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THERMAL AND PHASE-CHANGE PROPERTIES OF THE BLUBBER OF SHORT-
FINNED PILOT WHALES (Globicephala macrorhynchus) AND PYGMY SPERM
WHALES (Kogia breviceps)
Laura E. Bagge
A Thesis Submitted to the
University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of
Master of Science
Department of Biology and Marine Biology
University of North Carolina Wilmington
2011
Approved by
Advisory Committee
Antje Pokorny Almeida Richard M. Dillaman
Heather N. Koopman Sentiel A. Rommel
D. Ann Pabst
Chair
Accepted by
Dean, Graduate School
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The thesis has been prepared in the style and format consistent with the journal:
The Journal of Experimental Biology
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TABLE OF CONTENTS
ABSTRACT CHAPTER 1 ..................................................................................................v
ABSTRACT CHAPTER 2 ................................................................................................ vi
ACKNOWLEDGEMENTS ............................................................................................. viii
DEDICATION ................................................................................................................... ix
LIST OF TABLES ...............................................................................................................x
LIST OF FIGURES ........................................................................................................... xi
CHAPTER 1
INTRODUCTION .........................................................................................................1
MATERIALS AND METHODS .................................................................................10
Specimens ..............................................................................................................10
Samples ..................................................................................................................10
Lipid Extraction, Classification, and Analysis ......................................................13
Integument Thermal Experiments .........................................................................14
Statistical Analyses ................................................................................................21
RESULTS ....................................................................................................................24
Lipid Content, Thermal Conductivity and Conductance
of Entire Integument ..............................................................................................24
Lipid Content and Thermal Conductivity Across Blubber’s Depth ......................26
Comparison of Standard Material and Heat Flux Disc Methods ...........................29
DISCUSSION ...............................................................................................................31
Blubber’s Quantity and Quality ............................................................................31
Depth Specific Thermal Conductivities Measured
Using Standard Material Method ...........................................................................35
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Blubber As a Heat Storing Material.......................................................................37
Conclusions ............................................................................................................38
REFERENCES ............................................................................................................40
CHAPTER 2
INTRODUCTION .......................................................................................................45
MATERIALS AND METHODS .................................................................................50
Specimens ..............................................................................................................50
Samples ..................................................................................................................50
Lipid Extraction, Classification, Analysis .............................................................51
Thermal Analysis by DSC .....................................................................................53
Statistical Analyses ................................................................................................55
RESULTS ....................................................................................................................57
Lipid and Fatty Acid Composition ........................................................................57
Differential Scanning Calorimetry .........................................................................62
DISCUSSION ...............................................................................................................67
Fatty Acid Composition .........................................................................................67
Differential Scanning Calorimetry .........................................................................69
Functional Consequences of a Phase-changing Blubber .......................................72
Conclusions ............................................................................................................76
REFERENCES ............................................................................................................77
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v
ABSTRACT CHAPTER 1
Cetacean blubber’s insulative ability depends upon both its quantity and quality.
This study specifically sought to investigate blubber’s quality by exploring the influence
of lipid storage class – triacylglycerols (TAG) in short-finned pilot whales (Globicephala
macrorhynchus; n=7) and wax esters (WE) in pygmy sperm whales (Kogia breviceps;
n=7) – on blubber’s thermal properties. Although the blubber of both species had similar
total lipid contents, the thermal conductivities of the entire integument (0.1935 ± 0.0076
Wm-1°C
-1) and of the blubber (0.2022 ± 0.0086 Wm
-1°C
-1) of G. macrorhynchus were
significantly higher than those of K. breviceps (0.1445 ± 0.0070 and 0.1495 ± 0.0074
Wm-1°C
-1 respectively; P=0.0006). These results suggest that lipid class significantly
influenced blubber’s ability to resist heat flow. In addition, because blubber’s lipid
content is known to be stratified across its depth, this study also measured blubber’s
depth-specific thermal conductivities. In K. breviceps blubber, the depth-specific
conductivity values tended to vary inversely with lipid content. In contrast, G.
macrorhynchus blubber displayed unexpected depth-specific relationships between lipid
content and conductivity, which suggested that temperature-dependent effects, such as
melting, may be occurring. Differences in heat flux measurements across the depth of all
blubber samples utilized in this study provided evidence that both species of cetaceans
were capable of storing heat in their blubber, an observation first noted in bottlenose
dolphin (Tursiops truncatus) blubber by Dunkin et al. (2005). These results suggest that
blubber’s function as an insulator is complex and may rely upon its stratified composition
and dynamic heat storage capabilities.
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ABSTRACT CHAPTER 2
This study tested the hypothesis that cetacean blubber functions as a phase change
material by using differential scanning calorimetry (DSC) to elucidate the melting
profiles of full-depth and layer-specific blubber lipids. Blubber from two cetacean
species, the short-finned pilot whale (Globicephala macrorhynchus) and the pygmy
sperm whale (Kogia breviceps), whose blubber is composed of two different lipid classes,
was investigated. G. macrorhynchus possessed blubber composed of triacylglycerols
(TAG; 99.5 ± 0.07%), and K. breviceps possessed blubber composed predominately of
wax esters (WE; 82.1 ± 3.80%). In addition, layer-specific fatty acid (FA) analyses were
performed to gain insight into whether these features of blubber’s composition correlated
with phase change behavior in blubber. DSC melting profiles of the lipids from G.
macrorhynchus and K. breviceps demonstrated that blubber lipids, regardless of whether
they were composed of TAG or WE, underwent a broad, endothermic phase transition
within a physiologically relevant temperature range (5 – 37°C). The WE of K. breviceps
displayed a melting profile that was shifted slightly to the left relative to the TAG of G.
macrorhynchus. This result suggested that lipid class did play a role in the overall
melting behavior of blubber. There were also subtle depth-specific differences that were
observed across blubber layers in G. macrorhynchus; the deep blubber lipids, with a
higher concentration of long-chain PUFA, had better-defined secondary melting peaks
than the melting profiles of the middle and superficial layers. Although there were no
significant differences in the measured depth-specific FA patterns in K. breviceps, DSC
revealed that there were differences in the melting profiles of the superficial and deep
layers. DSC, therefore, provided a direct assessment of the melting behavior for the
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complex lipid mixtures found in G. macrorhynchus and K. breviceps blubber, and in
conjunction with FA analysis, resulted in new insights into the phase change behavior of
these lipids. These results demonstrate that under a physiologically relevant temperature
range, blubber is not only acting as a typical insulator, by simply resisting heat flow, but
is also acting as a dynamic, temporal, thermal buffer by absorbing or releasing heat.
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ACKNOWLEDGEMENTS
I would like to offer my sincerest thanks to my advisor Dr. Ann Pabst for her
endless support and excellent mentorship. The long hours that she has spent guiding me
in this project have helped me become a better student and a better scientist. I would also
like to thank Bill McLellan for all of his support, for always being available to help me
think of ways to improve my experiments, and for making blubber fun.
I would like to acknowledge my committee members for all their guidance. Dr.
Heather Koopman, thank you for welcoming me into your lab and for teaching me
everything about lipids. Dr. Antje Pokorny Almeida, thank you for helping me use the
DSC and for teaching me how to be a chemist. Dr. Butch Rommel, thank you for the
lively discussions about my project and for teaching me how to think on a higher plane.
Dr. Richard Dillaman, thank you for sharing your knowledge about histology and science
with me.
I am grateful to past and present members of the VABLAB - Jamie Dolan, Caitlin
Kielhorn, Brandy Velten, Erin Cummings, Ryan McAlarney, and Robin Dunkin. My
sincerest thanks go to my family, friends, and loved ones who helped me throughout this
project. Thank you for your support, Margaret Sironko, Phil Sironko, Richard Sironko,
Leah Fleenor, and Michael and Sharlene Friel.
I am eternally grateful to Jack Smith for the hours he spent setting up the thermal
experiments with me, for never tiring of listening to me talk about this project, for his
excellent technological support and blubber drawings, and for his love and friendship.
Finally, I would like to thank the UNCW Department of Biology and Marine
Biology for providing this opportunity for a wonderful graduate research experience.
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DEDICATION
I would like to dedicate this work to my mother, Anne Sironko Bagge. From
introducing me to the ocean as a little girl, to scuba-diving with me, to helping me move
to Wilmington, my mother always supported me in my goal of becoming a marine
biologist, and she worked tirelessly to ensure that I had every opportunity to follow my
dreams. I can’t express enough my gratitude for her love and support.
This is for you, Mom.
Return in peace to the ocean my love,
I too am part of that ocean, my love, we are not so much separated,
Behold the great rondure, the cohesion of all – how perfect!
But as for me, for you, the irresistible sea is to separate us,
As for an hour, carrying us diverse, yet cannot carry us diverse forever;
Be not impatient – a little space – know you I salute the air, the ocean, and the land,
Every day at sundown for your dear sake, my love.
~Walt Whitman
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LIST OF TABLES
Table Page
1. Thermal conductivity (k) values for blubber from various marine mammals
and for other materials .............................................................................................3
2. Specimens used in this study for lipid composition and thermal analyses ............11
3. Morphological, compositional, and thermal data for blubber
from G. macrorhynchus and K. breviceps ............................................................25
4. Specimens used in this study with a subset selected for
DSC analyses ........................................................................................................54
5. Blubber’s lipid characterization for G. macrorhynchus and
K. breviceps by depth .............................................................................................58
6. Most abundant FA in G. macrorhynchus and K. breviceps
blubber by depth ....................................................................................................59
7. Most abundant fatty alcohols in K. breviceps
blubber by depth ....................................................................................................60
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LIST OF FIGURES
Figure Page
1. The square represents the blubber sample sites .....................................................12
2. Thermal chamber showing the placement of
thermocouples and heat flux discs .........................................................................16
3. Experimental set-up demonstrating placement of thermocouples
and heat flux discs for G. macrorhynchus blubber samples .................................17
4. Experimental set-up demonstrating placement of thermocouples
and heat flux discs for K. breviceps blubber samples ...........................................18
5. Example outputs of heat flux discs and thermocouples
in G. macrorhynchus blubber samples..................................................................22
6. Example outputs of heat flux discs and thermocouples
in K. breviceps blubber samples ...........................................................................23
7. The percentage of lipid and thermal conductivities in the superficial,
middle, and deep blubber layers of G. macrorhynchus. .......................................27
8. The percentage of lipid and thermal conductivities in the
superficial and deep blubber layers of K. breviceps ..............................................28
9. Comparison of three methods used to calculate the integument
conductivity values for G. macrorhynchus and K. breviceps ................................30
10. Thermal conductivities of the integument of
G. macrorhynchus and K. breviceps shown in
relation to percentage of lipid in the blubber ........................................................33
11. Differential scanning calorimetry outputs for soybean oil ....................................56
12. Differential scanning calorimetry outputs for full-depth lipids
of G. macrorhynchus and K. breviceps ..................................................................64
13. Differential scanning calorimetry outputs for superficial,
middle, and deep blubber lipids of G. macrorhynchus ..........................................65
14. Differential scanning calorimetry outputs for
superficial and deep blubber lipids of K. breviceps ..............................................66
Page 12
CHAPTER 1: LIPID CLASS AND DEPTH-SPECIFIC THERMAL PROPERTIES IN
THE BLUBBER OF TWO SPECIES OF ODONTOCETE CETACEANS, THE SHORT-
FINNED PILOT WHALE (GLOBICEPHALA MACRORHYNCHUS) AND THE
PYGMY SPERM WHALE (KOGIA BREVICEPS)
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INTRODUCTION
Cetacean blubber, the specialized hypodermal layer of their integument, is a
complex biocomposite, composed of adipocytes and structural connective tissues fibers,
which functions in buoyancy control, serves as a site of energy storage, and streamlines
and insulates the body (e.g. Parry, 1949; Doidge, 1990; Koopman et al., 1996; Pabst et
al., 1999; Hamilton et al., 2004; Struntz et al., 2004; Dunkin et al., 2005; Koopman,
2007; Dunkin et al., 2010). Blubber’s insulative function is important to these marine
endotherms because water can conduct heat away from the body at a rate approximately
25 times faster than air of the same temperature (Parry, 1949; Schmidt-Nielsen, 1997).
Blubber’s insulative ability depends upon both its quality and its quantity (e.g.
Parry, 1949; Worthy and Edwards, 1990; Koopman et al., 1996; Pabst et al., 1999;
Hamilton et al., 2004; Struntz et al., 2004; Dunkin et al., 2005; Montie et al., 2008).
