Comparative Biochemistry and Physiology, Part B 143 (2006) 486 – 499

www.elsevier.com/locate/cbpb

Stratification and intra- and inter-specific differences in fatty acid

composition of common dolphin (Delphinus sp.) blubber:
Implications for dietary analysis
Heather R. Smith ⁎, Graham A.J. Worthy 1
Physiological Ecology and Bioenergetics Laboratory, Texas A&M University at Galveston, Galveston, TX 77551, USA
Received 31 May 2005; received in revised form 19 October 2005; accepted 31 December 2005
Available online 24 February 2006

Abstract

Sixty-five fatty acids were quantified in the blubber of common dolphins (Delphinus delphis, D. capensis) incidentally caught off the coast of
southern California. Dolphins were grouped by sex, reproductive status and species, and a blubber sample was collected at a mid-lateral site
located caudal to the trailing edge of the dorsal fin. Samples were divided horizontally into inner, middle and outer layers and gradients in fatty
acid content (mass percent) were observed across the depth of the blubber. Levels of monounsaturated fatty acids were greatest in the outer layer,
whereas levels of saturated and polyunsaturated fatty acids were greatest in the inner layer. Degree of stratification was greatest in sexually mature
dolphins. Blubber of sexually immature, but physically mature, male dolphins was also highly stratified, suggesting that this difference may be
attributed to differences in diet. Classification and regression tree analysis resulted in the fewest misclassifications when dolphins were grouped by
species, possibly indicating that these closely related animals forage on different prey species. Dietary-derived fatty acids were typically selected
as splitting criteria in classification and regression tree analyses, suggesting that the observed differences in fatty acid composition between the
various groups of dolphins may be attributed to differences in diet.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Blubber; Cetacean; Diet; Dolphin; Fatty acid; Feeding habits; Layer; Stratification

