MICROVASCULAR PATTERNS OF BLUBBER IN SHALLOW AND DEEP DIVING

ODONTOCETES

Sara J. McClelland

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

2010

Approved by

Advisory committee

Richard M. Dillaman D. Ann Pabst

Heather N. Koopman

Chair

Accepted by

Dean, Graduate School

TABLE OF CONTENTS

ABSTRACT...................................................................................................................................iii

ACKNOWLEDGMENTS...............................................................................................................v

DEDICATION...............................................................................................................................vii

LIST OF TABLES.......................................................................................................................viii

LIST OF FIGURES........................................................................................................................ix

INTRODUCTION...........................................................................................................................1

METHODS......................................................................................................................................6

Tissue Collection.................................................................................................................6

Histological Analysis...........................................................................................................7

Lipid Analysis....................................................................................................................12

Fatty Acid Stratification.....................................................................................................13

Statistics.............................................................................................................................13

RESULTS......................................................................................................................................14

Histological Analysis.........................................................................................................14

Lipid Analysis....................................................................................................................15

DISCUSSION................................................................................................................................16

Vascular Distribution and Morphology.............................................................................17

Relationship of Vascular Patterns to Biochemical Characteristics of Blubber..................18

Potential Influences on Blubber Vasculature Structure.....................................................22

Conclusions and Future Directions....................................................................................27

LITERATURE CITED..................................................................................................................29

ABSTRACT

Blubber is the hypertrophied subdermal adipose layer surrounding the bodies of marine

mammals and is an important and dynamic living tissue. Typically, adipose tissue is perfused by

networks of capillaries. Surprisingly, there is little information on the vascularization of blubber.

The goal of this study was to describe and characterize, for the first time, the microvasculature

(capillaries, microarterioles and microvenules) of the blubber of marine mammals, using toothed

whales (Odontoceti) as models. Marine mammals experience vasoconstriction during a dive as

part of the dive response, resulting in decreased blood flow to peripheral tissues (including

blubber). I predicted that deeper divers would have reduced vasculature in their blubber, since

they theoretically would not perfuse their blubber vasculature while on a dive. I used

histochemical techniques and digital image analysis to estimate the density and distribution of

the microvasculature in the blubber of two species of odontocetes, the shallow-diving bottlenose

dolphin (Tursiops truncatus; n=6), and deeper-diving pygmy sperm whale (Kogia breviceps;

n=6). Tursiops blubber showed significant differences in the density of the microvasculature

among the superficial (3.26±0.39%), middle (8.40±0.65%) and deep (9.31±0.72%) layers. Kogia

blubber differed, with its microvascular density more uniformly distributed across the blubber

(superficial 3.26±0.53%, middle 4.38±0.55%, deep 4.50±0.54%). Overall, Tursiops had

significantly (P<0.001) higher microvascular densities than Kogia, with this difference largely

due to the higher values in the inner and middle layers. Two beaked whales (Mesoplodon

densirostris, n=1; Ziphius cavirostris, n=1) examined had microvascular densities much lower

than either Tursiops or Kogia , with a relatively uniform distribution (all layers with values

between 1.5 and 2.8%). To place the data in a broader mammalian context, I also examined

microvascular density in the subcutaneous adipose tissue of the domestic pig (Sus scrofa, n=4).

The pig subdermal adipose exhibited no stratification and comparatively low vessel densities

(superficial 1.75±0.22%, middle 1.87±0.22%, deep 1.88±0.46%). Vascular data were then

compared with biochemical features of the blubber (lipid content and composition) to identify

any links between vessel distribution and lipids. Overall, the species that had the lowest lipid

content (Tursiops ~46% overall) had the highest microvascular densities. The species with the

highest lipid content (beaked whales ~80% overall) had the lowest microvascular densities, and

Kogia was intermediate in both categories. Lipid class also appeared to correlate with the

microvascular densities in blubber; Kogia and the beaked whales had blubber composed mainly

of wax esters and had the lowest microvascular densities. Pigs had low microvascular densities

but had no wax esters; the relationship between lipid class and vascularity is likely not direct.