Thermal conductivity (Wm-1
ºC-1
) is a constant material property that is a measure of how
well heat flows through a given material, and is, thus, a measure of thermal quality
(reviewed in Schmidt-Nielsen, 1997). Thermal conductivity can be calculated through
use of the Fourier equation:
Equation 1 k = dQ / A(T2-T1)
where d is the blubber thickness (m), Q is the rate of heat transfer (W), A is the surface
area of the material (m2), and T2 – T1 (°C) is the temperature differential across the depth
of the blubber sample (Parry, 1949; Kvadsheim et al., 1994; Schmidt-Nielsen, 1997).
The quantity, or thickness of a material, as well as its quality, dictates its thermal
conductance (W/m-2
ºC-1
), and, thus, provides an absolute value of the heat transfer across
a given material. The equation for thermal conductance is:
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2
Equation 2 C = H / (T2-T1)
where the H denotes heat flux (Wm-2
), a rate of energy transfer per unit area, and T2-T1
(°C) is the temperature differential across the depth of the blubber sample. The inverse of
conductance is insulation, R (m2°CW
-1), which measures the absolute resistance to heat
flow (Parry, 1949; Kvadsheim et al., 1994; Schmidt-Nielsen, 1997).
In past studies on the thermal properties of cetacean blubber, the amount (% wet
weight) of lipid present has been the compositional metric of quality, and has been found
to be inversely related to thermal conductivity (e.g. Worthy and Edwards, 1990;
Kvadsheim et al., 1996; Dunkin et al., 2005). For instance, the blubber of harbor
porpoises (Phocoena phocoena; Family Phocoenidae) has high lipid content
(81.6 ± 3.6%) and low thermal conductivity (0.10 ± 0.01 W/mºC), while the blubber of
spotted dolphins (Stenella attenuata; Family Delphinidae) has low lipid content (54.9 ±
2.8%) and high thermal conductivity (0.20 ± 0.02 W/mºC) (Worthy and Edwards, 1990).
This significant inverse relationship between lipid content and thermal conductivity has
also been found in the blubber of bottlenose dolphins (Tursiops truncatus; Family
Delphinidae) and minke whales (Balaenoptera acutorostrata; Family Balaenopteridae)
(Kvadsheim et al., 1996; Dunkin et al., 2005).
Most cetaceans, and all those for which thermal properties of blubber have been
investigated to date (Table 1), store their blubber lipids as triacylglycerols (TAG), three
fatty acids attached to a glycerol backbone (reviewed in Koopman, 2007). In contrast,
kogiids, physeterids, and ziphiids (sperm whales and beaked whales) store their blubber
lipids primarily as wax esters (WE), a long chain fatty acid attached to a long-chain fatty
alcohol (Lockyer, 1991; Koopman 2007).
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Table 1. Thermal conductivity k values for blubber from various marine mammals and for other materials.
Material k (W m-1
°C-1) Source Method
Metals
Aluminum [Al] 237 www.efunda.com Unknown
Copper [Cu] 410 www.efunda.com Unknown
Lead 35 Schmidt-Nielsen, 1997 Unknown
Iron 80 Schmidt-Nielsen, 1997 Unknown
Water (Freshwater at 25°C) 0.61 Boily, 1995 Unknown
Water (Salt water at 25°C) 0.6 Boily, 1995 Unknown
Pinniped Blubber
Mirounga leonina 0.07 Doidge, 1990 (Bryden, 1964) Unknown
Phoca vitulina 0.18 Worthy, 1985 Heat flux disc
Phoca groenlandica 0.18 Worthy, 1985 Heat flux disc
Phoca groenlandica 0.19 Kvadsheim et al., 1994 Standard material
Phoca hispida 0.2 Scholander et al., 1950 Hot plate
Halichoerus grypus 0.18 Worthy, 1985 Heat flux disc
Cetacean Blubber
Balaenoptera acutorostrata 0.20 - 0.28 Kvadsheim et al., 1996 Standard material
Balaenoptera acutorostrata 0.18 Folkow and Blix, 1992 Hot plate
Balaenoptera physalus 0.21 Parry, 1949 Hot plate
Delphinapterus leucas (blubber) 0.102 Doidge, 1990 Heat flux plate
Delphinapterus leucas (epidermis) 0.249 Doidge, 1990 Heat flux plate
Phocoena phocoena 0.06 Yasui and Gaskin, 1986 Heat flux disc
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Phocoena phocoena 0.1 Worthy and Edwards, 1990 Heat flux disc
Stenella attenuata 0.2 Worthy and Edwards, 1990 Heat flux disc
Tursiops truncatus: Dunkin et al., 2005 Standard material
Fetus 0.12
Neonate 0.13
Juvenile 0.12
Sub-adult 0.11
Adult 0.18
Pregnant female 0.12
Emaciated adult 0.24
Other lipids
Human Fat 0.21 Doidge, 1990 (Hensel, 1973) Unknown
Olive Oil 0.17 oliveoilsource.com Unknown
Fatty Acids
Stearic acid (C18:0) 0.16 Doidge, 1990 (Weast, 1989) Unknown
Palmitic Acid (C16:0) 0.17 Doidge, 1990 (Weast, 1989) Unknown
Oleic Acid (C18:1) 0.23 Doidge, 1990 (Weast, 1989) Unknown
Wood
Eastern White Pine Wood 0.09 Simpson and TenWolde, 1999 Unknown
White Oak Wood 0.16 Simpson and TenWolde, 1999 Unknown
White pine wood 0.104 Liley, 1996 Unknown
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The overall lipid content of the blubber in these species is similar to that of species which
store their lipids as TAG (Koopman, 2007). The thermal consequences of having a
“waxy” blubber layer, though, have not yet been investigated.
In addition to variations in lipid content and lipid class found in the blubber of
different species, histological and biochemical data have revealed that cetacean blubber’s
composition is also stratified across its depth (e.g. Parry, 1949; Lockyer, 1991 and 1993;
Koopman et al., 1996; Evans et al., 2003; Struntz et al., 2004; Koopman 2007; Montie et
al., 2008). Struntz et al. (2004) and Montie et al. (2008) described the blubber of the
bottlenose dolphin as having three layers – superficial, middle, and deep. The superficial
layer contained the highest density of structural fibers and fewer adipocytes; the middle
layer contained the most large adipocytes and the least density of structural fibers; and
the deep layer contained a high structural fiber density and smaller adipocytes (Struntz et
al., 2004; Montie et al., 2008). Montie et al. (2008) also observed larger adipocytes in
the middle and deep blubber layers of bottlenose dolphins from a colder geographic
location, which suggests that differences in adipocytes across blubber’s depth may affect
insulation.
There are also differences in lipid and fatty acid composition across blubber’s
depth. Koopman et al. (1996) described the fatty acid profiles of the innermost (0.5g
blubber sample trimmed from the deep hypodermis) and the outermost (0.5g blubber
sample trimmed from the superficial blubber) blubber layers in male harbor porpoises.
The inner blubber layer had higher concentrations of dietary fatty acids, while the outer
blubber layer contained more endogenous fatty acids. In addition, during periods of
extreme nutritional stress, harbor porpoises withdrew lipids from their inner blubber
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layer, but adipocyte size and shape were maintained in the outer blubber layer (Koopman
et al., 2002). These combined results suggest that the inner layer is more metabolically
active, while the outer blubber is metabolically inert and serves a structural role (e.g.
Koopman et al., 1996 and 2002).
The stratified anatomy and biochemistry of the blubber of the bottlenose dolphin
and harbor porpoise, which are composed of TAG, have been well-described (Koopman
et al., 1996; Struntz et al., 2004; Montie et al., 2008). Few studies, though, have
investigated other cetacean species, especially those species that deposit blubber lipids as
WE. Koopman (2007) examined biochemical stratification in 30 species of cetaceans,
including those in the Family Kogiidae, which possess blubber composed predominately
of WE. In this study, the pygmy sperm whale (Kogia breviceps) possessed a significantly
higher percentage of lipid in the inner blubber layer (67.3±3.5%) than the outer layer
(31.3±8.2%). The dwarf sperm whale (Kogia. sima) had inner blubber lipid content
(65.5±3.9%) that was similar to K. breviceps, but showed a surprisingly large outer
blubber lipid content (79.3±4.5%) (Koopman, 2007). Neither of these two species
displayed a large degree of stratification in fatty acid composition across the blubber
depth (Koopman, 2007).
Because blubber’s adipocytes, lipid content, and fatty acid composition can vary
across its depth (e.g. Koopman et al., 1996 and 2002; Struntz et al., 2004; Koopman
2007; Montie et al., 2008), and blubber’s insulative properties are thought to rely upon
both its quantity and quality (reviewed in Pabst et al., 1999), these layer-specific
differences in composition suggest that blubber’s thermal properties should similarly vary
across its depth. No study has yet investigated this feature.
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Blubber’s thermal conductivity and conductance have been measured using a
variety of different experimental approaches, the two most recent of which are the heat
flux disc and standard material methods (Table 1; reviewed in Dunkin et al., 2005). The
heat flux disc method directly measures heat flow through one surface of a blubber
sample. In this method, a heat flux disc is placed in series with a constant heat source
and a blubber sample. Once the amount of heat flowing through the blubber has reached
a steady state, Equation 1 can be used to calculate its thermal conductivity. Kvadsheim et
al. (1994) introduced a new method for measuring conductivity that relies on an indirect
measure of heat flux, and requires placing a standard material of known conductivity in
series with the heat source and the blubber. At steady state, the rate of heat flowing
through the standard material and the blubber must be equal. Therefore, conductivity can
be calculated using the Fourier equation (Eq. 1) by setting the heat flow through the
blubber (Qblubber) equal to the heat flow through the standard material (Qstandard).
Each method has advantages and disadvantages. The heat flux disc method
provides a direct measure of heat flow but can introduce error because the placement of
the disc on the surface of the blubber causes a local increase in insulation, which can
lower heat flux values (Ducharme et al., 1990). This error can be minimized when the
insulation of the heat flux disc is lower than that of the material being tested (Frim and
Ducharme, 1993; Willis, 2003). In addition, Dunkin et al. (2005) found that the position
of the heat flux disc (i.e. whether it was placed superficial to the epidermis or deep to the
blubber) can have a profound effect on the calculated conductivity value. Conductivity
values for bottlenose dolphin blubber calculated using the deep heat flux disc were
approximately 57% higher than those calculated using the superficial heat flux disc.
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Since previous studies have used a single heat flux disc placed between the heat source
and the blubber (e.g. Yasui and Gaskin, 1986; Worthy and Edwards, 1990), the calculated
conductivity values that have been reported are likely higher than those values that would
have been reported if the disc had been placed on the epidermis (Dunkin et al., 2005).
The standard material method allows for the avoidance of the errors associated
with the use of a heat flux disc, and is accurate to ±4% (Kvadsheim et al., 1994).
However, since the standard material method is a relatively new technique, few studies
have measured blubber’s thermal conductivity with this method. Dunkin et al. (2005) did
use both the standard material and heat flux disc methods simultaneously in their study of
bottlenose dolphin blubber, which allowed for a comparison of these techniques. These
authors found that blubber conductivity values calculated using the standard material
method were similar to those calculated using the output of the superficial heat flux disc
(mean conductivity values were within 2% of each other). The deep heat flux disc
yielded higher conductivity values than both these methods.
Therefore, my study used both the standard material method and two heat flux
discs to permit comparison among techniques in measuring blubber’s thermal properties.
Because cetacean blubber is a complex tissue that can be morphologically and
biochemically stratified across its depth (Koopman et al., 1996; Struntz et al., 2004;
Dunkin et al., 2005; Koopman, 2007; Montie et al., 2008), blubber’s depth-specific
thermal conductivities were investigated for the first time using the standard material
method. Past studies have focused on percent lipid content as a metric for insulative
quality, but no study has yet attempted to tease apart the specific effects that lipid class
has on blubber’s thermal properties.
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My study directly compared blubber’s depth-specific thermal properties in two
species of cetaceans that are known to possess blubber composed of different lipid
classes. The pygmy sperm whale (Kogia breviceps) was used because this species
possesses blubber containing high percentages of wax esters, or WE (Koopman, 2007).