1. Introduction et al., 2001). In order to effectively use FA composition of

Blubber is a biochemically dynamic tissue, in which fatty
acid (FA) composition is potentially influenced by diet. Ma-
rine mammals rely on their blubber layer for thermoregulation,
streamlining, buoyancy, and energy storage (Parry, 1949;
Worthy and Edwards, 1990; Pabst et al., 1999), and researchers
have begun to take advantage of its potential as a record of
dietary intake (Iverson et al., 1997b; Walton et al., 2000; Hooker
⁎ Corresponding author. Washington Cooperative Fish and Wildlife Research
Unit, School of Aquatic and Fishery Sciences, University of Washington, Box
355020, Seattle, WA 98195-5020, USA. Tel.: +1 206 221 5453; fax: +1 206 616
9012.
E-mail address: smithh@u.washington.edu (H.R. Smith).
1
Present address: Physiological Ecology and Bioenergetics Laboratory,
Department of Biology, University of Central Florida, Orlando, FL, 32816-
2368, USA.
1096-4959/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpb.2005.12.025
blubber in diet analysis techniques, the biochemical structure of
this tissue must be considered.
Biochemical stratification, or layering, has been observed in
the blubber of Pacific walrus (Odobenus rosmarus divergens)
(West et al., 1979b), polar bears (Ursus maritimus) (Grahl-
Nielsen et al., 2003), and several species of phocid seals (Fred-
heim et al., 1995; Best et al., 2003). Other studies, however,
have shown that phocid blubber is homogeneous and therefore
not layered (Jangaard and Ke, 1968; Käkelä and Hyvärinen,
1993). In contrast, biochemical stratification has consistently
been observed in baleen whale blubber (Ackman et al., 1965,
1975a,b; Lockyer et al., 1984; Olsen and Grahl-Nielsen, 2003).
Biochemical layering has also been noted in the blubber of
toothed whales such as bottlenose dolphins (Tursiops truncatus)
(Shoda et al., 1993; Samuel and Worthy, 2004), harbor porpoise
(Phocoena phocoena) (Koopman et al., 1996), killer whales
(Orcinus orca) (Worthy et al., 2003; Krahn et al., 2004) and
numerous other odontocetes (Koopman, 2001).
 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
While it is evident that stratification exists, the number of
distinct layers within cetacean blubber remains unclear. Koop-
man et al. (1996) found differences in FA composition in por-
poise blubber that was divided into inner and outer layers, and
suggested the existence of a continuous gradient in composi-
tion. Ackman et al. (1965, 1975a) noted differences between
inner and outer layers in mysticete whales. Evidence for the
existence of three distinct layers in bottlenose dolphins has been
suggested based on histological (Cowan and Worthy, 1991),
toxicological (Shoda et al., 1993), and biochemical (Samuel and
Worthy, 2004) evidence. This stratification has been attributed
to different levels of metabolic activity within layers (Ackman
et al., 1975a,b; Koopman et al., 1996; Koopman, 2001).
Differences in FA composition within and between species
have been attributed to diet (West et al., 1979a; Borobia et al.,
1995; Smith et al., 1996; Iverson et al., 1997a,b). Intraspecific
differences have also been attributed to age (Koopman et al.,
1996; Koopman, 2001), body site (Koopman et al., 1996),
season (Samuel and Worthy, 2004), reproductive status (Samuel
and Worthy, 2004) and starvation (Koopman, 2001). Interspe-
cific differences have been attributed to phylogeny (Koopman,
2001) and thermal regime (Koopman, 2001; Worthy et al.,
2003). With few exceptions (West et al., 1979a,b; Koopman,
2001; Samuel and Worthy, 2004), the possible influences of sex
and reproductive status on FA composition within a single
species have received little attention.
Examination of FA stratification in marine mammal blubber
is confounded by the type of ester prepared from lipid extracted
from the tissue of interest. Methyl esters are commonly used to
quantify medium- and long-chain FA in order to make dietary
inferences (Smith et al., 1997; Walton et al., 2000; Iverson et al.,
2004). These FAs have been used as “biomarkers” in diet studies
as they are generally passed from the prey to predator adipose
storage tissues without modification, whereas ingested short-
chain FAs are generally oxidized as a source of energy (Pond,
1998). Unlike methyl esters, butyl esters allow for the quanti-
fication of short-chain fatty acids (Christie, 1972), and have been
used in studies where levels of short-chain FA such as isovaleric
acid (iso 5 : 0) are of interest (Koopman et al., 1996, 2003).
Common dolphins (Delphinus sp.) are relatively small,
toothed whales (Suborder Odontoceti) that are easily identified
at sea by their black or dark grey V-shaped saddle. Adult
animals weigh an average of 80 kg, and are sexually dimorphic
with males being larger than females. Common dolphins are
distributed worldwide in temperate, tropical and subtropical
oceans. They are found along most coasts over the continental
shelf, and are commonly associated with prominent bathymetric
features such as seamounts and ridges (Evans, 1994). The
distribution of common dolphins has also been correlated with
the distribution of preferred prey species (Young and Cockcroft,
1994), water temperature, and possible competitive exclusion
by spinner (Stenella longirostris) and spotted (S. attenuata)
dolphins (Evans, 1975). Delphinus sp. forage on numerous
species of fish, cephalopods and crustaceans throughout the
water column (Fitch and Brownell, 1968; Jones, 1981; Evans,
1994; Silva, 1999). Stomach content analysis has indicated that
the relative proportions of prey types vary with several factors
487
including season and age of individual (Evans, 1975; Young
and Cockcroft, 1994; Chou et al., 1995; Silva, 1999).
Prior to 1994, common dolphins off the southern coast of
California were grouped into short- and long-beaked popula-
tions. These sympatric populations have not been observed to
form mixed herds (Evans, 1975), and were later recognized as
distinct species which could be differentiated based on aspects
of color pattern, external morphology and skeletal characters
(Heyning and Perrin, 1994). Rosel et al. (1994) examined mi-
tochondrial DNA sequences and found no DNA haplotypes
common to both forms, providing further support for the exis-
tence of two species.
These two species of common dolphins are typically para-
patrically distributed, with D. delphis (short-beaked) found in
pelagic waters, and D. capensis (long-beaked) in nearshore
waters less than 100 fathoms deep (Evans, 1975). As noted by
Heyning and Perrin (1994), these two species must be exploit-
ing the environment differently as their distributions overlap off
the southern coast of California. It is reasonable to expect that
foraging strategies, and therefore diet, influenced by differences
in body size and skull morphology, would differ between these
two species. There is little published data supporting this hy-
pothesis, however, as previous diet composition studies have
failed to distinguish between or compare the diets of these two
species.
As with other cetaceans, common dolphins spend much of
their time away from shore and below the surface, making direct
observation of dietary intake near impossible. Other dietary
analysis techniques such as scat and stomach content analyses
are of limited use with cetaceans as their scats are virtually
impossible to collect, they do not haul-out, and stomach con-
tents are typically only available from a small segment of the
population such as stranded animals or those associating with
and incidentally caught in fishing nets. Fatty acid analysis
shows great promise for gaining insight into the diet of free-
ranging cetaceans as only a small piece of blubber is required
for analysis. However, in order to design appropriate sampling
protocols for this type of work, factors that may influence the
fatty acid composition of cetacean blubber must be considered.
The overall aim of this research was to examine the fatty acid
composition of common dolphin blubber in the context of
making dietary inferences for these species. The first objective
of this study was to determine if Delphinus blubber could be
divided into biochemically distinct layers. The second objective
was to examine the intraspecific differences in blubber FA
composition with respect to sex and reproductive status. The
final objective was to determine if D. delphis could be dis-
tinguished from D. capensis on the basis of blubber FA
composition.
2. Materials and methods
2.1. Sample collection
Blubber samples (n = 84) and accompanying biological data
(species, sex, body size, reproductive condition) were made
available by the Southwest Fisheries Science Center (SWFSC)
488 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
Table 1
Species, sex and sexual maturity of Delphinus delphis and D. capensis blubber
samples used in fatty acid analysis
Species Sex Sexual maturity Notes
D. delphis Male (58) Mature (48)
Immature (10) Long a (5), Short (5)
Female (17) Mature (10) PL b (1), PnL (2),
nPL (3), nPnL (4)
Immature (7)
D. capensis Male (4) Mature (2)
Immature (2)
Female (5) Mature (3) nPL (1), nPnL (2)
Immature (2)
Numbers in parentheses indicate sample sizes.
a
Long (i.e., physically mature) animals had a mean body length of 174 ± 2 cm,
short (i.e., physically immature) animals had a mean body length of 137 ± 2 cm
(mean ± SEM).
b
PL = pregnant and lactating; PnL = pregnant, not lactating; nPL = not
pregnant, lactating; nPnL = not pregnant, not lactating.
of the National Marine Fisheries Service (NMFS), and the Los
Angeles County Museum of Natural History (LACM) (Table 1).
Samples were collected from common dolphins caught inciden-
tal to commercial fisheries operating along the coast of southern
California from 1990 to 2000. NMFS observers aboard gillnet
vessels collected blubber samples from dead, by-caught com-
mon dolphins on the left or right lateral side of the body, just
caudal to the trailing edge of the dorsal fin (Jefferson et al.,
1994). The entire blubber layer was collected such that the
epidermis and underlying muscle were still attached to the
blubber. Blubber samples were wrapped in foil or placed in
whirl-pak bags, and frozen until later analysis. Skin and gonads
were also collected, and geographic location, sex, and total body
length were recorded (Perrin et al., 1976; Jefferson et al., 1994).
Reproductive condition was determined using a variety of
criteria. Males were classified as sexually mature if the weight of
the right testis without epididymis was greater than 200 g (Ferrero
and Walker, 1995). Reproductive tracts of female dolphins were
examined in the laboratory to determine state of sexual maturity
and pregnancy (Akin et al., 1993). Females were classified as
sexually mature if either corpus albicans or corpus lutea were
present in either ovary (Perrin et al., 1976; Akin et al., 1993;
Ferrero and Walker, 1995). Mammary glands were palpated to
determine if the female was lactating (Jefferson et al., 1994).
NMFS observers performed the test for lactation in the field if the
whole carcass could not be brought back to the laboratory.
Dolphins were grouped by species, sex, and level of sexual
maturity for this study. Sexually mature females were further
grouped according to state of pregnancy and lactation. Sexually
immature male D. delphis could easily be divided into two
groups on the basis of body length. “Long” animals had a mean
body length of 174 ± 2 cm and were termed physically mature,
while “short” animals had a mean body length of 137 ± 2 cm and
were termed physically immature.
2.2. Sample analysis
To reduce the loss of lipid during sample preparation, blub-
ber was cut while still partially frozen. Freezer burn, identified
by a dark yellowish color, was trimmed from the edges of the
sample and discarded. With no visible layering apparent, each
sample was divided into five layers of approximately equal
thickness. The outer (adjacent to dermis), middle and inner
(adjacent to muscle) layers were retained, while the two
transitional layers were discarded (Fig. 1). Duplicate subsam-
ples (ca. 0.5 g) were taken from each of the three layers.
Lipid was extracted from blubber according to Folch et al.
(1957) and Iverson et al. (2001a,b). Fatty acid methyl esters
(FAME) were prepared from the lipid extract using the Hilditch
reagent (0.5 N H2SO4 in methanol) according to Iverson et al.
(1992). FAME were analyzed using temperature-programmed
gas-liquid chromatography (GLC). A Perkin Elmer Autosystem
XL gas chromatograph (capillary injector, FID) linked to a
computerized integration system (Turbochrom 4 software, PE
Nelson) was fitted with a 30 m × 0.25 mm i.d. column coated
with 50% cyanopropyl methylpolysiloxane (0.25 μm film
thickness; liquid phase DB-23; J and W Scientific Inc., Folsom,
CA). Helium was used as the carrier gas. The temperature
program employed was developed by Iverson et al. (1992) as
further modified by S.J. Iverson (personal communication).
Identifications of FAME peaks were determined using
commercially prepared standards (Nu-Chek Prep., Elysian,
MN) and known reference mixtures. Reference mixtures
contained the full suite of FA quantified, and peaks were
identified using silver nitrate (argentation) chromatography and
gas-chromatography/mass spectrometry according to Iverson
et al. (1997b, 2001a,b). All chromatograms were individually
examined to ensure that peak names were applied correctly.
Individual FAs were converted to mass percent of total FA
and unknowns by applying theoretical response factors rela-
tive to 18 : 0 (Ackman, 1972, 1991). FAs were named using
the IUPAC shorthand nomenclature of C : Dn − x, where C is
the number of carbons in the FA, D is the number of double
bonds, and n − x denotes the position of the first double bond
as numbered from the terminal methyl end of the FA.
epidermis
O — outer layer
x — discarded layer
~15 mm M — middle layer
x — discarded layer
I — inner layer
muscle
Fig. 1. Division of blubber into layers prior to fatty acid composition analysis.
Horizontal lines indicate where blubber was cut with a scalpel. Inner, middle,
and outer layers were retained for analysis, whereas the two transitional layers,
indicated with an “x”, were discarded.
 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
2.3. Data analysis
Mean values for individual FA were calculated from du-
plicate subsamples for each layer for each individual dolphin.
Except as noted, and with the exception of 16 : 4n − 3 and
18 : 3n − 6, these values were used in all subsequent calculations
and analyses. FA 16 : 4n − 3 and 18 : 3n − 6 often co-eluted with
adjacent peaks, preventing accurate measurement, and were
thus omitted. Values for total saturated FA (∑SFA), monounsat-
urated FA (∑MUFA), n − 3 polyunsaturated FA (∑(n − 3)
PUFA), n − 6 polyunsaturated FA (∑(n − 6)PUFA), and the
ratio ∑(n − 3)PUFA / ∑(n − 6)PUFA were calculated for each
layer for each individual dolphin. Mean values for individual
FA, summed FA and the ∑(n − 3)PUFA / ∑(n − 6)PUFA ratio
were calculated for each layer for each group of dolphins.
2.4. Blubber stratification
Stratification of FA in common dolphin blubber was pri-
marily examined in sexually mature, male D. delphis, as this
group of dolphins had the largest sample size (n = 48).
Differences in FA composition between the inner, middle, and
outer layers were examined using multivariate and univariate
approaches. A subset of 15 FA commonly present in quantities
N 1% by mass was often analyzed in place of the entire data set
to facilitate the use of statistical methods sensitive to the number
of variables being examined: 14 : 0, 14 : 1n − 9, 14 : 1n − 7,
14 : 1n−5, 16:0, 16:1n−9, 16:1n−7, 18:0, 18:1n−9, 18:1n−7,
18 : 2n − 6, 20 : 1n − 9, 20 : 5n − 3, 22 : 5n − 3, and 22 : 6n − 3.
Mass percent data were arcsin transformed (Steele and Torrie,
1980) prior to most analyses.
2.4.1. Multivariate approach
Principal components analysis (PCA) was used to explore
the possibility that sexually mature, male D. delphis blubber
may be divided into three layers on the basis of FA composition.
Principal components (PC) were generated from the subset of
15 arcsin transformed FA listed above, and PC with eigenvalues
≥ 1 were retained for interpretation. The varimax rotation was
applied to enhance interpretation of the PC. In an attempt to
reduce the effects of a priori variable selection, PC were also
generated from an additional subset of 23 arcsin transformed FA
commonly present in quantities N 0.5% by mass: 12 : 0, 14 : 0,
14 : 1n − 9, 14 : 1n − 7, 14 : 1n − 5, iso 15 : 0, 16 : 0, 16 : 1n − 9,
16:1n −7, 16 :2n− 4, 17 :1,18:0, 18 :1n− 9, 18 :1n − 7, 18 :2n − 4,
18:2n−6, 18:3n−3, 20:1n−9, 20:4n−6, 20:4n−3, 20:5n−3,
22 : 1n − 11, 22 : 5n − 3, and 22 : 6n − 3. Scatter plots of PC1
vs. PC2 were constructed for each PCA completed.
As the PC could be roughly interpreted as the summary
variables calculated earlier (e.g., ∑SFA), these summary
variables were used in the discriminant analysis (DA) instead
of the PC as they encompassed nearly all the original data and
did not require a priori variable selection. Discriminant analysis
(DA) was employed to determine the extent to which the layers
could be distinguished from one another using ∑SFA,
∑MUFA, ∑(n − 3)PUFA, and ∑(n − 6)PUFA as discriminating
variables. Jack-knifed classification rates were computed,
489
and differences in mean canonical scores between layers were
examined using ANOVAs with α = 0.025 (0.05 / 2 = 0.025).
Post hoc comparisons were made using Scheffé's test. A
scatter plot of the mean canonical scores for the two DF was
constructed.
2.4.2. Univariate approach
To test for differences between the three layers, a MANOVA
was performed on the data using all 15 arcsin transformed FA.
Differences in mass percent of individual FA were then exam-
ined using individual ANOVAs with α = 0.0033. The Bonferroni
method of modifying the alpha value (α = 0.05 / 15 = 0.0033) to
reduce the risk of committing a Type I error was employed
(Johnson and Wichern, 1998). Scheffé's test (α = 0.05) was used
post hoc to examine the nature of the differences for each FA as
indicated by the ANOVAs. The summary variables ∑SFA,
∑MUFA, ∑PUFA, ∑(n − 3)PUFA and ∑(n − 6)PUFA (all arcsin
transformed), and ∑(n − 3)PUFA / ∑(n − 6)PUFA were tested for
layering differences using MANOVAs, ANOVAs with modified
alpha values, and Scheffé's test.
2.4.3. Classification and regression tree analysis approach
The ability to distinguish among the three layers on the basis
of FA composition in mature, male D. delphis blubber was
examined using classification and regression tree (CART)
analysis (Smith et al., 1997; Iverson et al., 1997b; Brown et al.,
1999; Walton et al., 2000; Samuel and Worthy, 2004). All FA
data, including summary variables were used in CART analysis.
The classification and regression tree was generated using the
TREES function in SYSTAT (version 10 for Windows, SPSS
Inc., Chicago, IL). The default stopping criteria were used with
the exception of the minimum proportion reduction in error,
which was set at 1%.
2.4.4. Blubber stratification in other dolphin groups
Small sample sizes for the other groups of dolphins
precluded similar statistical analysis. The existence of FA
stratification in these other groups was examined visually using
bar graphs of the 15 most abundant FA.
2.5. Degree of stratification
Koopman (2001) developed a stratification index (SI) in
order to compare overall FA stratification in blubber among
groups of odontocetes. Koopman's SI was calculated as the
sum of the absolute values of the differences in mass percent
between the inner and outer blubber layers for the 16 most
abundant FA. SI values were similarily calculated for
Delphinus in this study, using the mass percent values for
the 15 FAs commonly present in quantities N1% by mass.
Mean SI values were then calculated for each dolphin group.
Mean SI values were compared for male D. delphis using an
ANOVA with α = 0.05. Scheffé's test was used post hoc to
examine differences among groups. Mean SI values were com-
pared visually among female D. delphis, male D. capensis, and
female D. capensis as small sample sizes precluded statistical
testing.
490 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499