The deepest divers had the lowest microvascular densities and the shallow-divers had the most

microvascular densities of the odontocetes analyzed to date. While dive depth and microvascular

densities appear to be correlated, the conserved nature of the microvascularity in the blubber of

beaked whales suggests that the microvascular densities in blubber is not a direct consequence of

dive depth. It is likely that other factor(s), such as energetics or thermoregulation, may be

influencing the vessel densities in the blubber of marine mammals.

ACKNOWLEDGEMENTS

I could not have finished this thesis without the help and support of many people, all of

whom deserve to be acknowledged and to receive a very special thank you. First and foremost, I

want to thank my advisor, Dr. Heather Koopman. Her guidance and support have carried me

through the difficulties that come with graduate school and completing a thesis. She has gone

above and beyond the duties of a mentor in helping me think through ideas, work on writing,

become better at public speaking, and just overall teaching me about science. More important

than all of that, she has helped me to learn how to think, which is a skill I am still trying to

master. Her advice and friendship are worth more than gold to me and without her help I would

never have been able to do this.

I would also like to express my deepest gratitude to Mark Gay. He spent countless hours

teaching me exciting, new, and useful laboratory techniques, helped me track down mounds of

information, took the time to think through numerous problems with me and was always there to

talk about my project with eagerness and intelligence. Without his assistance, I never would

have found a method to complete this work, nor would I have had the patience to sustain myself

through frustrating days in the lab when 'science just would not work'.

I want to acknowledge and thank Dr. Ann Pabst for all of her time and attention. She

always treated me as one of her own, and was always available to sit down and think about what

all of my data actually meant. She is an expert at asking the right questions and her input has

been invaluable in the process of completing this project.

Many more thanks go to Dr. Richard Dillaman for helping me to learn about histological

processes beyond the scope of my work, and in doing so, helping me to understand my own

work that much better. Without his time and help, this work would have suffered severely.

I would also like to acknowledge Dr. Andrew Westgate, who should have been a

committee member on this project; he has devoted the time and intellectual input above and

beyond what would be expected of a committee member. I am grateful for all of the time he has

devoted to this project, his patience, and his thoughtful comments on my work. I'd also like to

thank both him and Heather for genuinely caring about me and spending countless hours

encouraging me, without which I would still be floundering.

I would like to acknowledge the other students in my lab, both past and present, as well

as everyone who is a member of the VABLAB. It is rare to find one lab that cares so much

about each other and gets along so well, but to find two and have both accept me is something I

have been blessed with. I'd like to thank Zach Swaim, Hillary Lane, Caitlin McKinstry, Sandy

Camilleri, Bill McLellan, Dr. Sentiel "Butch" Rommel, Brian Balmer, Marina Piscitelli, Laura

Bagge, Caitlin Kielhorn, Ryan McAlarney, and Peter Nielsen for always providing a wonderful

audience for presentations and for being an important sounding board for ideas. I'd also like to

thank them for the friendship they've provided, which has enriched my life and kept me sane

while working on my project.

For sample collection, I'd like to thank the UNCW Marine Mammal Stranding Network,

the Cape Cod stranding Network, the Marine Mammal Center, and Ron and Shannon at Wells

Pork Products in Burgaw, NC. For funding I'd like to thank ONR for funding this project and

Prescott Grants for funding tissue collection. For additional help with methods and advice for

working with blubber, I'd like to thank D.J. Struntz and Eric Montie.

Lastly I would like to point out that the most important acknowledgement now and

forever, as well as all thanks, should go directly to God. He blessed me with a good mind and

has surrounded me by even better minds. I fail daily in my attempts to be a Christian, but He

continues to shower me with blessings, the most recent of which is this thesis.

DEDICATION

This thesis is dedicated to my family. My parents, Thomas William Rager and Carolyn

Jeanine Rager, have always stood beside me and given me their utmost support in every

adventure I have chosen to undertake no matter the emotional or financial cost to them. My

brother, Joshua Clarence Rager, has filled my life with friendship, laughter, and inspiration.

From the very first sentence my brother taught me to write, my family has worked to instill in me

a love of education and a belief that I could accomplish anything if I worked hard enough.