In contrast, the short-finned pilot whale (Globicephala macrorhynchus) possesses
blubber composed entirely of triacylglycerols, or TAG (Koopman, 2007). My study
sought to further elucidate blubber’s thermoregulatory function by (1) characterizing
blubber’s lipid composition by calculating the amount of lipid present (% wet weight),
and determining the predominant lipid classes present in full-depth and layer-specific
blubber samples from K. breviceps and G. macrorhynchus, and by (2) performing
standardized experiments, measuring overall conductance and both full-depth and layer-
specific thermal conductivity values, on intact blubber samples from K. breviceps and G.
macrorhynchus.
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MATERIALS AND METHODS
Specimens
Integument samples [epidermis, dermis, hypodermis (blubber), and some
underlying cutaneus trunci muscle] were obtained from seven adult female short-finned
pilot whales (Globicephala macrorhynchus) that stranded during a multi-species mass
stranding event on Bodie Island, NC on January 15, 2005 (Hohn et al., 2006). In
addition, integument samples were obtained from seven adult pygmy sperm whales
(Kogia breviceps) that stranded as part of a mass stranding event on September 2, 2006,
or as individuals during the years 2005 through 2010 (Table 2).
All specimens used in this study were in fresh to moderate carcass condition
(Smithsonian Institution Code 1 through 3; Geraci and Lounsbury, 2005) and normal to
robust body condition (Cox et al., 1998; McLellan et al. 2002). For each animal, a
standard set of external morphometrics (adapted from Norris 1961) was collected and a
necropsy was performed. Full-depth samples of the integument (with dimensions ranging
from 15cm X 15cm to 24cm X 24cm) were taken from a dorsal, mid-thoracic site on each
whale (Fig. 1). The blubber samples were vacuum-sealed (Koch 1700, Kansas City, MO,
USA) and stored at –20°C until analysis.
Samples
The integument thickness, including the hypodermis, dermis, and epidermis, was
measured at each side of the sample using precision digital calipers (Absolute Digimatic
calipers, Mitutoyo, Tylertown, MS, USA), and the mean thickness was used in all
calculations. The blubber of G. macrorhynchus did not appear grossly stratified, so a
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Table 2. Specimens used in this study for lipid composition and thermal analyses.
Species ID # Sex Total body length (cm)
Globicephala macrorhynchus RT 102 Female 386
RT 103 Female 349
RT 105 Female 375
RT 107 Female 357
RT 13 Female 297
RT 19 Female 358
RT 63 Female 359
Kogia breviceps BRF 092 Female 267
MLC 003 Female 262
WAM 611 Male 301
KMS 427 Female 267
KMS 429 Male 283
MDB 056 Male 263.5
VGT 221 Male 250
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(A)
Fig. 1. The square represents the blubber sample sites from (A) short-
finned pilot whales (Globicephala macrorhynchus) and (B) pygmy sperm
whales (Kogia breviceps) used in thermal experiments and lipid
composition analyses.
(B)
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standardized method was used to investigate the depth-specific lipid composition and
thermal properties. This method may not fully represent the morphological or
biochemical stratification of the blubber layer. Because previous studies had identified
delphinid blubber to be stratified into superficial, middle, and deep layers, the thickness
of G. macrorhynchus blubber (excluding the epidermis) was measured, and that thickness
was divided into equal thirds. In contrast, K. breviceps blubber was grossly stratified into
two distinct layers. The superficial layer (which represented approximately 38% of the
blubber layer’s thickness) appeared more densely fibrous, tough, rubber-like, and white
in color than the thicker, soft, buttery deep layer. Therefore, this blubber was divided at
the interface between the fibrous superficial layer and the deep layer.
Lipid extraction, classification, and analysis
Lipids were extracted and analyzed from full-depth blubber samples and from
layer-specific blubber samples (deep, middle, and superficial layers for G.
macrorhynchus and deep and superficial layers for K. breviceps). Approximately 0.5g
full-depth sections of blubber, in addition to 0.5g sections of each layer, were excised,
weighed, and placed in 9ml of 2:1 chloroform : methanol with 0.01% butylated
hydroxytoluene (BHT). A modified Folch procedure was then used for lipid extraction
(Folch et al., 1957; Iverson, 1988; Koopman et al., 1996), and a wet weight % of lipid in
the blubber was calculated. The lipids were then re-suspended in hexane at 100mg
lipid/ml of hexane, flushed with nitrogen to avoid oxidation, and stored at -20ºC until
further analysis.
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14
Lipid classes were separated and quantified using thin layer chromatography with
flame ionization detection (TLC-FID) (Iatroscan®
Mark VI Mitsubishi Kagaku Iatron,
Inc, Tokyo, Japan). Samples were analyzed in duplicate by spotting 1 µl on chromarods
followed by development in hexane : ethyl acetate : formic acid (94:6:1) and
quantification in the Iatroscan. Peaks were identified and integrated using PeakSimple
329 Iatroscan software (SRI Instruments, Torrance, CA), based on known lipid class
standards (Nu Chek Prep, Elysian, MN). Levels of lipid classes were calculated by
applying standard curves generated from known concentrations.
Integument thermal experiments
Approximately 24 hours prior to measuring the integument’s thermal properties,
the large vacuum-sealed integument sample was taken from the freezer and an 8cm X
8cm frozen square was excised using a Stryker® saw. This sub-sample was thawed on
ice and any remaining cutaneus trunci muscle was removed prior to the thermal
experiment. The methods by which the blubber samples were collected and stored may
influence the thermal properties; however, the methods also permit a standardized sample
treatment and the ability to compare results to other published studies where the blubber
was frozen prior to the time of experimentation. For instance, Dunkin et al. (2005)
collected and stored blubber samples from bottlenose dolphin (Tursiops truncatus) in this
same manner.
The thermal properties of blubber were measured using an experimental set-up
similar to that used by Dunkin et al. (2005) (see Figs. 2-4). This set-up utilized both the
standard material method (Kvadsheim et al., 1994) and the heat flux disc method (e.g.
Page 27
15
Worthy and Edwards, 1990) to estimate blubber’s thermal properties (Figs. 2-4). In this
study, a dual compartment heat flux chamber (69-quart Coleman Cooler, Albany, NY,
USA) was used; the lower compartment consisted of an insulated heat source of a
constant temperature (37°C), and the upper compartment consisted of a cooled (15-19°C)
air space (Fig. 2). This set-up created a temperature differential that simulated the
conditions of the temperature difference between the warm inner body of a whale and the
colder environment.
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16
(A)
(B)
Fig. 2. (A) Thermal chamber showing the placement of thermocouples (ovals) and heat
flux discs (rectangles) for G. macrorhynchus and (B) for K. breviceps. Because K.
breviceps blubber was grossly stratified into two layers, the superficial layer is colored
grey to represent the difference between the two layers. The figure is not drawn to scale
and certain aspects have been exaggerated for clarity.
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17
Fig. 3. Experimental set-up demonstrating placement of the 12 thermocouples and 2 heat
flux discs for G. macrorhynchus blubber samples. Figure drawn to scale, although
thermocouple tips have been exaggerated for clarity.
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18
Fig. 4. Experimental set-up demonstrating placement of the 11 thermocouples and 2 heat
flux discs for K. breviceps blubber samples. Because K. breviceps blubber was grossly
stratified into two layers, the superficial layer is colored white to represent the color
distinction observed between the two layers. Figure drawn to scale, although
thermocouple tips have been exaggerated for clarity.
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19
The constant temperature heat source was an aluminum box in the lower
compartment through which heated water (37°C) was continuously circulated from a
water bath (RE-120 Lauda Ecoline, Brinkmann Instruments, Inc., Toronto, Ontario,
Canada), and the upper compartment was filled with ice packs. The upper part of the
aluminum box was an open platform upon which the standard material and blubber were
placed. The insulating foam in the lower compartment, placed around the standard
material and the blubber, ensured unidirectional heat flow through the blubber sample
(Fig. 2). Air temperature was measured using a thermocouple suspended approximately
10cm above the blubber sample. For all of the experiments, the air temperature remained
between 15°C and 19°C. All temperatures were measured using copper-constantan
(Type T) thermocouples (Omega Engineering, Inc, Stamford, CT, USA).
The thermal properties of the entire integument and the depth-specific thermal
properties of blubber were investigated in G. macrorhynchus and K. breviceps. The
mean integument thickness was used to calculate the conductivity of the whole
integument sample. Because G. macrorhynchus blubber was considered to be stratified
into three layers, one side of the blubber sample was arbitrarily chosen, and deep, middle,
and superficial thermocouples were inserted into the blubber at those three equidistant
positions (Figs. 2A and 3). The tip of each thermocouple was inserted approximately
5mm into the lateral face of the blubber sample (Figs. 2A and 3). The superficial
thermocouple was placed approximately 1mm below the pigmented epidermis (Figs. 2A
and 3). The depth-specific thermal properties of K. breviceps blubber were investigated
in a similar manner (Figs. 2B and 4). One thermocouple was inserted at the interface
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20
between the fibrous superficial layer and the deep layer and another thermocouple was
inserted approximately 1mm below the pigmented epidermis (Figs 2B and 4).
The standard material was an elastomer (Plastisol vinyl, Carolina Biological
Supply, Burlington, NC, USA; k=0.109±0.01Wm-1
°C-1
) that was 8cm X 8cm X 0.67cm.
This elastomer was placed flush against the surface of the heated aluminum box. Two
thermocouples (probes 1 and 2; Fig. 2) monitored the temperature at the interface
between the aluminum and elastomer standard material. Three more thermocouples
(probes 3, 4, and 5; Fig. 2) monitored the temperature between the standard material and
the deep hypodermal surface of the sample. The sample, with the thermocouples already
inserted at the deep, middle, and superficial positions for G. macrorhynchus blubber (Fig.
2A) and at the deep and superficial positions for K. breviceps blubber (Fig. 2B), was then
positioned on the standard material. A final four thermocouples (probes 6-9; Fig. 2) were
positioned on the epidermis and held in place with thin pieces of medical tape (Johnson
and Johnson All Purpose Cloth Tape®).
Two heat flux discs (Thermonetics Corp., San Diego, CA, USA) were used to
directly measure heat flow (Figs. 2 - 4). The deep heat flux disc was placed at the
interface between the standard material and the deep hypodermis. The superficial heat
flux disc was positioned on the epidermis and also held in place with thin strips of the
same medical tape. The tape was carefully placed on the outer silicone edges of the disc
so that it did not contact the active thermal surface.
All thermocouples and heat flux discs were connected to a Fluke Hydra Data
Logger (Model 2625A, Fluke Inc., Everett, WA, USA). The outputs of the
thermocouples (°C) and the heat flux discs (mV) were recorded at a continuous interval.
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21
When the heat flux values at both the deep and superficial heat flux discs became stable
(a standard deviation of less than ±5Wm-2
around the mean for a period of at least 30
minutes) and the temperature values of all thermocouples became stable (a standard
deviation of less than ±0.1°C around the mean for a period of at least 30 minutes), the
experiment was concluded (Figs. 5 and 6). The data were then downloaded to a laptop
computer for analysis and heat flux readings were converted from mV to Wm-2
using the
calibration coefficients provided by the manufacturer. Conductivity and conductance
values were calculated according to Equations 1 and 2 as described in the introduction.
Statistical analyses
T-tests were used to determine if there were significant differences (P < 0.05)
between G. macrorhynchus and K. breviceps with regard to blubber thickness, epidermis
thickness, integument thickness, total and depth-specific lipid content, thermal
conductivities (for the entire integument; blubber only; and deep, middle, and superficial
layers), insulation, conductance, and heat flux disc outputs. In addition, t-tests were used
to compare the lipid content and thermal conductivities of the superficial and deep
blubber layers in K. breviceps. A one-way ANOVA with post-hoc Tukey HSD test was
performed to determine if there were significant differences in lipid content or thermal
conductivities across the superficial, middle, and deep blubber layers in G.
macrorhynchus. Thermal conductivities obtained using the three methods (standard
material, superficial heat flux disc, and deep heat flux disc) were also compared using a
one-way ANOVA with post-hoc Tukey HSD tests to identify significantly different
values.
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22
Fig. 5. (A) Example outputs from the deep and superficial heat flux discs using blubber
from RT 63, an adult female short-finned pilot whale. (B) Example of temperature
values recorded from the same animal. Al-SM (1-2) refers to the average temperature of
the two probes placed at the interface between the heat source and the standard material;
SM-B (3-5) is the average of the three probes placed between the standard material and
the deep blubber surface; Epi (6-9) is the average of the four probes that were taped onto
the surface of the epidermis and were in contact with the air. The blubber depth was
divided into thirds, and the deep, middle, and superficial probes were placed at these
positions. For both traces, data from the last 30 minutes of the experiment, during which
time temperature and heat flux values were stable, were used in thermal calculations.