2.6. Intraspecific differences in FA composition Table 2

Fatty acids (FAs) commonly present in quantities ≥0.5% by mass and summary
variables for inner, middle, and outer blubber layers of sexually mature, male, D.
The ability to distinguish between blubber samples on the delphis (n = 48)
basis of potential intraspecific differences in FA composition
FA Inner Middle Outer
was examined using CART analysis as described previously.
Analyses were conducted separately on each layer for both Saturated fatty acids
12 : 0 0.21 ± 0.01 0.36 ± 0.01 0.66 ± 0.02
short- and long-beaked common dolphins.
14 : 0 4.30 ± 0.07 3.77 ± 0.09 3.02 ± 0.08
Sex was used as the grouping variable for sexually immature Iso 15 0.25 ± 0.01 0.40 ± 0.02 0.78 ± 0.04
and mature animals, as well as for both mature and immature 16 : 0 13.89 ± 0.38 7.68 ± 0.38 4.06 ± 0.18
dolphins combined. Sexual maturity was used as the grouping 18 : 0 2.50 ± 0.09 1.28 ± 0.07 0.77 ± 0.03
variable for males and females (mature vs. immature), and ∑SFA 22.90 ± 0.41 15.13 ± 0.49 11.18 ± 0.27
reproductive status (i.e., pregnant — P, lactating — L; not
Monounsaturated fatty acids
pregnant — nP, not lactating — nL) was used for sexually 14 : 1n − 9 0.16 ± 0.01 0.66 ± 0.06 2.25 ± 0.13
mature females alone (PL, nPL, PnL, nPnL). Sexually mature 14 : 1n − 7 0.13 ± 0.01 0.61 ± 0.05 1.59 ± 0.07
D. delphis females were also simply categorized as pregnant 14 : 1n − 5 0.60 ± 0.06 1.95 ± 0.13 3.89 ± 0.13
or non-pregnant, and lactating or non-lactating, and CART 16 : 1n − 9 0.78 ± 0.04 1.92 ± 0.10 3.86 ± 0.13
16 : 1n − 7 10.80 ± 0.36 17.61 ± 0.54 24.03 ± 0.45
analysis was conducted on these two additional data sets. In
17 : 1 0.60 ± 0.01 0.73 ± 0.02 0.80 ± 0.02
addition to the modified stopping criteria listed earlier, minimum 18 : 1n − 9 31.38 ± 0.61 32.15 ± 0.53 30.79 ± 0.41
group size was altered to reflect group sizes smaller than five. 18 : 1n − 7 2.71 ± 0.04 2.42 ± 0.05 1.79 ± 0.06
20 : 1n − 9 3.13 ± 0.10 2.35 ± 0.08 1.13 ± 0.07
2.7. Interspecific differences in FA composition 22 : 1n − 11 0.77 ± 0.05 0.45 ± 0.03 0.15 ± 0.02
∑MUFA 53.37 ± 0.89 62.78 ± 0.91 71.81 ± 0.60

Potential interspecific differences in FA composition were Polyunsaturated fatty acids

also examined using CART analysis. Separate analyses were 16 : 2n − 4 0.55 ± 0.02 0.56 ± 0.02 0.51 ± 0.02
conducted for sexually mature males, sexually immature males, 18 : 2n − 6 1.18 ± 0.02 1.23 ± 0.02 1.19 ± 0.02
nPL females, nPnL females, and sexually immature females. 18 : 3n − 3 0.47 ± 0.02 0.49 ± 0.01 0.70 ± 0.06
20 : 4n − 6 0.71 ± 0.03 0.77 ± 0.02 0.65 ± 0.02
Data from each layer were analyzed separately, and minimum
20 : 4n − 3 0.57 ± 0.02 0.54 ± 0.02 0.36 ± 0.02
group size was altered to reflect groups smaller than five. 20 : 5n − 3 2.68 ± 0.18 3.01 ± 0.14 2.15 ± 0.10
22 : 5n − 3 3.26 ± 0.18 2.49 ± 0.15 1.11 ± 0.11
3. Results 22 : 6n − 3 9.74 ± 0.35 8.27 ± 0.30 4.51 ± 0.30
∑(n − 3)PUFA 17.41 ± 0.69 15.40 ± 0.57 9.20 ± 0.47
∑(n − 6)PUFA 3.12 ± 0.06 3.06 ± 0.05 2.55 ± 0.06
Sixty-five individual FA (excluding 16 : 4n − 3 and 18 : 3n − 6) ∑(n − 3)PUFA / ∑(n − 6)PUFA 5.49 ± 0.14 4.97 ± 0.12 3.51 ± 0.11
were regularly identified in all blubber samples examined. The ∑(15 FA) 87.23 ± 0.20 87.39 ± 0.19 86.13 ± 0.28
majority of these FAs were present in quantities ≤0.5% by mass
∑(15 FAs) is the sum of all FAs commonly present in quantities ≥1% by mass.
and only 15 FAs were commonly present in quantities ≥ 1% by Values are mean ± SEM, mass percent of total FAs and unknowns.
mass: 14 : 0, 14 : 1n − 9, 14 : 1n − 7, 14 : 1n − 5, 16 : 0, 16 : 1n − 9,
16 : 1n − 7, 18 : 0, 18 : 1n − 9, 18 : 1n − 7, 18 : 2n − 6, 20 : 1n − 9, 3.1. Blubber stratification
20 : 5n − 3, 22 : 5n − 3, and 22 : 6n − 3. In mature male D. delphis,
these 15 FAs accounted for total mean values ± SEM of 87.23 ± 3.1.1. Multivariate approach
0.20%, 87.39 ± 0.19%, and 86.13 ± 0.28% by mass in inner, In the first PCA (using 15 FAs), two PC were retained
middle and outer layers, respectively (Table 2). and interpreted. Cumulatively, they accounted for 83.58% of
∑MUFA accounted for the greatest proportion of all FA the total variance. In the second PCA (using 23 FAs), five
measured in all dolphin groups examined. In all dolphin groups PC, cumulatively accounting for 90.44% of the total variance,
combined, ∑MUFA ranged from 52.68 ± 0.71% by mass in the were retained. In general, the SFA and MUFA loaded
inner layer to 69.42 ± 0.67% by mass in the outer layer (mean ± heavily on PC1, and the PUFA loaded heavily on PC2, PC3,
SEM). ∑SFA, ∑(n − 3)PUFA, and ∑(n − 6)PUFA each accounted PC4 or PC5. Scatter plots of the first 2 PC showed that
for less than 30% by mass of all FA and unknowns in all samples. the data points tended to group together according to layer
In contrast to ∑MUFA, ∑SFA was greatest in the inner layer (Fig. 2).
(22.85 ± 0.28% by mass), and least in the outer layer (13.19 ± Discriminant analysis resulted in the three layers being
0.41% by mass). ∑(n − 3)PUFA and ∑(n − 6)PUFA were also classified correctly 83% of the time (jack-knifed classification)
greatest in the inner layer, and least in the outer layer. ∑(n − 3) (Table 4). The middle layer was misclassified more frequently
PUFA ranged from 18.10 ± 0.55% by mass in the inner layer to than either the inner or outer layer (Table 4). Two significant
9.68 ± 0.47% by mass in the outer layer, while ∑(n − 6) was fairly discriminant functions (DF) were produced (Wilks' lambda =
consistent across all blubber layers, ranging from 3.13 ± 0.05% by 0.1830, P b 0.001). The first mean canonical score was
mass in the inner layer to 2.56 ± 0.05% by mass in the outer layer. significantly different for all three layers; the inner layer had
∑(n − 3)PUFA / ∑(n − 6)PUFA ratios ranged from 5.70 ± 0.11 in the highest score, whereas the outer layer had the lowest score.
the inner layer to 3.64 ± 0.10 in the outer layer (Table 3). The second mean canonical score only differed significantly
 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499 491

Table 3
Fatty acid composition summary variables for all common dolphin groups
Layer SFA MUFA (n − 3) PUFA (n − 6) PUFA (n − 3) PUFA / (n − 6)PUFA
Delphinus delphis
Male, s. mature c Ib 22.90 ± 0.41 53.37 ± 0.89 17.41 ± 0.69 3.12 ± 0.06 5.49 ± 0.14
n = 48 M 15.13 ± 0.49 62.78 ± 0.91 15.40 ± 0.57 3.06 ± 0.05 4.97 ± 0.12
O 11.18 ± 0.27 71.81 ± 0.60 9.20 ± 0.47 2.55 ± 0.06 3.51 ± 0.11
Male, s. mature, strand I 23.57 ± 0.33 45.73 ± 0.61 23.80 ± 0.80 3.64 ± 0.10 6.55 ± 0.41
n=2 M 16.79 ± 1.94 51.26 ± 3.74 24.86 ± 2.03 3.72 ± 0.01 6.68 ± 0.54
O 12.55 ± 1.87 62.54 ± 4.16 17.37 ± 2.72 3.24 ± 0.23 5.33 ± 0.46
Male, s. immature, long I 21.21 ± 1.15 58.30 ± 2.26 14.68 ± 1.89 2.82 ± 0.13 5.14 ± 0.43
n=5 M 13.57 ± 0.51 68.46 ± 1.63 11.51 ± 1.10 2.70 ± 0.09 4.22 ± 0.29
O 13.49 ± 0.65 72.33 ± 0.95 6.52 ± 0.34 2.18 ± 0.06 2.98 ± 0.09
Male, s. immature, short I 21.85 ± 0.92 57.38 ± 1.20 15.41 ± 0.93 2.88 ± 0.08 5.33 ± 0.20
n=5 M 18.80 ± 0.65 62.97 ± 1.21 12.71 ± 0.74 2.61 ± 0.04 4.85 ± 0.23
O 20.34 ± 0.91 66.02 ± 1.03 7.65 ± 0.56 2.17 ± 0.09 3.52 ± 0.16
Female, s. mature, PL a I 25.19 58.50 11.69 2.32 5.05
n=1 M 17.27 64.93 12.73 2.57 4.95
O 12.75 74.07 6.40 2.01 3.18
Female, s. mature, PnL I 24.85 ± 0.40 50.85 ± 5.64 18.60 ± 4.85 2.81 ± 0.25 6.51 ± 1.14
n=2 M 14.88 ± 0.11 64.93 ± 4.23 14.19 ± 3.90 2.80 ± 0.34 4.96 ± 0.78
O 11.80 ± 0.44 72.91 ± 0.62 7.60 ± 1.20 2.36 ± 0.22 3.20 ± 0.22
Female, s. mature, nPL I 23.91 ± 0.44 53.32 ± 2.82 16.61 ± 2.71 3.01 ± 0.24 5.44 ± 0.50
n=3 M 15.59 ± 0.59 65.47 ± 0.97 12.41 ± 1.24 2.89 ± 0.13 4.28 ± 0.31
O 14.56 ± 2.69 65.34 ± 7.33 12.33 ± 4.87 2.85 ± 0.33 4.08 ± 1.13
Female, s. mature, nPnL I 22.45 ± 2.30 52.76 ± 4.61 18.48 ± 3.15 3.08 ± 0.25 5.89 ± 0.64
n=4 M 15.04 ± 1.84 62.14 ± 5.41 16.24 ± 3.37 3.03 ± 0.29 5.18 ± 0.61
O 11.05 ± 0.80 71.88 ± 2.09 9.18 ± 2.34 2.48 ± 0.29 3.51 ± 0.56
Female, s. immature I 23.04 ± 0.44 53.84 ± 1.19 17.11 ± 1.01 2.95 ± 0.11 5.79 ± 0.21
n=7 M 17.62 ± 0.76 64.89 ± 1.04 11.46 ± 1.17 2.56 ± 0.09 4.44 ± 0.35
O 18.29 ± 1.21 66.70 ± 1.51 8.04 ± 1.50 2.22 ± 0.12 3.50 ± 0.47

