 1965). Koopman et al. (1996) alsofound high levels of PUFAs in the innermost layer of harborporpoises (Phocoena phocoena (L., 1758)). West et al. (1979b)found more SFAs in the outermost blubber layer of walrus(Odobenus rosmarus rosmarus (L., 1758)), which suggestssimilarities with baleen whales. Other research on cetaceanshas indicated that dietary fatty acids may be first deposited

in the innermost blubber layer (Ackman et al. 1975b;Koopman et al. 1996, 2003). Deposition of PUFAs in the in-ner layer first is important energetically, as it would not beefficient to deposit or sequester these fatty acids in a less ac-tive outer layer (Koopman et al. 1996). High concentrationsof particular groups of fatty acids could originate from alarger proportion of these classes in the diet, but selectivemobilization/deposition of particular fatty acids from each ofthe blubber layers to serve other functions within the bodycould also be a factor. Because of layering effects present inbottlenose dolphin blubber and the potential for differentturnover rates in each layer, the influence of diet and physi-ology must be further explored to provide accurate informa-tion for future FASA.

Reproductive-state differencesLarge differences in fatty acid composition were evident

in the present study not only between male and female ani-mals, but also between nonlactating and lactating females(Fig. 5). It is not known whether these differences resultfrom dietary or physiological factors or a combination of thetwo. Bottlenose dolphins in the Indian Ocean and Gulf ofMexico showed gender-specific feeding differences, with adultmales consuming both larger and different fish species thanare taken by adult females (Barros and Odell 1990; Cock-croft and Ross 1990). Along the gulf coast of the UnitedStates, females with calves have been shown to inhabit a rel-atively limited core habitat area, whereas males encompass amuch wider range incorporating several female areas (Barrosand Odell 1990; Scott et al. 1990), allowing them to feed ondifferent prey items from each habitat. Bottlenose dolphinsfeeding on different prey items, as well as on different ageclasses within each prey species, could result in differencesin blubber fatty acid composition (Iverson et al. 1997).

Fatty acid composition of blubber was also impacted bythe reproductive state of the female bottlenose dolphins.CART analyses determined differences in blubber fatty acidcomposition between reproductive states with 97% accuracy.Energy expended during reproduction, especially lactation,is very high, with heavy fat depletion occurring in the blub-ber of lactating females (Ackman et al. 1975b; Lockyer 1986;Beck et al. 2003). Pregnant whales had the thickest blubber(Pond 1978; Lockyer 1986; Aguilar and Borrell 1990; Ryget al. 1990; Vikingsson 1995), while lactating whales havebeen shown to have the lowest body fat condition and blub-ber lipid content (Lockyer 1986; Aguilar and Borrell 1990).Aguilar and Borrell (1990) determined that male fin whalesshow no variation in blubber lipid content between repro-ductive states, but female fin whales showed changes in bothblubber lipid content and stratification, which suggests thatthese differences resulted from physiological changes im-posed on animals during each reproductive stage. It is possi-ble that specific fatty acids are mobilized or sequestered toaccommodate the physiological requirements of pregnancyand lactation.

It is not known how long the female bottlenose dolphinsin this study had been lactating, because milk compositioncould not be evaluated. Since the fat content of the milk ofmost marine mammals decreases over the lactation period,the fatty acids mobilized from the blubber will also changeover the same time frame (Iverson 1993; Iverson et al. 1995;

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Koopman et al. 1996). It has also been shown that as lactat-ing females increase their energy intake to match theirhigher energy expenditure, they eat different prey (Cockcroftand Ross 1990), and their fatty acid composition becomescloser to that of males (Ackman et al. 1975b). It is also con-ceivable that nonlactating females were a distinct group be-cause they were storing fat in anticipation of reproductiverequirements.