(A)
(B)
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23
Fig. 6. (A) Example outputs from the deep and superficial heat flux discs using blubber
from BRF 092, an adult female pygmy sperm whale. (B) Example of temperature values
recorded from the same animal. Al-SM (1-2) refers to the average temperature of the two
probes placed at the interface between the heat source and the standard material; SM-B
(3-5) is the average of the three probes placed between the standard material and the deep
blubber surface; Epi (6-9) is the average of the four probes that were taped onto the
surface of the epidermis and were in contact with the air. The blubber depth was divided
into the two grossly observable layers, and deep and superficial probes were placed at
these positions. For both traces, data from the last 30 minutes of the experiment, during
which time temperature and heat flux values were stable, were used in thermal
calculations.
(A)
(B)
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24
RESULTS
Lipid content, thermal conductivity and conductance of entire integument
Globicephala macrorhynchus and Kogia breviceps possessed blubber composed
of different lipid classes. G. macrorhynchus blubber was composed of 99.5 ± 0.07%
triacylgylcerols (TAG), and K. breviceps blubber was composed of 82.1 ± 3.80% wax
esters (WE). The total percentage of lipid present in the entire blubber layer tended to be
higher in G. macrorhynchus than in K. breviceps, but this difference was not significant
(P=0.07; Table 3). The blubber was thinner in G. macrorhynchus than in K. breviceps
(P=0.006; Table 3), but the epidermal thickness was similar across species (Table 3).
The thermal conductivity (k; Wm-1
ºC-1
) of the integument was compared across
species using values obtained from both the standard material and the heat flux disc
method; the heat flux disc measurements will be described in more detail below. Using
the standard material method, the thermal conductivities of the entire integument and of
the blubber of G. macrorhynchus were significantly higher (P<0.001 for both tissues)
than those of K. breviceps (Table 3). Although the epidermal conductivity was typically
higher in G. macrorhynchus than in K. breviceps, this value was not significantly
different between the species (P=0.08; Table 3).
Conductance and insulation values were calculated using the data obtained from
the superficial heat flux disc, and both differed significantly across species (Table 3).
The conductance of the entire integument of G. macrorhynchus was significantly higher
than that of K. breviceps (P=0.0009; Table 3). K. breviceps blubber was approximately
one and a half times more insulative than that of G. macrorhynchus (P=0.0019; Table 3).
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25
Table 3. Morphological, compositional, and thermal data for blubber from G. macrorhynchus and K. breviceps.
G. macrorhynchus (n=7) K. breviceps (n=7)
Blubber thickness (mm) 19.61 ± 0.96* 27.98 ± 1.35
Epidermis thickness (mm) 1.04 ± 0.02 0.98 ± 0.11
Total lipid content (% wet weight) 62.17 ± 2.02 56.64 ± 1.73
Deep lipid content (% wet weight) 67.60 ± 3.40 75.29 ± 2.61
Middle lipid content (% wet weight) 75.67 ± 2.20 ------
Superficial lipid content (% wet weight) 43.26 ± 2.85 26.22 ± 3.02
Conductance C (W m-2
°C-1
) using superficial heat flux disc 10.79 ± 0.71* 6.91 ± 0.44
Insulation R (m2 °C W
-1) using superficial heat flux disc 0.097 ± 0.007* 0.150 ± 0.010
Conductivity k using standard material method (W m-1
°C-1
)
k integument 0.1935 ± 0.0076* 0.1445 ± 0.0070
k blubber 0.2022 ± 0.0086* 0.1495 ± 0.0074
k epidermis 0.1942 ± 0.0408 0.1027 ± 0.0259
k deep blubber 0.2294 ± 0.0174 0.1381 ± 0.0051
k middle blubber 0.2131 ± 0.0083 ------
k superficial blubber 0.1642 ± 0.0135 0.1994 ± 0.0032
Conductivity k using heat flux disc method (W m-1
°C-1
)
k integument using deep heat flux disc 0.3541 ± 0.0133 0.3216 ± 0.018
k integument using superficial heat flux disc 0.2187 ± 0.0052* 0.1891 ± 0.010
Heat flux (Wm-2
)
Deep heat flux disc (Wm-2
) 156.66 ± 7.48 148. 93 ± 5.05
Superficial heat flux disc (Wm-2
) 96.73 ± 3.54 87.67 ± 3.26
Δ Heat flux discs (Wm-2
) 59.93 ± 5.85 61.26 ± 4.53
Values are means ± S.E.M. For all measurements across rows, significant differences are marked with an asterisk (P < 0.05).
Depth-specific lipid contents and thermal conductivities were not compared across species, as these were not homologous positions.
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26
Lipid content and thermal conductivity across blubber’s depth
The amount of TAG or WE present in each species did not differ significantly
across the blubber depth; however, the total percentage of lipid did. In G.
macrorhynchus, the deep and middle blubber layers had higher lipid contents than did the
superficial layer (P<0.0001; Table 3 and Fig. 7). In K. breviceps, the deep layer
contained more lipid than the superficial layer (P<0.0001; Table 3 and Fig. 8).
All depth-specific conductivity values were calculated using the standard material
method (Table 3). G. macrorhynchus blubber did not display the expected inverse
relationship between thermal conductivity and lipid content. The deep blubber layer,
with a higher lipid content, also had a significantly higher conductivity value than the
superficial layer (P=0.008; Fig. 7). The conductivity of the middle blubber layer was not
significantly different than either the deep or superficial layers. In contrast, the depth-
specific conductivity values in K. breviceps did display the expected inverse relationship
to lipid content. The superficial layer, with a lower lipid content, tended to have a higher
conductivity than the deep layer, although these values were not significantly different
(P=0.08; Fig 8).
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27
Fig. 7. The percentage of lipid and thermal conductivities (k) in the superficial,
middle, and deep blubber layers of G. macrorhynchus. Values represent mean ±
S.E.M.
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28
Fig. 8. The percentage of lipid and thermal conductivities (k) in the superficial
and deep blubber layers of K. breviceps. Values represent mean ± S.E.M.
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29
Comparison of standard material and heat flux disc methods
In all blubber samples, regardless of species, there was a substantial difference
between the heat flux values recorded by the deep and the superficial heat flux discs. The
deep disc recorded heat flux values that were higher than those recorded by the
superficial disc in G. macrorhynchus and in K. breviceps (both P < 0.0001; Table 3). The
mean difference in heat flux across the blubber of G. macrorhynchus was 58.93Wm-2
(range = 31.4 - 80Wm-2
), while that across K. breviceps blubber was 61.26Wm-2
(range =
46 - 81Wm-2
). These mean differences in heat flux across blubber’s depth did not differ
across species (P=0.86; Table 3).
Because the deep disc recorded higher heat flux values, calculations using the
deep heat flux disc data resulted in higher integument conductivity values than those
calculated using either the superficial disc or the standard material method in both species
(P < 0.0001; Table 3, Fig. 9). In contrast, for both species, the integument conductivity
values calculated using the superficial heat flux disc outputs and the standard material
method were statistically indistinguishable. Between species, the conductivity values for
the entire integument obtained using values from the deep heat flux disc were similar
(P=0.17; Table 3). However, there were significant difference in conductivity values
between species when either the superficial heat flux disc (P=0.03) or the standard
material method (P= 0.0006; Table 3; Fig. 9) was used.
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30
Fig. 9. Comparison of three methods (standard material method, superficial heat flux
disc, and deep heat flux disc) used to calculate the integument conductivity (k) values for
G. macrorhynchus and K. breviceps. Values represent mean ± S.E.M.
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31
DISCUSSION
This study investigated the thermal properties of the integument in two cetacean
species, Globicephala macrorhynchus and Kogia breviceps. While past studies have
focused on lipid content (% wet weight) as the compositional metric of blubber’s quality,
this study specifically sought to investigate the influence of lipid storage class -
triacylglycerols (TAG) in G. macrorhynchus and wax esters (WE) in K. breviceps - on
blubber’s thermal properties. In addition, because blubber is known to vary both
histologically and biochemically across its depth (e.g. Koopman et al., 1996; Struntz et
al., 2004; Koopman, 2007; Montie et al., 2008), this study measured blubber’s thermal
properties across its depth. The results of this study suggest that blubber composed
predominately of WE may provide enhanced insulation relative to that composed
predominately of TAG. Blubber’s thermal conductivity did vary across its depth, but the
relationship between lipid content and conductivity was complex.
Blubber’s quantity and quality
Blubber’s insulative ability depends upon both its quantity, or thickness, and its
quality, or thermal conductivity (e.g. Parry, 1949; Worthy and Edwards, 1990; Koopman
et al., 1996; Pabst et al., 1999; Hamilton et al., 2004; Struntz et al., 2004; Dunkin et al.,
2005; Montie et al., 2008). K. breviceps blubber was thicker and had a lower thermal
conductivity than G. macrorhynchus blubber (Table 3). Thus, K. breviceps blubber
provided significantly better insulation, approximately 1.5 times that of G.
macrorhynchus blubber.
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32
Although the thermal conductivities of the blubber of G. macrorhynchus and K.
breviceps differed significantly, lipid content was similar across species (Table 3).
However, another measure of quality, lipid storage class, did differ across species. G.
macrorhynchus possessed blubber composed almost entirely of TAG and K. breviceps
possessed blubber composed predominately of WE. G. macrorhynchus blubber typically
had higher conductivity values, even when its blubber had higher lipid content than that
of K. breviceps blubber (Fig. 10). These results suggest that lipid class may significantly
influence blubber’s ability to resist heat flow.
In blubber, TAG and WE storage lipids can contain any combination of saturated,
monounsaturated, or polyunsaturated fatty acids. However, TAG consist of three fatty
acids esterified to a glycerol backbone (reviewed in Pond, 1998), while WE consist of a
single fatty acid attached to a fatty alcohol (Lockyer, 1991; Koopman, 2007). Thus, one
half of any WE is a fatty alcohol, and the fatty alcohol portion of a WE is almost always
saturated or monounsaturated (Sargent et al., 1976; reviewed in Budge et al., 2006).
These differences in the chemical composition of the TAG and WE lipids may affect the
way in which these lipids are packed together within an adipocyte, and thus, may affect
the lipid’s thermal properties. The number of “kinks” in the carbon chain, due to the
addition of double bonds, appears to affect an individual fatty acid’s conductivity. For
example, stearic (18:0) and palmitic (16:0) acids are saturated fatty acids that have lower
thermal conductivity values (0.16 and 0.17 Wm-1
°C-1
respectively) than oleic acid (18:1n-
9), which is a monounsaturated FA with a thermal conductivity of 0.23 Wm-1
°C-1
(Weast,
1989). Because saturated FA have no double bonds, they may pack more tightly together
than monounsaturated or polyunsaturated FA, and, thus, may better resist heat flow.
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33
Fig. 10. Thermal conductivities of the integument of G. macrorhynchus and K. breviceps
shown in relation to percentage of lipid in the blubber of each of these individuals.
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34
All differences in composition, such as the individual fatty components that make
up each lipid molecule, should be investigated before the overall impact of lipid class on
thermal conductivity can be evaluated. Previous thermal experiments have, for example,
shown that other species of cetaceans with blubber composed of TAG have thermal
conductivity values that are equal to or lower than that of the K. breviceps blubber
composed of WE (Table 1). Worthy and Edwards (1990) found that the thermal
conductivity of harbor porpoise blubber (Phocoena phocoena), composed of TAG
(Koopman, 2007), was 0.10 ± 0.01Wm-1
ºC, which is lower than that of K. breviceps (see
Table 1 and 3). However, P. phocoena blubber also possessed a much higher lipid
content (81.6 ± 3.6%; Worthy and Edwards, 1990) than did the K. breviceps blubber used
in this study. The overall insulation value, which takes both quantity and quality into
account, of P. phocoena blubber was 0.15Wm-2
ºC (Worthy and Edwards, 1990), exactly
the same insulation as that of K. breviceps blubber (Table 3). These examples
demonstrate that accurately determining the quality of blubber is more complicated than
simply measuring the percentage of lipid present in the blubber, as quality can also
depend upon lipid class and other differences in composition.