Delphinus capensis
Male, s. mature I 21.75 ± 1.56 47.20 ± 0.46 22.15 ± 1.42 3.68 ± 0.50 6.20 ± 1.24
n=2 M 17.44 ± 3.64 55.62 ± 6.51 19.17 ± 1.54 3.47 ± 0.45 5.56 ± 0.28
O 12.35 ± 2.09 67.39 ± 8.10 11.90 ± 5.00 3.13 ± 0.74 3.63 ± 0.74
Male, s. immature I 22.81 ± 0.79 39.82 ± 0.22 28.54 ± 0.87 3.90 ± 0.13 7.33 ± 0.02
n=2 M 17.36 ± 1.07 54.31 ± 3.76 19.18 ± 5.51 3.67 ± 0.26 5.15 ± 1.13
O 15.00 ± 1.21 62.29 ± 3.75 12.14 ± 6.28 3.18 ± 0.46 3.61 ± 1.46
Female, s. mature, nPL I 25.90 38.57 27.73 3.41 8.13
n=1 M 22.02 42.75 27.50 3.50 7.87
O 16.63 53.68 21.18 3.58 5.92
Female, s. mature, nPnL I 21.49 ± 0.47 45.74 ± 3.99 25.33 ± 2.82 3.58 ± 0.06 7.05 ± 0.66
n=2 M 19.42 ± 0.41 49.82 ± 6.72 22.86 ± 5.69 3.57 ± 0.13 6.36 ± 1.36
O 19.13 ± 2.65 57.45 ± 3.19 14.30 ± 5.44 3.24 ± 0.42 4.27 ± 1.12
Female, s. immature I 23.81 ± 0.80 43.44 ± 0.22 25.27 ± 0.43 3.80 ± 0.03 6.65 ± 0.17
n=2 M 21.18 ± 1.11 49.95 ± 0.52 21.88 ± 0.27 3.52 ± 0.02 6.21 ± 0.12
O 20.86 ± 0.61 54.24 ± 1.25 17.72 ± 1.79 3.04 ± 0.12 5.82 ± 0.36
All dolphins I 22.85 0.28 52.68 0.71 18.10 0.55 3.13 0.05 5.70 0.11
n = 86 M 15.95 0.35 61.97 0.77 15.43 0.52 3.02 0.05 5.02 0.10
O 13.19 0.41 69.42 0.67 9.68 0.47 2.56 0.05 3.64 0.10
Values are means ± SEM, mass percent of total fatty acids and unknowns.
a
PL = pregnant and lactating; PnL = pregnant, not lactating; nPL = lactating, not pregnant; nPnL = not pregnant, not lactating. Long (i.e., physically mature)
animals had a mean body length of 174 ± 2 cm, short (i.e., physically immature) animals had a mean body length of 137 ± 2 cm (mean ± SEM).
b
I = Inner, M = middle, O = outer.
c
s. mature = sexually mature; s. immature = sexually immature.

between the inner and middle, and middle and outer layers
(ANOVA, both P b 0.001; Scheffé's test with α b 0.05) (Table 4).
The scatter plot of mean canonical scores showed that the DF
could be used to differentiate between the three layers;
however, there was some overlap between groups. The scatter
plot also demonstrated that the first DF was much more
effective than the second DF at separating the three groups
(Fig. 3).
3.1.2. Univariate approach
FA composition was found to differ significantly between the
three layers when all 15 FAs in the subset were considered
together (MANOVA, P b 0.001). Twelve of the 15 FAs ex-
amined were present in significantly different concentrations in
all three layers (ANOVA, all P b 0.001; Scheffé's test with
α b 0.05). Levels of 20 : 5n − 3 differed significantly only
between the inner and outer, and middle and outer layers. No
492 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499

a) b)
3 3
I
M I O II
M M I 2 O I
2 O M I II
O MI I
I I
O M I I OOO
O M I I III
M 1 I I
I
1 O M MM II I OO OO MO O
OO M M MI II
O M M I I I OOO
OOOO MMM
M
M
O IMI
M MM I MM II I
PC2 OOO MO OO
O M M M M
M M I II I 0 O
OO OO
OO
O OO OO
OO M M I I

PC2
OO M MO M M MO M III OO O O M M MMM MI I
0 O O OO O
O M O M M I III I M OM M
MM M IM MM II
M III
OO OO O OMM
O M M
IM M I I OO M M
M
M I
OO O OM O M I M I II -1 M
OM M
OOO M I I M M I
I MI I I
O O O M I I
OO M
M I II I O
-1
OO
O
M M III M I
I
I -2 M I I
O M M I I M
-2 M I -3
I I I

-3 -4
-2 -1 0 1 2 -2 -1 0 1 2
PC1 PC1

Fig. 2. Scatter plots of the first two principal components (PC) for sexually mature, male D. delphis (n = 144) calculated from the subset of a) 15 arcsin transformed fatty
acids (FAs), and b) 23 arcsin transformed FAs. The first 2 PC cumulatively account for 83.6% of the total variance in a), and 68.8% of the total variance in b). In both
plots, data points corresponding to the inner, middle, and outer layers are marked with the letters I, M and O, respectively.

significant differences were found for 18 : 1n − 9 or 18 : 2n − 6. A
significant increase in mass percent of 14 : 1n − 9, 14 : 1n − 7,
14 : 1n − 5, 16 : 1n − 9, and 16 : 1n − 7 was observed across the
blubber when moving from the inner to outer layer. In contrast
to this, 14 : 0, 16 : 0, 18 : 0, 18 : 1n − 7, 20 : 1n − 9, 22 : 5n − 3, and
22 : 6n − 3 decreased across the blubber from the inner to outer
layer (Fig. 4).
Significant layering differences were also found when the
summary variables were examined. The three layers differed
significantly when ∑SFA, ∑MUFA, ∑(n − 3)PUFA and ∑(n − 6)
PUFA were considered together (MANOVA, P b 0.001). ∑SFA
and ∑MUFA were present in significantly different quantities in
all three layers (ANOVA, all P b 0.001); ∑SFA decreased across
the blubber from the inner to outer layer, while ∑MUFA
increased across the blubber from the inner to the outer layer.
∑(n − 3)PUFA and ∑(n − 6)PUFA also decreased from the inner
layer to the outer, however; only the differences between the
inner and outer, and middle and outer layers were significant.
∑(n − 3)PUFA / ∑(n − 6)PUFA was also found to differ between
all three layers (ANOVA, P b 0.001), decreasing from the inner to
the outer layer.
14 : 1n − 7, and 14 : 0 as splitting variables (Fig. 5). The initial split
separated the majority (45 / 48) of the outer layer samples from the
inner and middle layer samples. Terminal nodes with misclassi-
fications always included at least one middle layer sample: three
inner samples were misclassified as middle, one middle sample
was misclassified as inner, three outer samples were misclassified
as middle, and two middle samples were misclassified as outer.
The tree had an overall misclassification rate of 9 / 144.
3.1.4. Blubber stratification in other dolphin groups
Visual examination of the most abundant FA revealed
noiceable layering differences in the other groups of common
dolphins. Fatty acid gradients were apparent across the blubber
in all groups. In general, 14 : 1n − 9, 14 : 1n − 7, 14 : 1n − 5,
16 : 1n − 9, and 16 : 1n − 7 increased in mass percent from the
inner to outer layer, while 14 : 0, 16 : 0, 18 : 0, 18 : 1n − 7,
20 : 1n − 9, 22 : 5n − 3, and 22 : 6n − 3 decreased in mass percent
from the inner to outer layer. In contrast, 18 : 2n − 6 tended to be
5

3.1.3. Classification and regression tree approach

CART analysis effectively separated sexually mature, male 2
D. delphis blubber into three layers, selecting 20 : 0, 14 : 1n − 9, LAYER
FACTOR(2)