Finally, this thesis is also dedicated to my husband, Jeffrey Bronson McClelland. He is the love

of my life. He has always been there to hold on to me during my darkest moments and to pick

me up and dust me off afterwards. His support and his help in all things have enabled me to

write this thesis and to be where and who I am today. I will forever be grateful that we get to

share our lives together and I look forward to each new day we get to spend together.

LIST OF TABLES

Table Page

1. Specimen information for individual animals used in this study.......................................40

2. Microvascular characteristics of blubber/adipose tissue....................................................41

3. Biochemical characteristics of blubber/adipose tissue......................................................42

4. Average stratification Index for fatty acids in the blubber/adipose tissue.........................43

5. Select fatty acids and fatty alcohols found in blubber/adipose tissue................................44

6. Mean percent area consisting of microvascularity in a diversity of adipose

tissues of terrestrial mammals............................................................................................46

LIST OF FIGURES

Figure Page

1. Sampling site of blubber/adipose tissue.............................................................................47

2. Determining total number of terminal branches/vessel.....................................................48

3. Images of microvasculature in blubber/adipose tissue......................................................49

4. Percent area of microvessels analyzed by depth (Tursiops truncatus)..............................52

5. Percent area of microvessels analyzed by depth (Kogia breviceps)..................................53

6. Percent area of microvessels analyzed by depth (Sus scrofa)............................................54

7. Comparison of Tursiops truncatus, Kogia breviceps, beaked whales,

and Sus scrofa percent area of microvessels analyzed by depth........................................55

8. Microvasculature diameter of microvessels in blubber/adipose tissue..............................56

9. Microvascular branching of microvessels in blubber/adipose tissue.................................57

10. Percent lipid (wet weight) of blubber/adipose tissue.........................................................58

11. Percent of wax ester in blubber/adipose tissue..................................................................59

12. Fatty Acid Stratification Index...........................................................................................60

INTRODUCTION

Adipose tissue is a specialized form of loose connective tissue that is composed mainly of

adipocytes (Ross and Pawlina, 2006). Adipocytes are specialized lipid storage cells. The center

of these cells is filled with lipids; the cell’s nucleus and cytoplasm are pushed to one side

forming a signet ring shape (Ross and Pawlina, 2006). Small aggregations of adipocytes as well

as loose lipid droplets can commonly be found in numerous types of tissue throughout the body,

but the majority of fat in the vertebrate body is located in adipose tissue depots (Pond, 1998).

Adipose tissue can experience large changes in size, with most of the expansion or shrinkage of

adipose depots being due to changes in adipocyte dimensions (Young, 1976; Miller et al., 1983;

Groscolas, 1990; Ramsay et al., 1992; Koopman et al., 2002). Rather than new cell formation or

loss of existing cells (apoptosis), adipocytes may undergo tenfold changes in volume depending

on the amount of resources available and the energetic demands, such as migration or lactation,

of the animal (Young, 1976; Miller et al., 1983; Ryg et al., 1988; Groscolas, 1990; Pond, 1998;

Koopman et al., 2002). With increased food intake, lipid can be stored in adipose tissue to be

used during times of inadequate energy intake.

Marine mammals (cetaceans, pinnipeds, and sirenians) possess a highly modified type of

adipose tissue in the form of blubber. Blubber, a specialized hypodermal layer, forms the bulk of

lipid storage in these animals and is distributed almost solely subcutaneously around the body

(Bryden, 1968; Pond, 1978). Blubber is more rigid and organized than the adipose tissue of

terrestrial mammals (Parry, 1949; Ling, 1974; Ackman et al., 1975; Lockyer et al., 1984; Pond,

1998; Pabst et al., 1999; Ross and Pawlina, 2006). The structural difference between blubber

and adipose tissue of terrestrial mammals is due to the networks of collagen and elastin

surrounding the adipocytes (Parry, 1949; Ling, 1974; Ackman et al., 1975; Lockyer et al., 1984;

Pond, 1998; Pabst et al., 1999; Hamilton et al., 2004; Struntz et al., 2004; Ross and Pawlina,

2006; Montie et al., 2008b). In addition, the bodies of marine mammals contain more fat than

terrestrial mammals. Healthy terrestrial mammals in the wild average between 4-8% dissectible

adipose tissue (Pond, 1998), and most healthy marine mammals average around 30% dissectible

adipose tissue (Pabst et al., 1999; McLellan et al., 2002). It is not unusual to see even higher

values: harbour porpoise calves 37% fat; right whales 43% (Lockyer, 1991; McClellan et al.,

2002).