Future studies might also consider whether other structural features of blubber,
not measured in this study, could be contributing to the difference in thermal conductivity
between these two species. For instance, K. breviceps blubber was grossly stratified,
with a fibrous outer blubber layer that differed in both color and texture from the deep
blubber. It is possible that some structural component of the outer layer in K. breviceps
could have affected the overall measure of blubber’s conductivity as compared to G.
macrorhynchus blubber, which lacked this grossly observable, fibrous, outer blubber
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35
layer. Investigating the thermal properties of another species, such as K. sima, which
possesses blubber composed of WE, but lacks the grossly stratified blubber that is seen in
K. breviceps, would help identify how stratification in K. breviceps affects the thermal
measurements, and whether WE blubber is a better insulator.
Depth-specific thermal conductivities measured using the standard material method
Past studies have found histological and biochemical stratification across
blubber’s depth (e.g. Koopman et al., 1996; Struntz et al., 2004; Koopman, 2007; Montie
et al., 2008); therefore, this study examined whether there was any stratification of lipid
content or lipid class across the blubber of G. macrorhynchus and K. breviceps, and
whether any depth-specific difference in lipid correlated to variations in thermal
conductivity. The lipid content did vary across blubber’s depth in both species (Table 3;
Figs. 7 and 8). The deep layer possessed significantly more lipid than the superficial
layer in K. breviceps, and the deep and middle layers possessed more lipid than the
superficial layer in G. macrorhynchus (Table 3; Figs. 7 and 8).
Because an inverse relationship between blubber’s lipid content and conductivity
has been demonstrated in other species (e.g. Worthy and Edwards, 1990; Dunkin et al.,
2005), it was expected that the deep layer, with more lipid, would have a lower thermal
conductivity value. In K. breviceps blubber, the deep layer did tend to have a lower
thermal conductivity, although it was not significantly lower than that of the superficial
layer (Fig. 8). Most surprising, however, was that G. macrorhynchus blubber displayed a
pattern that was opposite of that which was expected; the lipid-rich deep layer had a
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36
higher thermal conductivity than the superficial layer, which contained significantly less
lipid (Fig. 7).
These depth-specific conductivities were measured using the standard material
method, which relies on the depth, or thickness, of the sample, and the difference in
temperature, or ΔT, across that depth. A homogenous material, such as polystyrene
foam, that is divided into three layers of equal thickness would have the same ΔT across
each of those three layers, and, thus, would have the same calculated conductivity value
for each layer. In G. macrorhynchus, the blubber was divided equally into thirds, so the
depth of each layer was the same. Thus, ΔT was the determining factor for the calculated
differences in conductivity across the blubber layers. If this blubber were acting as a
typical insulator and simply resisting heat flow, then a lipid-rich deep layer should slow
the rate of heat flow more than the less lipid-rich superficial layer, and result in a large
ΔT across that deep layer. However, the deep layer of G. macrorhynchus had a smaller
ΔT than the middle and superficial blubber layers, which suggests that heat that entered
the deep surface was not simply transmitted through the blubber, but was instead trapped
and warmed that entire layer.
This result is not surprising if we recall that blubber is a biological material
composed of lipids, and lipids can change phase when exposed to their melting
temperature (reviewed in Tan and Che Man, 2000). In this thermal experiment, the deep
blubber layer was exposed to near core body temperatures, and the outer layer was
exposed to cooler environmental temperatures. If another material composed of lipid,
such as a stick of butter, were placed on a hot pan, the butter directly touching that pan
would begin to melt, although melting would not take place all at once throughout the
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37
depth. In this same way, the temperature regime in the deep blubber layer was different
than that in the superficial blubber, suggesting that differential melting could have
occurred throughout the blubber depth. When a lipid changes phase from solid to liquid,
it can absorb energy, or store heat (reviewed in Suppes et al., 2003). If the deep blubber
layer possessed lipids that melted, heat would be absorbed, which could account for the
small ΔT across that layer. This observation is also supported by the heat flux disc
results, which will be explained in more detail below. Further investigation into
blubber’s depth-specific lipid composition and potential melting behavior are warranted
(see Ch. 2).
Blubber as a heat storing material
Two heat flux discs, which provided a direct measure of the rate of energy both
entering and exiting the blubber, were used simultaneously with the standard material
method in this study. The deep disc yielded much higher heat flux (Wm-2
) values than
the superficial disc, as was also observed in the blubber of bottlenose dolphins (Tursiops
truncatus) by Dunkin et al. (2005). The difference in heat flux values between the two
discs varied by a mean of 58.93Wm-2
in G. macrorhynchus and 61.26Wm-2
in K.
breviceps. As was observed by Dunkin et al. (2005), (1) conductivity values calculated
using the deep disc heat flux values were more than 50% higher than conductivity values
calculated using the other methods, and (2) the superficial heat flux disc yielded
conductivity values that were quite similar to the conductivity values obtained from the
standard material method.
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38
Both Dunkin et al. (2005) and this study examined whether these heat flux values
represented heat loss across other surfaces of the sample and found that no significant
differences in heat flux values occurred across control materials, such as foam, which
suggested that there was little heat loss to the sides of any sample. In total, these results,
along with the unexpected conductivity results and low ΔT across the deep blubber in G.
macrorhynchus, support Dunkin et al.’s (2005) observation that blubber has the capacity
to store heat.
Perhaps this observed heat storage is a function of the lipids present in the blubber
undergoing temperature dependent phase change. Dunkin et al. (2005) reviewed the
evidence that supports classifying the blubber of cetaceans as a phase change material,
which is a material that can temporarily store heat at certain temperatures because of
changes in molecular structure (reviewed in Suppes et al., 2003). Previous studies have
identified mixtures of fatty acids in cetacean blubber that yield phase change materials
with melting points within the range of mammalian body temperatures (e.g. Suppes et al.,
2003). The relationship between blubber’s capacity to store heat, its lipid content, and
the fatty acids present in the blubber layer warrant further investigation (see Ch. 2).
Conclusions
The blubber from G. macrorhynchus and K. breviceps differed with regard to
lipid class, thickness, conductivity, and conductance and insulation values. For the first
time, blubber’s thermal properties were also investigated across its depth using the
standard material method. In K. breviceps, the depth-specific conductivity values tended
to vary inversely with lipid content. Unexpectedly, however, the deep blubber layer in G.
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39
macrorhynchus possessed more lipid, but also a higher conductivity. This result
suggested that there were temperature-dependent effects on blubber’s lipids, possibly
including melting, which influenced blubber’s thermal properties. In addition, this study,
which simultaneously used the standard material and heat flux disc methods,
demonstrated that conductivity values calculated using the deep heat flux disc were
significantly higher than those calculated using either the superficial disc outputs or the
standard material method. The heat flux disc method also provided evidence that heat
was being stored in blubber. The mechanism behind this observed heat storage,
including the hypothesis that blubber’s lipids are functioning as a phase change material,
will be directly investigated in Chapter 2.
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Worthy, G. A. J. and Edwards, E. F. (1990). Morphometric and biochemical factors
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CHAPTER 2: PHASE CHANGE PROPERTIES OF THE BLUBBER LIPIDS OF TWO
SPECIES OF ODONTOCETE CETACEANS, GLOBICEPHALA MACRORHYNCHUS
AND KOGIA BREVICEPS
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INTRODUCTION
Blubber, the specialized hypodermis of cetaceans, functions to thermally insulate
the body (e.g. Parry, 1949; Doidge, 1990; Worthy and Edwards, 1990; Kvadsheim et al.,
1994 and 1996; Pabst et al., 1999; Koopman et al., 2002; Dunkin et al., 2005; Koopman,
2007). Historically, blubber has been thought of as a “standard” insulative material – that
is, one that functions by simply resisting the flow of heat to and from the body.
However, blubber is composed of lipids, which can undergo reversible changes in
physical states, between solid and liquid, according to changes in temperature and heat
input (reviewed in Pond, 1998). The amount of heat required to convert a unit mass of a
substance from solid to liquid without a change in the temperature is its latent heat
capacity (reviewed in Suppes et al., 2003). Once its latent heat capacity has been
reached, the material’s temperature will begin to rise again if more heat is added
(reviewed in Suppes et al., 2003). A material that can temporarily store latent heat due to
changes in molecular structure at certain temperatures is defined as a phase change
material (PCM) (Suppes et al., 2003). If blubber’s lipids change phase within a
physiologically relevant temperature range, then blubber may not be only resisting heat
flow to and from the cetacean body, it may also be acting as a PCM with the capacity to
store and release heat.
Dunkin et al. (2005) were the first to hypothesize that blubber may function as a
PCM. These authors measured the thermal properties of the blubber of bottlenose
dolphins (Tursiops truncatus) by the simultaneous use of the standard material
(Kvadsheim et al., 1994) and heat flux disc (e.g. Worthy and Edwards, 1990) methods.
Because previous studies had not explicitly stated the position of the heat flux disc
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relative to the blubber sample, Dunkin et al. (2005) placed heat flux discs at both
blubber’s deep hypodermal surface and on the epidermal surface during each experiment.
Dunkin et al. (2005) found that the amount of heat entering the deep hypodermal layer
was greater (as high as 87W/m2) than that exiting the epidermal surface per unit time, and
that this difference was positively correlated to the thickness of the blubber sample. This
difference in heat flux across the thickness of the test material was not found when
control materials (white pine wood and polystyrene foam) were tested (Dunkin et al.,
2005). The blubber of short-finned pilot whales (Globicephala macrorhynchus) and
pygmy sperm whales (Kogia breviceps) was similar to that of bottlenose dolphins; more
heat entered the deep hypodermis per unit time than exited the epidermis (difference
between deep and superficial heat flux discs ranged between 30 and 90W/m2).
Dunkin et al. (2005) reviewed four requirements of an efficient PCM: (1) the
melting point of the material must be in an appropriate temperature range, (2) the
material must have a large range of temperatures across which the material will change
phase, or have a large latent heat plateau, (3) the material must not stratify in the liquid
phase, and (4) an intermittent heat load must be present to deliver heat and permit heat to
be absorbed. Blubber meets all four of these requirements, as it is composed of lipids
that are formed by many individual fatty acids (FA), some of which are known to melt
and solidify in a range of biologically relevant temperatures (e.g. Koopman et al., 1996
and 2002; Suppes et al., 2003; Koopman, 2007). In addition, many of the FA that
comprise blubber have large specific latent heats of fusion, with values that are often
greater than 180Jg-1
(Suppes et al., 2003). Blubber lipids would not stratify in the liquid
phase because they are contained within adipocytes within the highly structured
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integument (e.g. Parry, 1949; Koopman et al., 1996; Struntz et al, 2004; Montie et al.,
2008). Finally, intermittent heat loads could be provided by blood vessels undergoing
vasodilation and shunting warmed blood to the blubber layer (e.g. Parry, 1949;
Scholander and Schevill, 1955; Pabst et al., 1999; Meagher et al., 2002 and 2008).
Cetacean blubber, therefore, theoretically meets the requirements of a PCM. Only
one study to date, though, has investigated the phase change behavior of any cetacean
lipid. Clarke (1978A-C) tested the physical properties of spermaceti oil, specialized lipid
composed of wax esters and triacylglycerols, found in the head of the sperm whale
(Physeter macrocephalus), to investigate how pressure and temperature affected the oil’s
density. Clarke (1978) found that this spermaceti oil melted over a broad temperature
range between 0°C and 30°C, and that latent heat storage occurred during this phase
change. No study has yet specifically investigated the phase change behavior of
blubber’s lipids or investigated how lipid class or fatty acid composition influences this
behavior.
A direct method for determining the phase behavior of lipids is Differential
Scanning Calorimetry (DSC). This thermal analysis technique allows for the study of
various heat-related phenomena by monitoring associated changes in enthalpy (Tan and
Che Man, 2000; Brown, 2001). DSC devices have the ability to measure a lipid
mixture’s melting (endothermic event) and freezing (exothermic event) points or profiles
by monitoring these enthalpy changes (Brown, 2001). Any phase changes that occur in a
blubber lipid sample would appear as deviations from the DSC baseline in either an
endothermic or exothermic direction, depending on whether more or less energy has to be
supplied to the lipid sample relative to a reference material (Brown, 2001). Individual
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FA often have sharp melting and freezing points (e.g. Cedeño et al., 2001; Suppes et al.,
2003). However, Tan and Che Man (2000) examined several edible oils, and found that
the presence of multiple lipids and FA in each mixture resulted in broad
melting/crystallization “profiles” rather than sharp phase change points.