Inner
Table 4
Middle
Jack-knifed classification matrix and mean canonical scores (± SEM) for two
significant discriminant functions generated from the arcsin transformed Outer
-1
summary variables (∑SFA, ∑MUFA, ∑(n − 3)PUFA, and ∑(n − 6)PUFA) for
sexually mature, male D. delphis (ni = 48)
Actual Predicted membership Mean canonical scores
Membership Inner Middle Outer % correct Score 1 Score 2 a
-4
Inner 45 3 0 94 2.4 ± 0.1 − 0.3 ± 0.2 -4 -1 2 5
Middle 6 32 10 67 − 0.3 ± 0.2 0.6 ± 0.1 FACTOR(1)
Outer 0 5 43 90 − 2.1 ± 0.1 − 0.4 ± 0.2
Total 51 40 53 83
Fig. 3. Canonical scores plot for the 2 discriminant functions generated from the
a
The second mean canonical score only differed significantly between the summary variables (arcsin transformed) for sexually mature, male D. delphis
inner and middle, and middle and outer layers (ANOVA, both P b 0.001; (n = 48). The centroids of the 3 ellipses are significantly different from one
Scheffé's test with α b 0.05). another (MANOVA, P b 0.001).
 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
35
30 Inner
Mass % Total FAs and Unknowns
Middle
Outer
25
20
15
10
0
14:1n-7
14:0
14:1n-9
16:0
14:1n-5
16:1n-9
16:1n-7
Fig. 4. Mass percent values of fatty acids (FAs) commonly present in quantities ≥ 1% in sexually mature, male Delphinus delphis blubber (n = 48). Blubber was divided
into inner, middle, and outer layers. Bar values are means; error bars are 1 SEM. With the exception of 18 : 1n − 9, 18 : 2n − 6, and 20 : 5n − 3, FAs were present in
significantly different quantities in all 3 layers.
relatively consistent across the blubber layer, while 18 : 1n − 9
and 20 : 5n − 3 tended to be present in the highest concentration
in the middle layer.
3.2. Degree of stratification
SI values ranged from a low of 30.21 ± 2.67 in short, sexually
immature, male D. delphis (n = 5) to a high of 58.58 ± 6.38 in
pregnant, non-lactating, female D. delphis (n = 2; Table 5). SI
values for male D. delphis differed significantly (ANOVA,
P b 0.001), with the short, sexually immature males being
significantly less stratified (30.21 ± 2.67; n = 5) than the
sexually mature males (50.36 ± 1.52; n = 48). SI values of
long, sexually immature males were not significantly less
stratified than the sexually mature males. In general, blubber of
the older animals was more stratified than the younger animals.
3.3. Intraspecific differences in FA composition
D. capensis were correctly grouped by sex 100% of the time
when sexually mature and immature animals were analyzed
separately. When sexually mature and immature animals were
analyzed together, the middle and outer layers resulted in no
misclassifications, while the inner layer had a MR = 1 / 9. In all
cases, the trees consisted of only one split, using a SFA or
MUFA with ≤17C as the splitting criteria (Table 6). D. delphis
also grouped by sex, but typically resulted in higher MRs and
more splits per tree. Longer chain FAs such as 22 : 6n − 3,
20 : 5n − 3, 20 : 4n − 6, 20 : 1n − 9, 20 : 0, and ∑(n − 3)PUFA
were used as splitting criteria in analyses involving sexually
mature animals (Table 6).
In general, females that were pregnant and/or lactating
tended to separate from animals that were not. D. delphis PL
females separated from all other females in all trees when
sexually immature and mature animals were analyzed together,
493
20:5n-3
20:1n-9
22:5n-3
18:1n-9
18:1n-7
22:6n-3
18:2n-6
18:0
and in the middle and outer layer trees when only sexually
mature females were analyzed. D. capensis nPL females
separated from the sexually immature and nPnL females in
both the middle and outer layer trees when sexually immature
and mature animals were analyzed together, and also from the
nPnL females in all trees when only sexually mature animals
were analyzed. When the simplified data sets were analyzed
(i.e., D. delphis females, P vs. nP, and L vs. nL) all trees had
lower MRs than the previous analyses of data sets with more
detailed reproductive states (Table 6). Of all CART analyses
performed, the highest MR (9 / 17) occurred in all three layers in
D. delphis females (Table 6). D. delphis sexually mature males
often grouped together with “long” sexually immature males,
while D. capensis sexually mature males always separated from
sexually immature males (Table 6).
3.4. Interspecific differences in FA composition
All dolphin groups examined were effectively classified to
the species level on the basis of differences in FA com-
position. All samples were correctly classified in all analyses
(i.e. MR = 0) with the exception of the outer layer in sexually
mature males, where MR = 2 / 50. Thirteen of the 15 trees
produced used only one split to classify the data points into
groups. Shorter chain (≤ 16 C) saturated and monounsatu-
rated FAs were selected as splitting criteria in all cases
(Table 6).
4. Discussion
FA composition of common dolphin blubber is comparable
to that of other marine mammal species previously studied
(Jangaard et al., 1963; Ackman and Eaton, 1966; West et al.,
1979a; Iverson et al., 1997b; Samuel and Worthy, 2004) with
16 : 0 as the predominant saturated FA (SFA), followed by 14 : 0
494 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499

LAYER

(48,48,48)

20:0<0.055

Inner Outer
(48,46,3) (0,2,45)
49/97 2/47

14:1n-9<0.230

Inner Middle
(43,3,0) (5,43,3)
3/46 8/51

14:1n-7<0.145 20:0<0.145

Inner Inner Inner Middle

(36,0,0) (7,3,0) (4,1,0) (1,42,3)
0/36 3/10 1/5 4/46

14:1n-9<0.205 14:0<4.250

Inner Middle Middle Middle

(5,0,0) (2,3,0) (1,35,0) (0,7,3)
0/5 2/5 1/36 3/10

Fig. 5. Classification tree for sexually mature, male D. delphis blubber samples (n = 144), grouped by layer. Each square represents a node in the tree and lists the
following node information: Mode (most abundant sample type present), number of samples in each group (Inner, Middle, Outer), and Misclassification Rate (MR) =
(number of samples from least abundant groups) / (total samples in node). The overall MR for the entire tree was 9 / 144. Fatty acids (FAs) used as splitting criteria for
each node are listed, as well as values (mass percent total FAs and unknowns) used to produce indicated splits.

and 18 : 0. Overall, ∑MUFA accounted for the greatest
proportion of FA measured, with 18 : 1n − 9 and 16 : 1n − 7
being predominant. PUFAs were predominated by the n − 3
class, primarily 22 : 6n − 3, 22 : 5n − 3, and 20 : 5n − 3. 18 : 2n − 6
and 20 : 4n − 6 were the most common n − 6 FA.
4.1. Blubber stratification
In the present study, common dolphin blubber was found to
be biochemically stratified, similar to the blubber of nume-
rous other cetacean species. Common dolphin blubber had

Table 5
Mean stratification index (SI) values (±SEM) for all dolphin groups
Species Male Female
a
Mature Immature Mature Immature
PL b nPL PnL nPnL
D. delphis 50.36 ± 1.52 44.33 ± 3.15 (long c) 49.03 54.16 ± 3.86 58.58 ± 6.38 51.70 ± 4.73 40.25 ± 4.79
(n = 48) (n = 5) (n = 1) (n = 3) (n = 2) (n = 4) (n = 7)
30.21 ± 2.67 (short)
(n = 5)
D. capensis 46.38 ± 19.40 53.76 ± 12.08 34.95 36.23 ± 3.47 35.03 ± 2.16
(n = 2) (n = 5) (n = 1) (n = 2) (n = 2)
SI was calculated as the sum of the absolute values of the differences in mass percent between the inner and outer blubber layers for the 15 FAs commonly present in
quantities N1% by mass: 14 : 0, 14 : 1n − 9, 14 : 1n − 7, 14 : 1n − 5, 16 : 0, 16 : 1n − 9, 16 : 1n − 7, 18 : 0, 18 : 1n − 9, 18 : 1n − 7, 18 : 2n − 6, 20 : 1n − 9, 20 : 5n − 3, 22 : 5n − 3,
and 22 : 6n − 3.
a
Mature = sexually mature, Immature = sexually immature.
b
PL = pregnant and lactating; PnL = pregnant, not lactating; nPL = not pregnant, lactating; nPnL = not pregnant, not lactating.
c
Long animals had a mean body length of 174 ± 2 cm, short animals had a mean body length of 137 ± 2 cm (mean ± SEM).
 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499 495