The increased fat stores in marine mammals serve multiple functions that enable marine

mammals to live in water. Marine mammals face higher thermoregulatory demands than those

living on land. Water conducts heat away from the body 25 times faster than air at the same

temperature (Parry, 1949; Scholander et al., 1950; Schmidt-Nielsen, 1997). Blubber provides

insulation that reduces heat loss, making it possible for marine mammals to expend less energy

to maintain their body temperature (Worthy and Edwards, 1990; Dunkin et al., 2005).

Movement in water is also associated with increased drag forces on the body, and blubber plays

a role in decreasing the amount of those forces experienced by marine mammals. Water is three

times denser than air and sixty times more viscous, leading to increased drag on the body (Webb,

1984; Fish, 1993; Vogel, 2003). Blubber sculpts the body, creating a more streamlined shape

and thereby reducing drag and conserving energy (Ryg et al., 1988; Pabst, 1990; Koopman et al.,

2002; Pabst et al., 1999; Hamilton et al., 2004). Blubber is also often less dense than water,

aiding the body by decreasing the overall density of the body and therefore increasing its

buoyancy (Taylor, 1994; Kipps et al., 2002).

There are two different types of blubber: metabolic and structural (Ackman et al., 1965;

Ackman et al., 1971; Koopman et al., 2002; Struntz et al., 2004; Montie et al., 2008b). Metabolic

blubber is used for energy storage (Young, 1976; Lockyer, 1986; Ryg et al., 1988; Aguilar and

Borrell, 1990; Pond, 1998; Koopman et al., 2002; Montie et al., 2008b). In certain locations of

the body, such as the thoracic region, blubber is stratified in terms of its structure and its

biochemical composition. Histological analysis of adipocytes and structural fibers suggests that

the inner layers of blubber from the thoracic region of a number of cetacean species are

metabolically active, but that the outer layer of blubber from this region is structural (Ackman et

al., 1965; Ackman et al., 1971; Koopman et al., 2002; Struntz et al., 2004; Montie et al., 2008b).

In the metabolic blubber layer of odontocetes in good body condition, the adipocytes are larger

than those of the metabolically inert stores (Koopman et al., 2002; Struntz et al., 2004; Montie et

al., 2008b). During starvation, the metabolically active adipose tissue shrinks significantly, but

the metabolically inert (“structural”) blubber shows no change from that of a healthy cetacean

(Koopman et al., 2002; Struntz et al., 2004).

Blubber is also stratified biochemically. Lipid content varies throughout the depth of

blubber, with the highest lipid content generally found in the innermost and middle layers

(Ackman et al., 1975; West et al., 1979; Kakela et al., 1993; Kakela and Hyvarinen, 1996;

Koopman, 2007). Data from bottlenose dolphins suggests that, during lipid mobilization, lipids

are only mobilized from the middle and deep layers, with fat from the deep layer being

mobilized first (Struntz et al., 2004; Montie et al., 2008b). The blubber that is metabolically

“active” contains more fatty acids that are from a dietary origin, while the possibly inert

structural layer of blubber has a higher degree of fatty acids from an endogenous origin; such a

pattern of fatty acid distribution has been observed across a wide range of marine mammal

species including seals, walruses and other cetaceans (Ackman et al., 1965; Ackman et al., 1971;

Ackman et al., 1975; West et al., 1979; Lockyer et al., 1984; Aguilar and Borrel, 1990; Lockyer

et al., 1991; Kakela et al., 1993; Kakela and Hyvarinen, 1996; Koopman et al., 1996; Koopman

et al., 2002; Cooper, 2004; Iverson et al., 2004; Koopman, 2007).