Most cetaceans store their blubber lipids in the form of triacylglycerols (TAG),
three FA esterified to a glycerol backbone (reviewed in Pond, 1998). Species within
three families of odontocete cetaceans (Kogiidae, Physeteridae, and Ziphidae) store their
blubber lipid in the form of wax esters (WE), a single FA esterified to a fatty alcohol
(Koopman, 2007). In all cetaceans, blubber’s lipids are formed by a complex mixture of
FA of different chain lengths and number of double bonds [i.e. saturated (SFA),
monounsaturated (MUFA), and polyunsaturated (PUFA)] (Koopman, 2007). Chain
length and degree of unsaturation affect the melting point of the FA (Gurr and Harwood,
1991). For example, the SFA, stearic acid (18:0), melts at 70°C, but a MUFA of the
same chain length with a single cis double bond, oleic acid (18:1n-9), melts at 6.35°C
[Cedeño et al., 2001; Pond, 1998; FA designated according to the International Union of
Pure and Applied Chemistry (IUPAC) nomenclature]. Branching in the carbon chain can
also have a profound impact on its melting point, as is the case for isovaleric acid (i-5:0),
an unusual 5-carbon, short-chained, branched FA. The melting point of isovaleric acid is
approximately -35°C (Weast, 1989). This particular FA is known to accumulate in the
outer blubber layer of harbor porpoises (Phocoena phocoena), and has been hypothesized
to function to maintain fluidity of the blubber lipids at low environmental temperatures
(Koopman et al., 1996).
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Thus, FA can also be stratified across the depth of the blubber. Koopman (2007)
examined biochemical stratification across blubber’s depth in 30 species of cetaceans
representing all families except the river dolphins, and found that levels of a MUFA,
palmitoleic acid (16:1n-7), were higher in the outer blubber in all families. A longer
chain, dietary PUFA, cetoleic acid (22:1n-11), was found in greater quantities in the inner
blubber layers (Koopman, 2007). This large-scale study supports earlier observations of
an overall pattern of long-chain, dietary PUFA being found in greater quantities in the
inner blubber while shorter-chained fatty acids and MUFA can be found in greater
quantities in the outer blubber layers (Koopman et al., 1996 and 2002; Samuel and
Worthy, 2004). FA structure (chain length and degree of unsaturation) and depth-specific
stratification patterns are likely important to blubber’s thermal function.
The primary goal of this study was to test the hypothesis that cetacean blubber
functions as a PCM across a physiologically relevant temperature range, defined here as
37°C (core body temperature) to 5°C (water temperature that an animal may experience
on a deep dive). The short-finned pilot whale (Globicephala macrorhynchus), which
stores blubber lipids as TAG (Koopman 2007), and the pygmy sperm whale (Kogia
breviceps), which stores blubber lipids as WE (Koopman, 2007), were compared. The
depth-specific FA compositions from the blubber of these two species of cetaceans were
quantified. DSC was used to elucidate the melting profiles of full-depth and layer-
specific blubber lipids to directly measure whether intact blubber lipids from G.
macrorhynchus and K. breviceps and went through phase transitions in the range
temperatures regularly experienced by these animals, and whether the lipid storage class
or fatty acid composition affected that behavior.
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MATERIALS AND METHODS
Specimens
Integument samples [epidermis, dermis, hypodermis (blubber), and some
underlying panniculus muscle] were obtained from seven adult female short-finned pilot
whales (Globicephala macrorhynchus) that stranded during a multi-species mass
stranding event on Bodie Island, NC on January 15, 2005 (Hohn et al., 2006). In
addition, integument samples were obtained from seven adult pygmy sperm whales
(Kogia breviceps) that stranded as part of a mass stranding event on September 2, 2006,
or as individuals during the years 2005 through 2010 (Table 2).
All specimens used in this study were in fresh to moderate carcass condition
(Smithsonian Institution Code 1 through 3; Geraci and Lounsbury, 2005) and normal to
robust body condition (Cox et al., 1998; McLellan et al. 2002). For each animal, a
standard set of external morphometrics (adapted from Norris 1961) was collected and a
necropsy was performed. Full-depth samples of the integument (with dimensions ranging
from 15cm X 15cm to 24cm X 24cm) were taken from a dorsal, mid-thoracic site on each
whale (Fig. 1). The blubber samples were vacuum-sealed (Koch 1700, Kansas City, MO,
USA) and stored at –20°C until analysis.
Samples
The integument thickness, including the hypodermis, dermis, and epidermis, was
measured at each side of the sample using precision digital calipers (Absolute Digimatic
calipers, Mitutoyo, Tylertown, MS, USA), and the mean thickness was used in all
calculations. The blubber of G. macrorhynchus did not appear grossly stratified, so a
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standardized method was used to investigate the depth-specific lipid composition. This
method may not fully represent the morphological or biochemical stratification of the
blubber layer. Because previous studies on delphinid blubber had identified it to be
stratified into superficial, middle, and deep layers, the thickness of G. macrorhynchus
blubber (excluding the epidermis) was measured, and that thickness was divided into
equal thirds. In contrast, K. breviceps blubber was grossly stratified into two distinct
layers. The superficial layer (which represented approximately 38% of the blubber
layer’s thickness) appeared more densely fibrous than the thicker, deep layer. Therefore,
this blubber was divided at the interface between the fibrous superficial layer and the
deep layer.
Lipid extraction, classification, and analysis
Lipids were extracted and analyzed from full-depth blubber samples and from
layer-specific blubber samples (deep, middle, and superficial layers for G.
macrorhynchus and deep and superficial layers for K. breviceps). Approximately 0.5g
full-depth sections of blubber, in addition to 0.5g sections of each layer, were excised,
weighed, and placed in 9ml of 2:1 chloroform : methanol with 0.01% butylated
hydroxytoluene (BHT). A modified Folch procedure was then used for lipid extraction
(Folch et al., 1957; Iverson, 1988; Koopman et al., 1996), and a wet weight % of lipid in
the blubber was calculated. The lipids were then re-suspended in hexane at 100mg
lipid/ml of hexane, flushed with nitrogen to avoid oxidation, and stored at -20ºC until
further analysis.
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Lipid classes were separated and quantified using thin layer chromatography with
flame ionization detection (TLC-FID) (Iatroscan®
Mark VI Mitsubishi Kagaku Iatron,
Inc, Tokyo, Japan). Samples were analyzed in duplicate by spotting 1 µl on chromarods
followed by development in hexane : ethyl acetate : formic acid (94:6:1) and
quantification in the Iatroscan. Peaks were identified and integrated using PeakSimple
329 Iatroscan software (SRI Instruments, Torrance, CA), based on known lipid class
standards (Nu Chek Prep, Elysian, MN). Levels of lipid classes were calculated by
applying standard curves generated from known concentrations.
For fatty acid/alcohol analysis, fatty acids were tranesterified into fatty acid butyl
esters (FABE) using 10% BF3 in a heating block at 100°C; this process also cleaved fatty
alcohols in WE, leaving them in free form. FABE and alcohols were separated and
analyzed using a Varian capillary (3800) gas chromatograph (Varian Inc., Division of
Agilent, Santa Clara, CA, USA) with flame ionization detector (FID) fitted with a silica
30m X 0.25mm FFAP (Free Fatty Acid Phase) column (Zebron FFAP; Phenomenex,
Inc., Torrance, CA, USA) with helium as the carrier gas; temperature programming
followed Swaim et al., 2009. Fatty acids were identified and integrated using Galaxie
(vers. 1.8.501.1, Varian, Inc., Palo Alto, CA, USA) in accordance with standards (Nu
Check Preparations, Elysian, Minn., USA) and known sample mixtures (Iverson, 1988;
Koopman et al., 1996), using theoretical response factors (Ackman, 1991) that had been
adjusted according to known values from standard mixtures (Iverson, 1988; Koopman et
al., 1996). Individual fatty acids were named according to IUPAC nomenclature (#
carbons : # double bonds; n-z denotes the position of the final double bond relative to the
methyl end of the fatty acid).
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Thermal analysis by Differential Scanning Calorimetry (DSC)
Differential Scanning Calorimetry has been used to characterize the phase change
behavior of complex lipid mixtures, such as edible oils (e.g. Tan and Che Man, 2000).
Lipids extracted from blubber for composition analysis (described above) were also used
for DSC experiments. For both cetacean species, blubber lipids obtained from the full-
depth sample, as well as from each of the blubber layers, were investigated. One
representative animal of each species was chosen for the full-depth blubber lipid DSC
analysis, and two representative animals of each species were chosen for the layer-
specific blubber lipid DSC analysis (Table 4).
Approximately 200mg of intact lipid in a suspension of 100mg of lipid per ml
hexane was added to DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine; Avanti Polar
Lipids, Inc., Alabaster, Alabama, USA) in chloroform to form a mixture consisting of
0.1% DLPC. The blubber lipid/DLPC/solvent mixture remained in a vacuum-chamber
for 4 hours to ensure complete evaporation of the solvent. Next, 1.0ml of distilled water
was added to the lipid/DLPC mixture, and the sample was extruded 10 times through a
PC membrane (pore size 4µm) to form a stable lipid emulsion of equal lipid droplet size.
This lipid emulsion (300µl) was placed into the sample chamber of the DSC (Nano DSC
microcalorimeter, TA Instruments, New Castle, DE, USA), and distilled water (300µl)
was placed in the reference chamber. The samples were subjected to the following
temperature program: 1°C for 1 hour, heated at 0.2°C per minute to 50°C, held at 50°C
for half an hour, then cooled at 2°C per minute back to 1°C. This program was repeated
at least two times to ensure reproducibility of the data.
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Table 4. Specimens used in this study, with a subset selected for DSC analyses.
Species ID # Sex Total body length (cm) DSC
Globicephala macrorhynchus RT 102 Female 386 lipids from superficial, middle, deep layers
RT 103 Female 349 lipids from superficial, middle, deep layers
RT 105 Female 375 lipids from entire layer
RT 107 Female 357
RT 13 Female 297
RT 19 Female 358
RT 63 Female 359
Kogia breviceps BRF 092 Female 267 lipids from entire layer
MLC 003 Female 262 lipids from superficial and deep layers
WAM 611 Male 301 lipids from superficial and deep layers
KMS 427 Female 267
KMS 429 Male 283
MDB 056 Male 263.5
VGT 221 Male 250
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Because DSC is a technique in which the differences in the amount of heat
required to increase the temperature of a sample (the blubber lipid) relative to a reference
(water) are measured as a function of temperature, any phase change that occurred in the
blubber lipid sample appeared as a positive endothermic peak on the graph. For example,
if blubber lipids melted, more heat was required to increase the lipid sample’s
temperature at the same rate as the reference, due to the lipid sample undergoing an
endothermic phase transition.
To ensure that the lipid sample emulsifier, DLPC, did not affect the melting
behavior of the sample, a control experiment was performed. Soybean oil was used as
the control because its melting point is -20ºC (MSDS); thus, this oil should not undergo
phase change within the physiologically relevant temperature range (5ºC - 37ºC). The
DSC profile of soybean oil emulsified with 0.1% DLPC appeared as a flat line with no
endothermic phase transition (Fig. 11). Therefore, the addition of DLPC did not alter the
phase behavior of soybean oil in the relevant temperature range.
Statistical analyses
Statistical analysis was conducted using JMP 7.0 (SAS Institute, Inc., Cary, NC,
USA) and a significance level of P<0.05 for each test performed. T-tests were used to
determine if there were significant differences between G. macrorhynchus and K.
breviceps with regard to total and depth-specific lipid content and lipid class. FA were
grouped into either long (greater than 18 carbons) or short-chains (less than 18 carbons),
and a one-way ANOVA with post-hoc Tukey HSD test was performed to determine if
there were significant differences in these percentages across the three blubber layers in
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G. macrorhynchus; additionally, a t-test was performed to determine if there were
significant differences in percentage across the two blubber layers in K. breviceps. FA
were also grouped according to degree of unsaturation (SFA, MUFA, and PUFA), and
one-way ANOVA and t-tests were performed to determine significant differences in
these groups across blubber’s depth in both species.
Fig. 11. DSC melting profile of soybean oil. No peak was generated because
soybean oil did not change phase in this temperature range.
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RESULTS
Lipid and fatty acid composition
Globicephala macrorhynchus and Kogia breviceps possessed blubber composed
of different lipid classes. G. macrorhynchus blubber was composed of 99.5 ± 0.07%
triacylgylcerols (TAG), and K. breviceps blubber was composed of 82.1 ± 3.80% wax
esters (WE). The total percentage of lipid present in the entire blubber layer tended to be
higher in G. macrorhynchus (62.17 ± 2.80%) than in K. breviceps (56.38 ± 3.01%) but
this difference was not significant (P=0.07).