Table 6
Summary of CART results
Grouping variable Species Individuals Layer n MR FA used as split criteria
Sex (M, F) D. delphis All I 75 9 / 75 Iso 15 : 0, 18 : 0, 22 : 6n − 3, iso 16 : 0, 12 : 0, 18 : 1n − 7, 18 : 1n − 9
M 75 4 / 75 20 : 4n − 6, 20 : 1n − 9, 12 : 0, 18 : 1n − 9, anti 15 : 0
O 75 7 / 75 16 : 1n − 5, 20 : 5n − 3, anti 15 : 0, ∑(n − 3)PUFA, iso 14 : 0
s a. mature I 58 7 / 58 22 : 6n − 3, iso 15 : 0, 18 : 1n − 7, iso 16 : 0
M 58 5 / 58 20 : 4n − 6, ∑(n − 3)PUFA, iso 14 : 0, 20 : 0
O 58 3 / 58 16 : 2n − 6, iso 14 : 0, 20 : 0, 16 : 1n − 7
s. immature I 17 2 / 17 16 : 1n − 11, 14 : 0
M 17 2 / 17 16 : 1n − 11
O 17 2 / 17 16 : 1n − 11, 10 : 0
Sex (M, F) D. capensis All I 9 1/9 17 : 0
M 9 0 14 : 1n − 7
O 9 0 14 : 1n − 5
s. mature I 5 0 15 : 0
M 5 0 14 : 1n − 9
O 5 0 12 : 0
s. immature I 4 0 10 : 0
M 4 0 13 : 0
O 4 0 13 : 0
Reproductive status D. delphis All females I 17 9 / 17 16 : 1n − 11
(PL b, PnL, nPL, nPnL, I) M 17 9 / 17 Iso 16 : 0
O 17 9 / 17 16 : 1n − 11
Reproductive status s. mature females I 10 4 / 10 15 : 1n − 8
(PL, PnL, nPL, nPnL) M 10 5 / 10 13 : 0
O 10 3 / 10 Iso 14 : 0, 10 : 0
Reproductive status s. mature females I 10 1 / 10 18 : 1n − 11
(L c, nL) M 10 2 / 10 14 : 0
O 10 1 / 10 14 : 0
Reproductive status s. mature females I 10 1 / 10 18 : 2n − 6
(P d, nP) M 10 2 / 10 13 : 0
O 10 0 Iso 14 : 0
Reproductive status D. capensis All females I 5 1/5 10 : 0
(nPL, nPnL, I) M 5 2/5 10 : 0
O 5 2/5 10 : 0
Reproductive status s. mature females I 3 0 12 : 0
(nPL, nPnL) M 3 0 10 : 0
O 3 0 10 : 0
Maturity D. delphis All males I 58 5 / 58 22 : 1n − 9, 16 : 1n − 7, 14 : 1n − 9
(M e, IL, IS) M 58 6 / 58 anti 15 : 0
O 58 5 / 58 16 : 1n − 9
Maturity D. capensis All males I 4 0 14 : 0
(M, I) M 4 0 Iso 14 : 0
O 4 0 Iso 14 : 0
Species Both nPL females I 4 0 12 : 0
M 4 0 10 : 0
O 4 0 Iso 14 : 0
nPnL females I 6 0 14 : 0
M 6 0 Iso 14 : 0
O 6 0 Iso 14 : 0
s. immature females I 9 0 14 : 0
M 9 0 10 : 0
O 9 0 14 : 0
s. mature males I 50 0 16 : 4n − 1
M 50 0 16 : 4n − 1, 16 : 1n − 11
O 50 2 / 50 15 : 0, 14 : 0
s. immature males I 12 0 13 : 0
M 12 0 13 : 0
O 12 0 14 : 1n − 5
Overall misclassification rate (MR) for each tree is listed. The fatty acid (FA) listed first was used to create the initial split.
a
s. mature = sexually mature.
b
PL = pregnant and lactating; PnL = pregnant, not lactating; nPL = not pregnant, lactating; nPnL = not pregnant, not lactating; I = sexually immature.
c
L = lactating; nL = not lactating.
d
P = pregnant, nP = not pregnant.
e
M = sexually mature, IL = immature long = sexually immature, physically mature; IS = immature short = sexually immature, physically immature.
496 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
decreasing levels of ∑SFA and increasing levels of ∑MUFA,
from the inner to the outer layer, as has also been observed in
bottlenose dolphins (Samuel and Worthy, 2004), killer whales
(Worthy et al., 2003; Krahn et al., 2004), and fin whales
(Balaenoptera physalus) (Ackman et al., 1965; Lockyer et al.,
1984). These gradients have been hypothesized to correspond to
the thermal gradient that exists across the blubber layer of
animals that inhabit cold water. As the outer blubber layer is
exposed to lower temperatures than the inner layer, a reduced
level of SFA, which pack close together at low temperatures, and
an increased level of MUFA, which do not pack together as
readily as SFA, will better allow this outer blubber to remain
fluid (Pond, 1998).
The decrease in ∑SFA across common dolphin blubber
observed in the present study is in contrast to the work of
Koopman et al. (1996), who found an increase in ∑SFA from
23.18 ± 3.90 to 31.16 ± 5.34 mass percent from the inner to the
outer layer in harbor porpoise blubber (data listed are for the “III
Dorsal” body site, i.e., dorsal location, approximately at the
trailing edge of the dorsal fin). It is likely that this discrepancy
may be attributed to the different methods used to prepare esters
from the lipid extract, rather than to species differences.
Koopman et al. (1996) were interested in quantifying short
chain FA such as isovaleric acid (iso 5 : 0), and therefore
prepared butyl esters, as they are much less volatile than the
methyl esters prepared in this study. As a result, ∑SFA as
calculated by Koopman et al. (1996) included values for iso 4 : 0
and iso 5 : 0, both of which increase from the inner to the outer
layer (0.56 ± 0.77 to 1.71 ± 0.90, and 3.11 ± 2.09 to 8.61 ± 4.05
mass percent, respectively). These short chain FAs were not
measured in this study, and were therefore not included in our
∑SFA calculation. Additionally, preparation and analysis of
different types of esters can result in different mass percent
values for FA in the same sample. Because FAs are expressed as
a percentage of all FAs identified in a given sample, when butyl
esters are prepared, a greater number of FA are identified (i.e.,
short chain FA), and mass percent values of FA are propor-
tionately lower than if methyl esters had been prepared. This
produces lower ∑SFA values overall, and may contribute to the
reduced magnitude of between layer differences.
Examination of FA commonly present in quantities ≥ 1% by
mass in mature male common dolphins revealed that levels of
most individual FAs differed significantly between all three
layers. All other FAs measured appear to follow the same pattern
as the more abundant FAs of the same class (e.g., SFA, MUFA).
The middle layer values were typically intermediate between the
inner and outer layer values (e.g., Table 2), supporting the
suggestion of Koopman et al. (1996) that a continuous gradient
in FA composition runs across the entire blubber layer.
Koopman et al. (1996) tested for significant differences in
levels of individual FAs between the inner and outer blubber
layers in mature, male, harbor porpoise. Similar to the results of
Koopman et al. (1996), 18 : 1n − 9 was not found to differ
significantly across the complete depth of blubber of mature,
male, short-beaked common dolphins. Animals are capable
of synthesizing 18 : 1n − 9 (Cook, 1985; Nelson, 1992) in ad-
dition to obtaining it from the diet. Consistently high levels
(approximately 30% by mass, this study) of 18 : 1n − 9 across
the complete depth of blubber suggests that this FA may
contribute to outer blubber fluidity, as well as being involved in
metabolic activities. Animals feeding on diets deficient in
essential FA (i.e., 18 : 2n − 6 and 18 : 3n − 3) have been shown to
desaturate and elongate 18 : 1n − 9 to produce 20 : 3n − 9. This
new FA is able to act as a partial substitute for 20 : 4n − 6, and
when incorporated into phospholipids, adequately performs
some membrane functions, but does not act as a precursor for
prostaglandins (Cook, 1985). As 18 : 1n − 9 may be involved in
such metabolic activities, it is reasonable that such high
quantities would be maintained in the inner blubber layer,
where they would be readily accessible in a time of need, in
addition to the high quantities contributing to membrane
fluidity in the outer layer.
The magnitude of 18 : 2n − 6 levels (approximately 1.2% by
mass) measured in mature, male, short-beaked common dol-
phins in this study was similar to the levels measured in mature,
male, harbor porpoise by Koopman et al. (1996). However,
unlike the situation found in harbor porpoise, no gradient across
the blubber layer was found in common dolphins. Because
18 : 2n − 6 is an essential FA, it is available only through the diet
(Cook, 1985). It is therefore logical to expect to find the highest
levels of 18 : 2n − 6 in the more metabolically active inner
blubber layer. It is possible that the lower than expected levels of
18 : 2n − 6 in the inner layer may be due to the physiological state
of the animals collected, with the 18 : 2n − 6 demand exceeding
current intake, thereby resulting in no gradient.
CART analysis effectively separated the inner blubber layer
samples from the outer blubber layer samples, but was less
successful at separating out the middle blubber layer samples.
The number of misclassifications involving inner and middle
samples was roughly equal to the number of misclassifications
involving outer and middle samples, and therefore does not
indicate that the middle layer is more similar to either the inner
or the outer layer. However, the initial split in the tree separated
the majority (45 / 48) of the outer layer samples from the inner
and middle layer samples, which suggests that the middle layer
may be most similar to the inner layer. This suggestion is further
supported by the ANOVA results, where 20 : 5n − 3 was not
found to differ significantly between the inner and middle layers
in sexually mature, male D. delphis.
Samuel and Worthy (2004) found that the inner and middle
layers tended to group together in male and non-lactating female
bottlenose dolphins, while the outer and middle layers tended to
group in lactating females. The tendency for the middle layer to
reflect either the inner or outer layer was attributed to the re-
productive status, and therefore the differing physiological and/
or dietary requirements of lactating animals. The suggestion of
variable FA composition in the middle layer with respect to state
of lactation is supported by the work of Aguilar and Borrell
(1990), who found that reproductive status affected the lipid
content of the inner and middle blubber layers in fin whales.
Iverson et al. (1995) found that lactating animals selectively
mobilize FA such as 20 : 5n − 3 from maternal blubber into the
milk. The ability of female mammals to forage while lactating
would maintain levels of such PUFA in the blubber during
 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
lactation and will influence the degree to which the layers
resemble one another. If stores of mobilized FA are not re-
plenished, the FA composition of the mobilized layer will
change over the course of lactation. Assuming that stores of
PUFA are depleted as the animal lactates, mobilization of FA
from the inner layer will result in the inner layer becoming more
similar to the middle layer. In contrast, if PUFA are mobilized
from the middle layer, the middle layer will become more
similar to the outer layer.
Variation in FA composition of the different blubber layers
may also be caused by changes in diet. Bernard and Hohn
(1989) noted differences in feeding habits between pregnant and
lactating spotted dolphins, and Young and Cockcroft (1994)
noted significant differences in the species of prey consumed by
lactating versus non-lactating common dolphins. Depending on