All of the above properties and functions of blubber point to its being an important and

dynamic living tissue. Typically, adipose tissue is perfused by networks of capillaries,

microarterioles and microvenules (Gersh and Still, 1945; Herd et al., 1968; Hausman and

Wright, 1996; Crandall et al., 1997) so that the vascular system can transport molecules to and

away from the tissue with both nutrient and gas exchange occurring at capillaries (Campbell et

al., 1987). In terrestrial animals the adipose tissue is highly vascularized (Gersh and Still, 1945;

Herd et al., 1968; Hausman and Wright, 1996; Crandall et al., 1997) and can experience high

rates of blood flow (Rossell et al., 1974). Surprisingly there is little information on the

vascularization of blubber. One of the few studies that tried to analyze capillaries in blubber

(Parry 1949) was unable to do so and instead only briefly described large vessels travelling

through the blubber to the epidermis; this description is based upon a single harbor porpoise.

The virtual absence of data on vascular density, morphology and anatomy in blubber is

remarkable given the importance of this tissue and the fact that other aspects of blubber have

been very well studied (e.g. Bryden, 1968; Pond, 1978; Ryg et al., 1988; Worthy and Edwards,

1990; Kipps et al., 2002; Koopman et al., 2002; Montie et al., 2008). Many factors may influence

the microvasculature of blubber, such as thermoregulation, habitat, metabolic demands,

reproductive strategies, or phylogeny. Another unique aspect of marine mammal life to consider

is diving and the mammalian dive response. During a dive, marine mammals undergo

bradycardia, apnea, and ischemia, in which vasoconstriction is believed to control blood flow to

the peripheral tissues (Irving et al., 1941). If the blubber experiences peripheral

vasoconstriction, the blood supply to the blubber could be significantly reduced, if not stopped.

However, our ability to evaluate blubber’s behavior during diving, or the metabolic activity of

blubber, is hampered by a lack of foundational information on the relationship between this

tissue and the circulatory system.

Therefore the first step in considering the relationship between blubber and blood is to

characterize the vascular patterns in blubber. The overall goal of this study was to provide the

first detailed descriptions of the microvasculature (capillaries, microarterioles and microvenules)

in blubber, using the bottlenose dolphin (Tursiops truncatus) as a model. Tursiops is often used

as a model cetacean as much is known about the species (e.g. Scholander and Schevill, 1955;

Ridgeway et al., 1969; Noren et al, 1999; Meagher et al., 2002; Rommel et al., 2002; Wells and

Scott, 2002; Houser et al., 2004; Gannon et al., 2005; Harper et al., 2008). The first objective of

this study was to determine microvascular characteristics (density, vessel size, branching

patterns) using histological analysis. Vascular characteristics were then compared with other

lipid content, lipid class and fatty acid composition to determine whether the distribution of

blood vessels was linked to the biochemical features of blubber. Since the blubber of Tursiops

has been determined to be stratified by histological examination of adipocytes and structural

fibers (Struntz et al., 2004; Montie et al., 2008b) as well as biochemically (Samuel and Worthy,

2004; Koopman, 2006), I measured the microvasculature density in three layers of blubber: the

superficial layer (nearest to the epidermis), the middle layer, and the deep layer (nearest the

muscle), with the working hypothesis that there would also be stratification of the

microvasculature.

Although there are many potential factors controlling or affecting patterns of blood vessel

distribution in blubber, one influence that unites all marine mammals is the act of diving. The

second objective of this study was to compare the blubber microvasculature of Tursiops, a short-

duration (approximately 20-40 seconds), shallow diving (approximately 1-10 meters) cetacean

(Wursig, 1978; Irvine et al., 1981; Shane, 1990; Bassos, 1993), with that of the blubber of the

pygmy sperm whale, Kogia breviceps. Kogia makes dives to approximately 400 meters for

likely around 18 minutes which is a moderate dive duration (Scott et al., 2001; Beatson, 2007)

allowing a possible view of how dive depth may affect microvasculature density. The third

objective of this study was to put the cetaceans into a larger mammalian context by comparing

microvasculature of blubber with that of the subcutaneous adipose depots of a terrestrial

mammal, the domestic pig (Sus scrofa).