Across the blubber depth, the amount of TAG or WE present in each species did
not significantly differ; however, the total percentage of lipid did. In G. macrorhynchus,
the deep and middle blubber layers had higher lipid content than did the superficial layer
(ANOVA; P<0.0001; Table 5). In K. breviceps, the deep layer contained more lipid than
the superficial layer (P<0.0001; Table 5).
A total of 83 different fatty acids (FA) were identified in G. macrorhynchus
blubber, with between 55 and 72 FA identified in each blubber layer. K. breviceps
blubber possessed a similarly complex fatty acid mixture, with a total of 65 FA (61 to 64
FA in each blubber layer) and eight fatty alcohols identified. Each FA has a specific
melting point, and these melting temperatures ranged from -57°C for stearidonic acid
(18:4n-3) to 80°C for behenic acid (22:0) (Weast, 1989). Table 6 lists the most prevalent
FA (those FA with greater than 1% concentrations) and their published melting points
(Weast, 1989) that were found in G. macrorhynchus and K. breviceps blubber. For each
species, ten FA met this criterion, and seven of these were common to both species
(Table 6).
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Table 5. Blubber lipid characterization for G. macrorhynchus and K. breviceps by depth. (1) Lipid content (% wet weight). (2) Fatty
acid composition (area %) grouped according to number of double bonds. (3) FA composition (area %) grouped according to chain
length (long chain ≥ 18C; short chain ≤ 17C). Each value in the table represents the means ± SE. Across depth and species
comparisons (i.e. within column); values that share the same letter are not significantly different (P > 0.05).
Lipid
Content PUFA MUFA SFA
SF
Alcohol MUF Alcohol
Long
Chain
Short
Chain
G. macrorhynchus
Superficial 43.26 ± 2.85
X
7.34 ± 0.44
A
67.46 ± 1.18
A
23.47 ± 0.89
A ---- -----
63.58 ± 1.46
α
36.17 ± 1.44
α
Middle 75.67 ± 2.20
Y
9.92 ± 0.99
A,B
66.87 ± 1.28
A
22.09 ± 0.38
A ---- ----
71.77 ± 1.39
β
27.90 ± 1.39
β
Deep 67.60 ± 3.40
Y
11.11 ± 1.27
B
66.06 ± 1.75
A
22.03 ± 0.48
A ---- ----
76.76 ± 1.15
γ
22.82 ± 1.09
γ
K. breviceps
Superficial 26.22 ± 3.02
Z
2.47 ± 0.28
C
53.18 ± 1.95
A
11.24 ± 0.82
B
12.38 ± 1.34
A
22.46 ± 1.37
A
63.24 ± 1.20
α
36.40 ± 1.16
α
Deep 75.29 ± 2.61
Y
3.35 ± 0.44
C
51.07 ± 2.50
B
12.33 ± 0.63
B
10.12 ± 1.06
A
20.25 ± 1.27
A
65.24 ± 1.20
α
34.25 ± 1.09
α
Abbreviations: PUFA, polyunsaturated fatty acid; MUFA, monounsaturated fatty acid; SFA, saturated fatty acid; SF Alcohol,
saturated fatty alcohol; MUF Alcohol; monounsaturated fatty alcohol.
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Table 6. Most abundant fatty acids in G. macrorhynchus and in K. breviceps blubber by depth.
All melting temperatures are from Weast, 1989; the * value for 18:1n-9 was obtained from Cedeño et al., 2001.
Superficial Middle Deep Superficial Deep
14:0 53.9°C 3.85 ± 0.21 2.85 ± 0.20 2.19 ± 0.19 3.17 ±0.08 3.54 ± 0.13
16:0 63.1°C 15.02 ± 0.69 13.58 ± 0.37 12.79 ± 0.43 5.63 ± 0.59 6.16 ± 0.48
16:1n-9 32.5°C 1.33 ± 0.08 1.05 ± 0.06 0.97 ± 0.05 < 1% < 1%
16:1n-7 0.5°C 9.19 ± 0.91 5.43 ± 0.92 2.78 ± 0.53 9.82 ± 0.61 7.40 ± 0.74
18:0 69.6°C 2.67 ± 0.19 3.75 ± 0.27 4.95 ± 0.24 0.98 ± 0.15 1.21 ± 0.15
18:1n-11 12.5°C < 1% < 1% < 1% 1.04 ± 0.30 1.13 ±0.33
18:1n-9 6.35°C 46.34 ± 1.76 47.93 ± 1.97 47.84 ± 2.19 23.74 ± 2.01 22.58 ± 1.36
18:1n-7 15.4°C 3.13 ± 0.08 3.23 ± 0.05 3.06 ± 0.04 1.10 ± 0.13 1.01 ± 0.15
20:1n-11 23.5°C < 1% < 1% < 1% 2.11 ± 0.53 2.47 ±0.58
20:1n-9 23.4°C 3.30 ± 0.16 5.08 ± 0.27 6.45 ± 0.24 5.23 ±0.86 6.59 ± 0.74
22:1n-11 33.5°C < 1% < 1% < 1% 4.53 ± 1.56 5.13 ± 1.54
22:5n-3 -23.0°C 0.57 ± 0.05 1.12 ± 0.11 1.46 ± 0.16 < 1% < 1%
22:6n-3 -44.0°C 1.91 ± 0.21 3.53 ± 0.50 4.26 ± 0.73 < 1% < 1%
K. brevicepsG. macrorhynchus
Fatty acid Melting point
*
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The most common FA found in both G. macrorhynchus (47.37 ± 0.51%) and K.
breviceps (23.16 ± 0.58%) blubber was oleic acid (18:1n-9), with a melting point of
6.35°C (Cedeño et al., 2001). In addition, because K. breviceps blubber contained WE, it
also contained fatty alcohols, six of which were found at 1% or greater concentrations
(Table 7). Fatty alcohol tridecylic (13:0) and myristic (14:0) were the only other fatty
alcohols found in K. breviceps blubber.
Table 7. Most abundant fatty alcohols in K. breviceps
blubber by depth.
K. breviceps
Fatty alcohols Superficial Deep
ALC16:0 7.82 ± 0.90 9.43 ± 0.98
ALC16:1n-7 2.10 ± 0.23 1.32 ±0.21
ALC18:0 1.54 ± 0.23 2.29 ±0.48
ALC18:1n-9 17.43 ±1.26 16.01 ±1.19
ALC18:1n-7 1.78 ± 0.24 1.75 ±0.29
ALC20:1n-9 1.15 ± 0.20 1.17 ± 0.18
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FA from each blubber layer of G. macrorhynchus and both FA and fatty alcohols
from each blubber layer of K. breviceps were grouped according to number of double
bonds [saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA)], and
chain length (long chain ≥ 18 carbons; Table 5). In G. macrorhynchus, PUFA were
stratified across the blubber depth (Table 5); the deep blubber layer possessed
significantly more PUFA than the superficial layer (P = 0.037; Table 5); however, the
middle blubber layer was not significantly different from any other layer (Table 5). The
MUFA and SFA % concentrations were not stratified in G. macrorhynchus blubber. G.
macrorhynchus blubber also displayed stratification of FA based upon chain length.
Each layer was significantly different from the others (P<0.0001). Longer chain FA
made up an increasingly larger percentage of the total from superficial to deep (Table 5).
In contrast to G. macrorhynchus, K. breviceps blubber did not display any FA
stratification across its depth based upon degree of unsaturation or chain length.
Within each layer, MUFA represented the largest % of all FA, followed by SFA
and PUFA (Table 5). This pattern was displayed in both species and for all blubber
layers. Within K. breviceps, there were higher concentrations of MUF alcohols than SF
alcohols in both the superficial and deep layers (Table 5). Long-chain FA predominated
regardless of species or blubber layer (Table 5).
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Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) melting profiles were generated for the
intact lipids from full-depth and layer-specific blubber samples for a small subset of G.
macrorhynchus and K. breviceps (see Table 4, Materials and Methods). An increase in
heat flowing into the sample is demonstrated by a positively oriented deviation in the
trace, which represents an endothermic phase transition. All lipid samples from the
TAG-rich blubber of G. macrorhynchus and the WE-rich blubber of K. breviceps
changed phase over a broad, physiologically relevant temperature range (Figs. 12-14).
The full-depth G. macrorhynchus lipid sample displayed a large endothermic
peak around 10°C, and a broad, right shoulder that indicated more lipids continued to
melt until approximately 30°C (Fig. 12A). The phase change profile of the full-depth K.
breviceps lipid sample was shifted to the left relative to that of G. macrorhynchus; phase
change began below 0°C, and lipids continued to melt until approximately 23°C, where
there was an abrupt end to the peak (Fig. 12B). The beginning of this phase transition
could not be observed in K. breviceps lipids because the DSC could only measure the
phase change behavior of the lipid sample within the range of temperatures that the water
reference did not change phase.
The superficial, middle, and deep blubber from G. macrorhynchus also possessed
lipids that changed phase across a broad, physiologically relevant temperature range (Fig.
13). In both individuals, lipids changed phase between 2ºC and 30ºC, although the
shapes of the melting curves and the end points of the peaks were subtly different across
layers and between individuals. The melting profile of the deep blubber lipids most
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clearly displayed a secondary peak between 24 - 26°C, but in one individual, RT 102,
that secondary peak was especially pronounced (Fig. 13).
Similarly, both the superficial and deep blubber lipids of K. breviceps underwent
phase change across a broad range of temperatures; lipids began melting below 0°C and
continued to melt until approximately 23°C (Fig. 14). There were also subtle differences
across layers (the deep layer displayed more pronounced peaks than did the superficial
layer) and between individuals (Fig. 14).
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(A)
(B)
Fig. 12. Differential scanning calorimetry outputs for full-depth blubber lipids
from (A) G. macrorhynchus (RT 105, adult female) and (B) K. breviceps
(BRF 092, adult female).
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Fig. 13. Differential scanning calorimetry outputs for superficial, middle, and deep
blubber lipids of G. macrorhynchus (left RT 102, adult female; right RT 103, adult
female).
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Fig. 14. Differential scanning calorimetry outputs for superficial and deep blubber lipids
of K. breviceps (left WAM 611, adult male; right MLC 003, adult female).
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DISCUSSION
The major goals of this study were to determine whether Globicephala
macrorhynchus blubber composed of triacylglycerols (TAG) and whether Kogia
breviceps blubber composed of wax esters (WE) would undergo phase change in a
physiologically relevant temperature range, and whether TAG and WE lipids from these
two cetacean species would have different melting profiles. Layer-specific fatty acid
(FA) analyses were performed to gain insight into whether any features of blubber’s
composition correlated with its phase change behavior. The results of differential
scanning calorimetry (DSC) studies clearly indicate that the lipids isolated from the
blubber of these species do change phase within a physiologically relevant temperature
range (5°C - 37°C).
Fatty acid composition
Because the specific melting point of an individual FA depends upon its chain
length and number of double bonds (Weast, 1989), this study grouped FA according to
these two physical properties and characterized the FA present across the blubber depth
in both G. macrorhynchus and K. breviceps. This method of grouping FA was based
upon Tan and Che Man’s (2000) study of the phase change behavior of edible oils.
These authors found, for example, that safflower oil, an edible oil with a high content of
TAG molecules formed of three unsaturated FA, changed phase at lower temperatures
than other edible oils with lower percentages of unsaturated TAG. While the manner in
which individual saturated and unsaturated FA form the specific TAG and WE lipid
molecules in blubber was not identified in this study, it was expected that overall
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differences in FA composition might influence the temperature at which the intact
blubber lipids melt. The deep blubber layer of G. macrorhynchus contained more long-
chained, polyunsaturated FA (PUFA) than did the superficial layer (see Table 5), which
led to the expectation that the deep layer might melt at lower temperatures than the
middle and superficial layers. K. breviceps blubber did not display any stratification in
FA composition across the blubber depth (Table 5), which suggested that the intact
blubber lipids might have similar melting behaviors across these two layers. Neither of
these expectations was realized (see discussion below).
However, blubber’s lipids are not as simple as those of an edible oil (e.g. Tan and
Che Man, 2000), because each blubber layer contained a complex mixture of between 55
and 72 FA (typical edible oils usually contain between 5 and 10 FA). G. macrorhynchus
and K. breviceps shared many predominant FA. In both species, oleic acid (18:1n-9) was
most abundant, and this FA melts within the physiologically relevant temperature range
(6.35ºC; Cedeño et al., 2001). However, the melting points of the 10 most abundant FA
ranged from -44ºC to 69.6ºC (Table 6).