the FA composition of consumed prey items, and assuming that
excess FAs are deposited in the inner blubber layer, the FA
composition of the inner layer may become even more different
than that of the middle layer, causing the middle and outer
layers to appear more similar.
4.2. Degree of stratification
The blubber of sexually mature, male common dolphins was
more highly stratified than that of sexually immature males, in
agreement with the results of Koopman et al. (1996), who
suggested that this difference is due to changes in lipid meta-
bolism, and therefore, changes in FA composition of the outer
blubber layer over time. PUFA, and n − 3 PUFA in particular,
were present in only very small quantities in the outer blubber
layer of older porpoises (Koopman et al., 1996).
It is also possible that differences in the degree of strat-
ification may be attributed to differences in diet, with prey items
rich in certain FA resulting in steep FA gradients across the
blubber. Sexually immature males in the present study were
further classified according to body length. “Short” animals
were significantly shorter (137 ± 2 cm) than “long” animals
(174 ± 2 cm) (ANOVA, P b 0.001; Scheffé's test with α = 0.05),
which were more similar in length to sexually mature animals
(186 ± 1 cm). While blubber of both groups of sexually
immature animals were less stratified than the sexually mature
animals, the “short” immature group was also less stratified than
the “long” immature group. Dietary differences were the most
likely causative factor since the longer immature dolphins were
physically mature and were thus capable of catching prey items
similar to those caught by the sexually mature animals. This
ability would likely be due to a combination of larger body size
and more foraging experience over the shorter, younger, and
probably less experienced physically immature dolphins.
An increase in the degree of stratification with sexual matu-
rity was also observed in female D. delphis, similar to harbor
porpoises (Koopman, 2001). Stratification index values for
mature females were quite variable with respect to reproductive
status, and by extension, dietary differences (e.g., Bernard and
Hohn, 1989). Unfortunately, small sample sizes precluded
statistical testing, and visual examination did not reveal any
correlations.
497
Stratification index values for D. delphis from the present
study (∼ 40.2 to 58.6) were higher than the SI values (27.5–
53.6, n = 13) calculated by Koopman (2001) for the same
species. This could potentially be a function of the method by
which blubber was divided into layers. Koopman (2001)
divided blubber into inner and outer layers, discarding a layer
of blubber between the two. In the present study, blubber was
divided into five layers, and blubber between the inner and
middle, and middle and outer layers was discarded (Fig. 1.). As
such, the inner and outer layers examined in the present study
were potentially thinner than the inner and outer layers exam-
ined by Koopman, which may have incorporated some of the
blubber considered “middle” in the present study. As concen-
trations of FA in the middle layer were intermediate between
inner and outer layer values (present study) then including
portions of middle blubber in outer or inner blubber samples
could potentially result in less pronounced differences between
the inner and outer layers. Until standardized methods of ana-
lyzing fatty acid composition are adopted, it will remain
difficult to determine whether such differences exist solely due
to method, or are the result of phylogenetic, environmental or
other factors.
4.3. Intraspecific differences in FA composition
Foraging differences between male and female common
dolphins have been observed (Young and Cockcroft, 1994;
Chou et al., 1995), and this was expected to result in gender
differences in FA composition. As dietary FAs are initially
deposited in the inner blubber layer (Lockyer et al., 1984;
Aguilar and Borrell, 1990; Koopman et al., 1996), it was
expected that this would be where the FA composition of males
and females would be most easily distinguished. Contrary to
this expectation, the highest misclassification rates occurred in
the inner layer analyses for both species of common dolphin.
FA selected as splitting criteria in the CART analyses with
sex as the grouping variable, and involving sexually mature
D. delphis often included long chain PUFA of dietary origin
(e.g., 22 : 6n − 3, 20 : 4n − 6, 20 : 5n − 3). In contrast to this,
trees produced in all other CART analyses grouped by sex
used FA with carbon chains ≤ 17C as splitting criteria.
However, the majority of these shorter chain FAs were odd-
chained (e.g., 13 : 0, 15 : 0, 17 : 0) and hence also of dietary
origin. Vertebrates are typically capable of biosynthesising
only even-chained FA (Cook, 1985). Regardless of chain
length, the selection of dietary FA as splitting criteria suggests
that differences in diet between the two sexes do exist. Of the
nine CART trees produced for D. capensis using sex as the
grouping variable, only the tree for the inner layer, with
sexually mature and immature animals combined, resulted in
a misclassification (1 / 9).
FA composition was expected to vary with reproductive
status, as differences in lipid metabolism and diet selection
have been observed in animals of different reproductive con-
dition. Based on evidence that female mammals selectively
mobilize certain FA from the blubber layer during lactation
(Iverson et al., 1995), differences in the blubber FA composition
498 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
of lactating and non-lactating females were anticipated. In the
present study, lactating females tended to, but did not always
separate from non-lactating females. Small cetaceans, including
common dolphins, forage throughout an extended (∼6 months
to 2 years) lactation period (Boyd et al., 1999). It is possible,
that while common dolphins may selectively mobilize particular
FA in milk production, the continued foraging by these animals
reduces the need to mobilize FA stores to the same extent as has
been observed in phocid seals (e.g., Iverson et al., 1995).
In the present study, sexually mature and immature long
D. delphis grouped together, while sexually immature short
animals formed their own group. This provides evidence that
differences in FA composition, as attributed to differences in
feeding habits, are more likely correlated with body size
(physical maturity) than sexual maturity, as discussed above.
4.4. Interspecific differences in FA composition
There was only one misclassification when species was used
as the grouping variable, supporting the idea that these two
species of dolphins may be feeding on different prey items in
order to co-exist in the same area (niche partititioning) (Heyning
and Perrin, 1994). Even female animals, with the same repro-
ductive status and therefore facing similar nutritional demands,
were consistently separated from one another.
In conclusion, this study demonstrates that common dolphin
blubber is biochemically stratified, with inner, middle and outer
blubber layers distinguishable from one another on the basis of
FA composition. The majority of FAs were distributed along
gradients across the blubber layer. Levels of MUFA were
highest in the outer layer, while SFA and PUFA were highest in
the inner layer. Blubber stratification was most pronounced in
mature dolphins. FA composition was found to differ between
dolphins grouped by sex, reproductive status and species.
Selection of dietary FA as splitting criteria in CART analyses
suggests that differences in FA composition observed in the
present study may be attributed to dietary differences between
these groups of animals. These results highlight the need to
collect full-depth blubber cores in future studies where the
intention is to use fatty acid data to make dietary inferences. It is
important that the innermost layer of blubber be analyzed for
this purpose. It is equally important that blubber be divided into
layers in a consistent manner such that between-study com-
parisons of data may be possible.
Acknowledgements
We would like to thank S. Chivers, K. Robertson, K. Danil
(SWFSC, NMFS), J. Heyning, and D. Janiger (LACM) for
providing archived blubber samples. We thank S. Iverson, S.
Lang, and D. Cowan for their technical expertise. We ap-
preciate the constructive comments of B. Fadely, M. Lander
and R. Watson on earlier versions of this manuscript. This
manuscript was also improved by comments provided by two
anonymous reviewers. Funding for this work was provided
by: The Mooney Graduate Student Travel Grant, the Office of
Graduate Studies (TAMU), the Research Management Office
(TAMUG), and a research award to GAJW from the Texas
Higher Education Coordinating Board Advanced Research
Program.
References
Ackman, R.G., 1972. The analysis of fatty acids and related materials by gas–
liquid chromatography. In: Holman, R.T. (Ed.), Progress in the Chemistry of
Fats and Other Lipids, vol. 12. Pergamon Press, Oxford, pp. 165–284.
Ackman, R.G., 1991. Application of gas–liquid chromatography to lipid
separation and analysis: qualitative and quantitative analysis. In: Perkins,
E.G. (Ed.), Analyses of Fats, Oils and Lipoproteins. American Oil Chemists'
Society, Champaign, pp. 270–300.
Ackman, R.G., Eaton, C.A., 1966. Lipids of the fin whale (Balaenoptera
physalus) from north Atlantic waters. III. Occurrence of eicosenoic and
docosenoic fatty acids in the zoolplankter Meganyctiphanes norvegica
(M. Sars) and their effect on whale oil composition. Can. J. Biochem. 44,
1561–1566.
Ackman, R.G., Eaton, C.A., Jangaard, P.M., 1965. Lipids of the fin whale
(Balaenoptera physalus) from North Atlantic waters. Can. J. Biochem. 43,
1513–1520.
Ackman, R.G., Hingley, J.H., Eaton, C.A., Logan, V.H., Odense, P.H., 1975a.
Layering and tissue composition in the blubber of the northwest Atlantic sei
whale (Balaenoptera borealis). Can. J. Zool. 53, 1340–1344.
Ackman, R.G., Hingley, J.H., Eaton, C.A., Sipos, J.C., Mitchell, E.D., 1975b.
Blubber fat deposition in mysticeti whales. Can. J. Zool. 53, 1332–1339.
Aguilar, A., Borrell, A., 1990. Patterns of lipid content and stratification in the
blubber of fin whales (Balaenoptera physalus). J. Mammal. 71, 544–554.
Akin, P.A., Peltier [Robertson], K.M., Miller, R.B., 1993. Techniques for the
preparation and examination of reproductive samples collected from dolphins
in the eastern tropical Pacific. NOAA Tech. Mem., NMFS-SWFSC-192.