I hypothesized that the deeper divers would have reduced microvascular densities in their

blubber, given that these tissues would likely be experiencing chronic reduced perfusion during a

dive. Furthermore, at depth, diving mammals experience high pressure and consequently

elevated nitrogen partial pressures in their tissues (Serway and Faughn, 1992). Nitrogen is a

lipophilic molecule that is five times more soluble in lipid than blood and as such will move into

fat (Ikels, 1964; Gerth, 1985). Therefore, I reasoned that having a reduced amount of

microvasculature could be a means to allow deep divers to limit the amount of nitrogen that

could potentially enter the blubber. However, it may be possible that adipose tissue vascularity

is conserved across species and, with our choice of three diverse species we may elucidate this

relationship.

Data from this project will provide new and much needed data concerning the anatomy of

the microvasculature in blubber. It will also enhance our understanding of diving physiology

and provide insight into the specialized adaptations of being a marine mammal.

MATERIALS AND METHODS

Tissue Collection

Measurements were made on the thoracic blubber of six Tursiops truncatus, six Kogia

breviceps, two beaked whales (Blainville’s beaked whale, Mesoplodon densirostris and Cuvier’s

beaked whale, Ziphius cavirostris), and the subcutaneous layer of back fat from four pigs, Sus

Scrofa (see Table 1 and Figure 1). All odontocetes sampled had either stranded or had been

killed incidentally in commercial fishing operations, and only blubber obtained from animals

with a Smithsonian stranding code of 1 (live stranded and died or euthanized) or 2 (fresh dead)

were used in this study (Geraci and Lounsbury, 1993). All samples were from adult specimens

that were classified as having a normal body condition on the basis of an examination during

necropsy (Cox et al., 1998). All pig samples were obtained from a porcine butcher (Wells Pork

Products, Burgaw, NC) from animals that had been sacrificed for human consumption. After

collection all samples were wrapped in plastic and frozen at -20°C prior to further analysis.

Freezing effects on blubber are negligible (Pond and Mattacks, 1985). However, to

eliminate any possible freezing artifacts in blubber or pig fat samples, pieces selected for either

histological or lipid analysis were taken from the centre of the sample (i.e. not the edges touching

the sample storage bag) of the tissue. The epidermis was removed and the blubber section was

then cut into equal thirds to allow analysis of the samples based on blubber depth: superficial

(nearest the epidermis), middle, and deep (nearest the subdermal connective tissue sheath and

muscle) (Samuel and Worthy, 2004; Struntz et al., 2004; Montie et al., 2008b). Four samples

were removed from each layer, two for histological analysis of vessels: one in the longitudinal

plane and one in the transverse plane, one for histological analysis of adipocytes, and one for

lipid biochemistry.

Histological Analysis of Vessels

As this was the first study to quantify the microvascular densities of blubber, I first had to

develop the methodology. While numerous techniques have worked to distinguish vessels from

surrounding tissue, not all mammalian tissues nor those from different species respond the same

to various staining and histological techniques requiring us to attempt numerous techniques to

find a suitable method for detecting blood vessels in blubber. I attempted to visualize the vessels

in blubber using various lectins to stain for specific carbohydrates found on the cell surfaces of

endothelial cells (Alroy et al., 1986). The lectins that were used to try to identify the vessels in

blubber were GSL-I, RCA, and WGA, all of which have been shown to stain vessels in cats,

cows, dogs, goats, horses, mice, pigs, rats, sheep and humans (Alroy et al., 1986; Hausman and

Wright, 1996 ). Discerning the vessels in blubber was not possible with the lectins because the

lectins bound to adipocyte cell membrane components in addition to the vasculature. When

Tursiops samples of muscle and spleen were used, staining occured the same as it had for

blubber, making visulization of the vasculature difficult. Each lectin worked as previously

described on mouse tissues; however this method did not work with any dolphin tissues.