Seasonal effectsSummer blubber samples were readily distinguishable

from winter samples, with fatty acids of both dietary and en-dogenous origins showing differences (Fig. 5). These differ-ences could be influenced by both external and internalconstraints. A multitude of studies have shown seasonalchanges in feeding habits that correlated with changes inblubber fatty acid composition (e.g., in bearded seals (Erig-nathus barbatus (Erxleben, 1777)) and ringed seals (Pusahispida (Schreber, 1775)); West et al. 1979a). Other studieshave shown seasonal changes in prey distribution, predationpressure, or productivity that would have ramifications con-cerning prey species availability and quality. There is defini-tive evidence of changes in the species composition ofbottlenose dolphin stomach contents between seasons(Cockcroft and Ross 1990; Barros 1993; Barros and Wells1998) as well as changes in prey composition, as lipid con-tent changes with both diet shifts and spawning cycles(Gallagher et al. 1991; Clavijo et al. 1999; Gámez-Meza etal. 1999; Aro et al. 2000; Shirai et al. 2001; Bradshaw et al.2003). Changes in prey type and composition between sea-sons for bottlenose dolphins could influence the individualfatty acids stored in the blubber layer.

Previous studies have concentrated on changes in thethickness or lipid content of blubber in relation to season(e.g., Kleivane et al. 1995; Vikingsson 1995; Käkelä andHyvärinen 1996), but little information exists on changes infatty acid composition. Pond (1978) summarized seasonaldifferences seen in fat of blue whales (Balaenopteramusculus (L., 1758)), gray whales (Eschrichtius robustus(Lilljeborg, 1861)), manatees (Trichechus manatus L., 1758),harp seals (Pagophilus groenlandicus (Erxleben, 1777)),California sea lions (Zalophus californianus (Lesson, 1828)),and southern elephant seals (Mirounga leonina (L., 1758)),and she noted changes in the amount of fat present betweensummer and winter. Polar bears (Ursus maritimus Phipps,1774) also exhibited large seasonal changes in body compo-sition, with adipose tissue expanding up to half of the totalbody mass (Pond et al. 1992). Lipid content of bottlenosedolphin blubber has been shown to vary between 14% and90% seasonally (L.K.M. Shoda, T.L. Wade, G.A.J. Worthy,and T.A.M. Worthy, unpublished data), and the thickness ofthe blubber layer has been shown to increase during expo-sure to lower temperatures (Kleivane et al. 1995). As theselarge changes in fat deposition and lipid content occur, theconcentration of individual fatty acids could change in theblubber to increase insulation in cold water. Fatty acid com-position has not been examined in this way for cetaceans,but could complicate dietary-history analysis because of thefunctional requirements of blubber tissue during differentseasons.

Another concern for cetaceans in cold water is membranefluidity. Fluidity of membranes can be maintained whentemperatures are low by increasing the degree of unsatu-ration and changing the chain length of fatty acids (i.e., low-ering the melting point) (Gurr and Harwood 1991; Pond etal. 1992; Fredheim et al. 1995). These changes have beenshown to occur in the extremities of pinnipeds (e.g., West etal. 1979b; Käkelä and Hyvärinen 1996). It is possible thatsimilar changes occur within cetacean blubber in response todecreasing or increasing water temperature. The fatty acidswithin the cells may also need to remain fluid because of in-creased insulation or energy requirements. Some of the ob-served changes in fatty acid composition could result fromthe constraints imposed on dolphins by lower temperaturescoupled with dietary shifts, making FASA difficult withoutfurther information on the properties of blubber.

In conclusion, the fatty acid composition of bottlenosedolphin blubber has been shown to be consistent across bodysites but variable among layers, reproductive states, and sea-sons. The individual fatty acids that change between thesegroups originate from both the diet and biosynthesis. Whileblubber samples for fatty acid analysis could be taken any-where on the body from the axilla to the anus of winter dol-phins, it is evident that these other factors need to beevaluated to construct an accurate dietary history. The influ-ences of blubber functions such as energy storage, insula-tion, structural support, hydrodynamics, and buoyancy couldmake dietary determination difficult. The use of FASA in thefuture to discern diet will require a complete understandingof the layer(s) that are most responsive to diet changes, theturnover rates for each blubber layer, and the influence ofphysiological requirements and feeding habits during eachseason and for each gender.

Acknowledgements

We thank the Advanced Research Program of the TexasHigher Education Coordinating Board and the National Ma-rine Fisheries Service for financial support to G.A.J.W.Thanks are extended to the Texas Marine Mammal StrandingNetwork for collecting the blubber samples used in thisstudy. Dr. Sara J. Iverson and Shelley Lang provided techni-cal aid. Special thanks are extended to Dr. Tuhin Giri andTamara Worthy for discussion and assistance. The authorsalso thank two anonymous reviewers for their comments andsuggestions.

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