Bernard, H.J., Hohn, A.A., 1989. Differences in feeding habits between pregnant
and lactating spotted dolphins (Stenella attenuata). J. Mammal. 70, 211–215.
Best, N.J., Bradshaw, C.J.A., Hindell, M.A., Nichols, P.D., 2003. Vertical
stratification of fatty acids in the blubber of southern elephant seals
(Mirounga leonina): implications for diet analysis. Comp. Biochem.
Physiol. B 134, 253–263.
Borobia, M., Gearing, P.J., Simard, Y., Gearing, J.N., Béland, P., 1995. Blubber
fatty acids of finback and humpback whales from the Gulf of St. Lawrence.
Mar. Biol. 122, 341–353.
Boyd, I.L., Lockyer, C., Marsh, H.M., 1999. Reproduction in marine mammals.
In: ReynoldsIII III, J.E., Rommel, S.A. (Eds.), Biology of Marine Mammals.
Smithsonian Institution Press, Washington, pp. 218–286.
Brown, D.J., Boyd, I.L., Cripps, G.C., Butler, P.J., 1999. Fatty acid signature
analysis from the milk of Antarctic fur seals and Southern elephant seals
from South Georgia: implications for diet determination. Mar. Ecol. Prog.
Ser. 187, 251–263.
Chou, L., Bright, A.M., Yeh, S., 1995. Stomach contents of dolphins (Delphinus
delphis and Lissodelphis borealis) from North Pacific Ocean. Zool. Stud. 34,
206–210.
Christie, W.W., 1972. The preparation of alkyl esters from fatty acids and lipids.
In: Gunstone, F.D. (Ed.), Topics in Lipid Chemistry, vol. 3. Logos Press Ltd,
London, pp. 171–197.
Cook, H.W., 1985. Fatty acid desaturation and chain elongation in eukaryotes.
In: Vance, D.E., Vance, J.E. (Eds.), Biochemistry of Lipids and Membranes.
Benjamin-Cummings Publishing Co, Inc., Menlo Park, pp. 181–211.
Cowan, D.F., Worthy, G.A.J., 1991. Body site variation in the structure of the
blubber in Tursiops truncatas. Abstracts of the Ninth Bienniel Conference on
the Biology of Marine Mammals. Chicago, IL, 5– 9 December 1991, p. 14.
Evans, W.E., 1975. Distribution, differentiation of populations, and other aspects
of the natural history of Delphinus delphis Linnaeus in the Northeastern
Pacific. Ph.D. dissertation, University of California, Los Angeles.
Evans, W.E., 1994. Common dolphin, white-bellied porpoise, Delphinus delphis
Linnaeus, 1758. In: Ridgeway, S.H., Harrison, R.J. (Eds.), Handbook of
Marine Mammals, vol. 5. Academic Press Ltd., London, pp. 191–224.
 H.R. Smith, G.A.J. Worthy / Comparative Biochemistry and Physiology, Part B 143 (2006) 486–499
Ferrero, R.C., Walker, W.A., 1995. Growth and reproduction of the common
dolphin, Delphinus delphis Linnaeus, in the offshore waters of the North
Pacific Ocean. Fish. Bull. 93, 483–494.
Fitch, J.E., Brownell Jr., R.L., 1968. Fish otoliths in cetacean stomachs and their
importance in interpreting feeding habits. J. Fish. Res. Board Can. 25,
2561–2574.
Folch, J., Lees, M., Sloan-Stanley, G.H., 1957. A simple method for the isolation
and purification of total lipides from animal tissues. J. Biol. Chem. 226,
497–509.
Fredheim, B., Holen, S., Ugland, K.I., Grahl-Nielson, O., 1995. Fatty acid
composition in blubber, heart and brain from phocid seals. In: Blix, A.S.,
Walløe, L., Ulltang, Ø. (Eds.), Whales, Seals, Fish and Man. Elsevier
Science, Amsterdam, pp. 153–168.
Grahl-Nielsen, O., Andersen, M., Derocher, A.E., Lydersen, C., Wiig, Ø.,
Kovacs, K.M., 2003. Fatty acid composition of the adipose tissue of polar
bears and their prey: ringed seals, bearded seals and harp seals. Mar. Ecol.
Prog. Ser. 265, 275–282.
Heyning, J.E., Perrin, W.F., 1994. Evidence for two species of common dolphins
(genus Delphinus) form the Eastern North Pacific. LACM Contr. Sci., vol.
442, pp. 1–35.
Hooker, S.K., Iverson, S.J., Ostrom, P., Smith, S.C., 2001. Diet of northern
bottlenose whales inferred from fatty-acid and stable-isotope analyses of
biopsy samples. Can. J. Zool. 79, 1442–1454.
Iverson, S.J., Sampugna, J., Oftedal, O.T., 1992. Positional specificity of gastric
hydrolysis of long-chain n − 3 polyunsaturated fatty acids of seal milk
triglycerides. Lipids 27, 870–878.
Iverson, S.J., Oftedal, O.T., Bowen, W.D., Boness, D.J., Sampugna, J., 1995.
Prenatal and postnatal transfer of fatty acids from mother to pup in the
hooded seal. J. Comp. Physiol., B 165, 1–12.
Iverson, S.J., Arnould, J.P.Y., Boyd, I.L., 1997a. Milk fatty acid signatures
indicate both major and minor shifts in the diet of lactating Antarctic fur
seals. Can. J. Zool. 75, 188–197.
Iverson, S.J., Frost, K.J., Lowry, L.F., 1997b. Fatty acid signatures reveal fine
scale structure of foraging distribution of harbor seals and their prey in
Prince William Sound, Alaska. Mar. Ecol. Prog. Ser. 151, 255–271.
Iverson, S.J., Lang, S., Cooper, M., 2001a. Comparison of the bligh and dyer
and folch methods for total lipid determination in a broad range of marine
tissue. Lipids 36, 1283–1287.
Iverson, S.J., MacDonald, J., Smith, L.K., 2001b. Changes in diet of free-
ranging black bears in years of contrasting food availability revealed through
milk fatty acids. Can. J. Zool. 79, 2268–2279.
Iverson, S.J., Field, C., Bowen, W.D., Blanchard, W., 2004. Quantitative fatty
acid signature analysis: a new method of estimating predator diets. Ecol.
Monogr. 74, 211–235.
Jangaard, P.M., Ke, P.J., 1968. Principal fatty acids of depot fat and milk lipids
from harp seal (Phoca groenlandica) and hooded seal (Cystophora cristata).
J. Fish. Res. Board Can. 25, 2419–2426.
Jangaard, P.M., Ackman, R.G., Burgher, R.D., 1963. Component fatty acids of
the blubber of fat from the common or harbour seal Phoca vitulina concolor
De Kay. Can. J. Biochem. Physiol. 41, 2543–2546.
Jefferson, T.A., Myrick Jr., A.C., Chivers, S.J., 1994. Small cetacean dissection
and sampling: a field guide. NOAA Tech. Mem., NMFS-SWFSC-198.
Johnson, R.A., Wichern, D.W., 1998. Applied Multivariate Statistical Analysis,
4th ed. Prentice Hall, Inc., Upper Saddle River, NJ.
Jones, R.E., 1981. Food habits of smaller marine mammals from Northern
California. Proc. Calif. Acad. Sci. 42, 409–433.
Käkelä, R., Hyvärinen, H., 1993. Fatty acid composition of fats around the
mystacial and superciliary vibrissae differs from that of blubber in the
Saimaa ringed seal (Phoca hispida saimensis). Comp. Biochem. Physiol. B
105, 547–552.
Koopman, H.N., 2001. The structure and function of odontocete blubber. Ph.D.
dissertation, Duke University, Durham, NC.
Koopman, H.N., Iverson, S.J., Gaskin, D.E., 1996. Stratification and age-related
differences in blubber fatty acids of the male harbour porpoise (Phocoena
phocoena). J. Comp. Physiol. B 165, 628–639.
Koopman, H.N., Iverson, S.J., Read, A.J., 2003. High concentrations of
isovaleric acid in the fats of odontocetes: variation and patterns of
499
accumulation in blubber vs. stability in the melon. J. Comp. Physiol. B
173, 247–261.
Krahn, M.M., Herman, D.P., Ylitalo, G.M., Sloan, C.A., Burrows, D.G., Hobbs, R.
C., Mahoney, B.A., Yanagida, G.K., Calambokidis, J., Moore, S.E., 2004.
Stratification of lipids, fatty acids and organochlorine contaminants in blubber
of white whales and killer whales. J. Cetacean Res. Manag. 6, 175–189.
Lockyer, C.H., McConnell, L.C., Waters, T.D., 1984. The biochemical
composition of fin whale blubber. Can. J. Zool. 62, 2253–2562.
Nelson, G.J., 1992. Dietary FAs and lipid metabolism. In: Chow, C.K. (Ed.),
Fatty Acids in Foods and Their Health Implications. Marcel Dekker, Inc.,
New York, NY, pp. 437–471.
Olsen, E., Grahl-Nielsen, O., 2003. Blubber fatty acids of minke whales:
stratification, population identification and relation to diet. Mar. Biol. 142,
13–24.
Pabst, D.A., Rommel, S.A., McLellan, W.A., 1999. The functional morphology
of marine mammals. In: ReynoldsIII III, J.E., Rommel, S.A. (Eds.),
Biology of Marine Mammals. Smithsonian Institution Press, Washington,
DC, pp. 15–72.
Parry, D.A., 1949. The structure of whale blubber, and a discussion of its thermal
properties. Q. J. Microsc. Sci. 90, 13–25.
Perrin, W.F., Coe, J.M., Zweifel, J.R., 1976. Growth and reproduction of the
spotted porpoise, Stenella attenuata, in the offshore eastern tropical Pacific.
Fish. Bull. 74, 29–269.
Pond, C.M., 1998. The Fats of Life. Cambridge University Press,
Cambridge,UK.
Rosel, P.E., Dizon, A.E., Heyning, J.E., 1994. Genetic analysis of sympatric
morphotypes of common dolphins (genus Delphinus). Mar. Biol. 119,
159–167.
Samuel, A.M., Worthy, G.A.J., 2004. Variability in fatty acid composition of
bottlenose dolphin (Tursiops truncatus) blubber as a function of body site,
season, and reproductive status. Can. J. Zool. 82, 1933–1942.
Shoda, L.K.M., Worthy, G.A.J., Wade, T.L., 1993. Lipid stratification and
contaminant distribution in the blubber of bottlenose dolphins. Abstracts of
the Tenth Biennial Conference on the Biology of Marine Mammals.
Galveston, TX, 11– 15 November 1993, p. 99.
Silva, M.A., 1999. Diet of common dolphins, Delphinus delphis, off the
Portuguese continental coast. J. Mar. Biol. Assoc. UK 79, 531–540.
Smith, R.J., Hobson, K.A., Koopman, H.N., Lavigne, D.M., 1996. Distinguish-
ing between populations of fresh- and salt-water harbour seals (Phoca
vitulina) using stable-isotope ratios and fatty acid profiles. Can. J. Fish
Aquat. Sci. 53, 272–279.
Smith, S.J., Iverson, S.J., Bowen, W.D., 1997. Fatty acid signatures and
classification trees: new tools for investigating the foraging ecology of seals.
Can. J. Fish Aquat. Sci. 54, 1377–1386.
Steele, R.G.D., Torrie, J.H., 1980. Principles and Procedures of Statistics: A
Biometrical Approach, 2nd ed. McGraw-Hill, Inc., New York.
Young, D.D., Cockcroft, V.G., 1994. Diet of common dolphins (Delphinus
delphis) off the southeast coast of Southern Africa: opportunism or
specialization? J. Zool. London 234, 41–53.
Walton, M.J., Henderson, R.J., Pomeroy, P.P., 2000. Use of blubber fatty
acid profiles to distinguish dietary differences between grey seals
Halichoerus grypus from two UK breeding colonies. Mar. Ecol. Prog.
Ser. 193, 201–208.
West, G.C., Burns, J.J., Modafferi, M., 1979a. Fatty acid composition of
blubber from the four species of Bering Sea phocid seals. Can. J. Zool.
57, 189–195.
West, G.C., Burns, J.J., Modafferi, M., 1979b. Fatty acid composition of Pacific
walrus skin and blubber fats. Can. J. Zool. 57, 1249–1255.
Worthy, G.A.J., Edwards, E.F., 1990. Morphometric and biochemical factors
affecting heat-loss in a small temperate cetacean (Phocoena phocoena)
and a small tropical cetacean (Stenella attenuata). Physiol. Zool. 63,
432–442.
Worthy, G.A.J., Samuel, A.M., Worthy, T.A.M., 2003. Composition of killer
whale (Orcinus orca) skin and blubber: implications for fatty acid
signature analysis. Fifteenth Biennial Conference on the Biology of
Marine Mammals. Greensboro, North Carolina, December 14–20, 2003.