Antibodies known to bind with endothelial cells were also used to try to visualize the vasculaure

of blubber; trials were conducted with CD-31, MECA-32, and vWF on Tursiops blubber and

kidney samples; these are antibodies that bind endothelial cells in humans (CD-31, MECA-32,

vWF), mice (CD-31, MECA-32, vWF), rats (CD-31, vWF), guinea pigs (CD-31, vWF), rabbit

(CD-31, vWF), monkeys (CD-31), pigs (CD-31), and chickens (CD-31) (Developmental Studies

Hybridoma Bank, Iowa City, Iowa; Vector Laboratories, Inc., Burlingame, CA; Abcam, Inc.,

Cambridge, MA: Hallmann et al., 1985; Wong et al., 2000; Zanetta et al., 2000; Norrby and

Ridell, 2003; Young and Black, 2004). The antibodies all demonstrated non-specific binding in

blubber and when secondary binding was blocked with BSA, no binding occurred. The same

results were obtained with Tursiops kidney tissue with the exception of MECA-32, which did

have light staining of Tursiops kidney tissue and may be a possibility for viewing endothelial

cells in this tissue. When all of these techniques failed to reliably stain for blood vessels, I

turned to standard histochemical techniques. Techniques such as H&E, H&E with alcian blue,

PAF, and PAS were all tried (Groat, 1949; Parry, 1949; Gomori, 1950; Hausman and Thomas,

1983; Hausman and Richardson, 1983; Presnell and Schreibman, 1997), and none provided

sufficient contrast with the surrounding tissue to enable accurate blood vessel

counts/measurements. Finally, samples were incubated with nitro blue tetrazolium chloride plus

5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). NBT/BCIP stains for alkaline phosphatase,

which is located in the microarterioles, microvenules, and capillaries (Foley et al., 1954;

McComb and Bowers, 1979; Hansen-Smith et al., 1992) and has been used to view blood vessels

in people, rats, rabbits, pigs and other mammals (Foley et al., 1954; Wachstein and Meisel, 1959;

Cros et al., 1980; Hausman and Richardson, 1983; Werner et al., 1987). NBT/BCIP proved to be

a viable option for fresh, frozen tissue samples. According to Gomori, samples fixed with

alcohol or acetone and paraffin embedded at 56-60°C still have alkaline phosphatase activity

(Gomori, 1939; Presnell and Schreibman, 1997). However, when attempted with acetone fixed,

paraffin embedded tissues ,no staining was visible requiring us to use fresh, frozen tissue

sections.

Frozen, unfixed tissue samples were placed in a Leica Cryocut 1800 (Leica Microsystems

Inc., Bannockburn, IL), covered with Optimal Cutting Temperature Compound (OCT) (Ted

Pella, Inc., Redding, CA), and allowed to freeze to approximately -27°C (temperature

determined through preliminary trials with blubber samples). The samples were sectioned at

30μm thick and placed on a Superfrost Gold Plus slide (Fisher Scientific, Pittsburgh, PA). The

slides were then stored in the freezer at -20°C in a slide box enclosed in a freezer bag until

staining could be completed (not longer than 12 hours).

Samples were rinsed in Sorensen’s phosphate-buffered saline (PBS) for 15 minutes

(Presnell and Schreibman, 1997), and then incubated in a nitro blue tetrazolium chloride plus 5-

bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution made out of NBT/BCIP ready-to-use

tablets dissolved in 10ml of distilled water (Roche Diagnostics, Mannheim, Germany). Different

parameters, such as temperature, pH, and time can all affect the binding of NBT/BCIP to the

alkaline phosphatase enzymes found in the microvasculature (McComb et al., 1979). Roche

Diagnostics advises to incubate samples at room temperature and a pH of 9.5 (Roche

Diagnostics, Mannheim, Germany). Preliminary incubations of 20-30 minutes, which should

provide good staining intensity (Vector Laboratories, Inc., Burlingame, CA), did not provide

optimal staining for all species. This was obvious due to the faint coloration of the samples for

all of the species in the study except Tursiops. According to Vector Laboratories, Inc.,

lengthening incubation times provides significantly more sensitivity of NBT/BCIP (Vector