In addition, because half of every WE is a fatty alcohol, there were large
percentages of saturated and monounsaturated fatty alcohols found in the blubber layers
of K. breviceps (Table 7). The influence that these fatty alcohols may have on melting
behavior warrants further investigation, although one study on intact wax ester lipids of
insect cuticle offers insight into their melting properties (Patel et al., 2001). These
authors found that the total chain length of the wax ester as well as placement of the ester
bond affected melting point, and the insertion of a double bond decreased the melting
point of the wax ester by approximately 30 ºC (Patel et al., 2001).
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The different temperatures over which blubber’s constituent FA melt and the fact
that different FA may be organized in various combinations as either TAG or WE mean
that it is difficult, if not impossible, to make predictions about the melting behavior of the
intact lipids. Nikolić et al. (2002) found that combining just two fatty acid methyl esters
could result in a mixture that displayed a wider temperature range over which phase
change occurred than for either single fatty acid methyl ester. Tan and Che Man (2000)
also observed broad melting profiles, rather than sharp melting points, in edible oil
samples, composed of far fewer FA than are found in cetacean blubber lipids. For
example, soybean oil contains only five FA (16:0, 18:0, 18:1n-9; 18:2n-6, and 18:3n-3).
These FA have different melting points that range between -11°C and 69.6°C (Weast,
1989). However, the melting point of soybean oil as a whole, with intact lipids, is -20°C
(MSDS; CAS#8001-22-7). Therefore, it is not only the individual FA components, but
also the different ways in which FA are organized into different lipid molecules that
ultimately determines the phase change behavior of a mixture. A physical chemistry
technique called differential scanning calorimetry (DSC) was therefore used to
investigate phase change in intact lipids from these two cetacean species.
Differential Scanning Calorimetry
The DSC melting profiles of the lipids from G. macrorhynchus and K. breviceps
demonstrated that blubber lipids underwent a broad, endothermic phase transition within
a physiologically relevant temperature range (5 – 37°C) (Figs. 12 - 14). Although all
samples from both species showed a primary peak at the lower end of the temperature
range, broad right shoulders and/or secondary peaks indicated that lipids were still
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absorbing heat and changing phase over an approximate temperature range of 25°C to
30°C (Figs. 12 - 14). This phase change behavior may reflect the transition from solid to
liquid; however, the endothermic peaks on the graph could have also included some
reversible solid-solid or liquid-liquid transitions, which reflect the chemical
reorganizations of the lipid molecules that result when heat is absorbed (Cedeño et al.,
2001). This known phenomenon of polymorphism makes it even more difficult to tease
apart any phase change effects of a specific FA or intact lipid molecule (e.g. Tan and Che
Man, 2000; Cedeño et al., 2001; Nikolić et al., 2002). For instance, Cedeño et al. (2001)
used DSC to demonstrate that oleic acid, the most abundant FA found in blubber of both
species, went through both a solid-solid and liquid-liquid reorganization process, and that
these molecular events overlapped with the melting process, resulting in a wider
endothermic peak. Therefore, blubber lipids may display endothermic peaks that are not
only the result of a phase change from solid to liquid, but are also a result of heat being
absorbed during molecular reorganization events.
The TAG and WE lipid classes both melted over a broad temperature range,
though there were differences in the phase change behavior between the two cetacean
species. The WE of K. breviceps displayed a melting profile that was shifted slightly to
the left relative to that of G. macrorhynchus. This result suggested that lipid class did
play a role in the overall melting behavior of blubber; K. breviceps blubber lipids,
composed predominately of WE, began melting below 0ºC. Because water was used as
the reference material, DSC could not be used to investigate the beginning of the phase
transition that occurred below 0ºC. Future studies could use a different reference
material (such as ethylene glycol) to observe a more complete phase transition. For the
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purposes of this study, however, only those phase changes that took place within the
physiologically relevant temperature range were investigated.
Across blubber’s depth, G. macrorhynchus and K. breviceps displayed different
FA stratification patterns, which led to the hypothesis that the different FA compositions
in the superficial and deep layers of G. macrorhynchus would be correlated to depth-
specific differences in melting behavior. There were subtle depth-specific differences
that were observed across the three blubber layers in G. macrorhynchus; the deep blubber
lipids, with a higher concentration of long-chain PUFA, had better-defined secondary
melting peaks than the melting profiles of the middle and superficial layers, though there
was some variation in this pattern between individuals as well (Fig. 13). Interestingly,
even though there were no significant differences in the measured depth-specific FA
patterns in K. breviceps, DSC revealed that there were differences in the melting profiles
of the superficial and deep layers (Fig. 14).
Because of the complexity of the FA present in the blubber, it is possible that the
methods used to group and differentiate FA across depth were not sensitive enough to
capture the variation that contributed to the differences in the melting profiles across
depth. Interestingly, Koopman (2007), using an index based upon differences in
concentration of the 16 most abundant FA across depth, also found low levels of FA
stratification in kogiids. Even if a more sensitive FA stratification analysis could be
performed, it still may not be possible to directly predict the melting profiles of such a
complex blubber lipid mixture that contains more than 55 different FA. Thus, DSC
provided a direct assessment of the melting behavior for the complex lipid mixtures
found in G. macrorhynchus and K. breviceps blubber, and in conjunction with FA
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analysis, resulted in the first insights into how these lipids, regardless of whether they
were TAG or WE, melted over a broad, physiologically relevant temperature range.
Functional consequences of phase-changing blubber
The DSC melting profiles demonstrated that blubber lipids absorb heat as they
change phase from solid to liquid under physiologically relevant temperature conditions.
Though only the melting curves were shown in this study (Figs. 12 - 14), phase change in
blubber lipids is a reversible, reproducible process, meaning that phase change also
occurs in the opposite direction. When blubber lipids cool and change from liquid to
solid, then an exothermic transition, or heat release, occurs. Thus, blubber is not only
acting as a typical insulator by simply resisting heat flow from the body core, but is also
acting as a dynamic, temporal, thermal buffer by absorbing and/or releasing heat. That is,
blubber is likely functioning as a phase change material (PCM), a hypothesis first
suggested by Dunkin et al. (2005).
The use of PCM as a thermal buffer has received growing interest from industry
as a mechanism to enhance thermal insulation in building and housing applications (e.g.
Nikolić et al., 2001; Sari and Kaygusuz, 2001; Sari, 2003; Sari et al., 2003; Suppes et al.,
2003). Historically, increasing the insulation, R (m2 °C W
-1), of a building material has
been achieved by either constructing a better quality (lower conductivity, see Ch. 1)
material or by making the material thicker. When space is limited, it is impractical to add
thicker insulation. In theory, only a small amount of PCM placed in the wall of a
building could act as a thermal buffer against changes in environmental temperatures by
melting and absorbing large amounts of heat during the hottest hours of the day, and then
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solidifying and releasing that heat at night when the temperature cools (e.g. Nikolić et al.,
2001; Sari et al., 2003; Suppes et al., 2003). Most commercial applications require a
PCM with a relatively narrow melting range matched to the specific and predicted
environmental temperatures. However, utilizing a PCM with a wider melting range may
be desirable if they are exposed to broad extremes of change in environmental
temperatures.
The broad temperature range over which phase change occurred in blubber lipids,
which indicates that heat is also absorbed over a large temperature range, may be a useful
feature for a cetacean that regularly experiences a large thermal gradient during the
course of a deep dive. G. macrorhynchus have been recorded to dive to a mean depth of
762m for an average of 15 min (Soto et al., 2008). Less information is known about K.
breviceps dive behavior, but dietary analyses suggest that they feed on deep-water squid
typically occurring between 500 and 1500m (reviewed in Piscitelli et al., 2010) and one
visual tracking study observed a maximum dive duration of 52 minutes (Barlow et al.
1997). Both species may experience as much as a 20ºC drop in ambient temperatures
over the course of a dive (Goold and Clarke, 2000), which is interesting because DSC
results demonstrated that blubber lipids of both cetacean species absorb or release heat
throughout that temperature range.
While the functional consequences of possessing a phase-changing blubber layer
are not yet known, there are several possible scenarios that may be considered. The first
explanation is that, while melting, phase-changing blubber may be acting as a temporary
heat storage compartment. If blubber were instead acting as a typical insulator, then the
heat produced by the body core would be transmitted through the blubber layer and lost
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to the environment at a rate dependent upon the temperature differential across the
blubber layer. As that temperature differential increased during a deep dive, more heat
would be lost to the environment. If the blubber could, instead, temporarily store a
portion of the heat entering it from the body core, the rate of energy loss to the
environment during the dive would be decreased. There is empirical evidence that the
amount of heat entering and exiting the blubber per unit time does differ; heat flux disc
experiments demonstrated that more heat entered the deep hypodermis than exited the
epidermis per unit time (see Ch. 1 and Dunkin et al., 2005). These results imply that heat
is transforming the blubber rather than simply being transferred through it.
Another way in which a phase-changing blubber layer could function as a thermal
buffer is by solidifying and releasing heat back to the body core. The outer blubber layer
of G. macrorhynchus or K. breviceps, when exposed to cooling environmental
temperatures, may undergo exothermic phase transitions where some lipids solidify
during a deep dive. If the lipids undergo this phase transition, heat would be released
omnidirectionally. This release of heat could warm adjacent lipids, which could delay
cooling of the entire blubber layer. These scenarios suggest that the rate at which heat is
lost through a phase-changing blubber is dynamically variable and dependent upon the
environmental temperatures to which the blubber is exposed. It is likely that a
combination of the deep blubber layer melting and storing heat and the superficial
blubber solidifying and releasing heat results in a thermal buffering function for the
diving cetacean.
This thermal buffering function could be further enhanced by the active delivery
of heat, via blood flow, to the well-vascularized blubber (Parry, 1949; Dunkin et al.,
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2005). Heath and Ridgway (1999) hypothesized that bottlenose dolphins (Tursiops
truncatus) can variably perfuse their blubber to redistribute large amounts of heat from
the body core to the periphery as a “preemptive strike” against overheating in warm,
tropical waters. By shunting blood, or redistributing heat, to the outer blubber, the
surface temperature of the animal increases, which reduces the amount of heat entering
the animal’s body from the hot environment (Heath and Ridgway, 1999). These authors
called blubber a “heat sink”, but meant this term in the sense of a well-vascularized tissue
that could accept heat loading via enhanced perfusion (Heath and Ridgway, 1999); they
did not consider how shunting blood to the blubber layer could result in blubber lipids
undergoing phase change and temporarily storing that heat. G. macrorhynchus and K.
breviceps may be able to utilize vascular controls as a way of dynamically controlling the
phase-change behavior of their blubber. If other cetacean species also possess a phase-
changing blubber layer, then whether a cetacean lives in cold or hot environments,
blubber’s buffering ability may be finely tuned by controlled delivery of blood to and
away from the blubber lipids.
A final factor to consider is that all DSC (this study) and intact blubber thermal
experiments (see Ch. 1) took place under atmospheric pressure conditions. Therefore,
this study could only provide insights into the phase change behavior of blubber lipids
based upon changes in temperature alone. However, a rise in pressure does increase both
the density and temperature of lipids, and both G. macrorhynchus and K. breviceps may
experience a rise in pressure over 100atm, possibly in less than 20 minutes (Clarke,
1978B; Goold and Clarke, 2000). The way in which increasing pressure and
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simultaneous decreasing environmental temperatures affects the temperature, density,
and, thus, phase change of blubber’s lipids warrants further investigation.
Conclusions
G. macrorhynchus and K. breviceps blubber lipids, regardless of whether they are
composed of TAG or WE, underwent a broad phase change in the physiologically
relevant temperature range (5 ºC to 37 ºC). The complexity of the lipid mixture made it
impossible to predict blubber’s phase change behavior, but use of the DSC allowed for
the characterization of the melting profiles of blubber’s intact lipids. Heat may be
absorbed or released over a wide temperature range, which means that blubber could be
acting as a dynamic, temporal thermal buffer. This buffering ability could be finely
controlled by how blood is shunted to or away from the blubber layer over the course of a
diving cycle. The functional consequences to the diving cetacean, especially the effects
of pressure, density, and temperature at depth on the phase-change behavior, still require
further investigation.
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