Laboratories, Inc., Burlingame, CA). Incubation times were therefore varied and optimal

incubation times were determined by a series of trials of increasing incubation times. Specific

times for each species were chosen based on qualitative analysis of stained sections. Incubation

times varied: 20 - 25 minutes for Tursiops, approximately two hours for Kogia and 36 hours for

beaked whales and pigs. This could be due to a number of reasons. There are different families

of alkaline phosphatases and these may vary among the test species. (McComb et al., 1979). The

species in the study may possibly have different concentrations of those enzymes in their

microvasculature (McComb et al., 1979). Furthermore, NBT/BCIP is "little soluble in water and

lipid" (Vector Laboratories, Inc., Burlingame, CA), and the incubation times needed for the

species analyzed in this study correlated with the percent lipid of the tissue samples (the tissue

with the higher percent lipid had the longest incubation times; see Table 3; Figure 10). The

samples were rinsed in PBS for seven minutes and placed under coverslips with trisglycerol

(Presnell and Schreibman, 1997).

Stained sections were viewed with an Olympus BX60 (Olympus America Inc., Center

Valley, PA) and digital pictures were taken of each section using a Diagnostic Instruments SPOT

RT digital camera (Diagnostics Instruments, Sterling Heights, MI). All images were analyzed

using Image ProPlus software (Media Cybernetics, Inc., Bethesda, MD). All vessel image

analyses were carried out on sections cut from two orthogonal body planes (longitudinal and

transverse) and then averaged together to eliminate any possible orientation bias between the two

planes.

To calculate percent vascularity and describe vascular branching, one image was captured

from five different, noncontiguous tissue sections for each layer of each sample using a 4x

objective. Percent vascularity of each layer was determined by counting each vessel that touched

the intersecting point of the horizontal and vertical lines of a proportional, orthogonal grid

consisting of eleven horizontal and 15 vertical grid lines, which was overlaid on each image in

Image ProPlus (Howard and Reed, 1998). This grid size allowed for a minimum count of 100

vessels when the counts from each of the 5 tissue sections were pooled.

Vascular branching was determined by counting the total number of terminal end points

for the first five vessels that touched a grid intersection for each of the five noncontiguous tissue

section images (see Figure 2). This resulted in the number of branches per vessel being counted

for 25 vessels, which were averaged to determine the number of branches per vessel for each

layer.

Images were captured with a 10x objective for all vessel diameter measurements and

diameters were measured for each layer by using the linear measurement tool in Image ProPlus.

Vessels were found under a microscope and then followed (in the x, y, and z planes) until the

vessel was cut. If the terminal point of the vessel had been cut in a cross section then the vessel

was used to determine vessel diameter. The first ten vessels that were found cut in cross section

were measured for each layer and the average of those measurements was used to determine

vessel diameter for that layer.

Lipid Analysis

Lipid was extracted from blubber subsamples weighing approximately 0.5 g using a

modified Folch method (Folch et al., 1957; Koopman et al., 1996) to yield percent lipid (wet

weight). Lipid classes [triacylglycerols (TAG), wax esters (WE), cholesterol and phospholipids]

were identified and quantified using a Mark VI Iatroscan TLC/FID (Mitsubishi Kagaku Iatron,

Inc, Tokyo, Japan). Samples were developed in a solvent system of 94/6/1 hexane/ethyl

acetate/formic acid. Lipid class peaks were integrated and quantified using Peak Simple 329

Iatroscan software (SRI Instruments, Torrance, CA). Identification of peaks was confirmed using

known standards (NuChek Prep, Elysian, MN) of WE, TAG, free alcohols, phospholipids,

cholesterol and diacylglycerols (see Koopman, 2007). Fatty acids and alcohols of lipid class

components were analyzed using temperature-programmed capillary gas-liquid chromatography

(GC) of butyl esters on a Varian 3800 GC (Varian, Inc., Palo Alto, CA). Components were

separated and analyzed with a flame ionization detector (ID) in a fused silica column (30 x

0.25mm ID) (Zebron FFAP; Phenomenex). Helium was used as the carrier gas and the gas line

was equipped with an oxygen/water scrubber. The following temperature program was used:

65°C for 2 min, hold at 165°C for 0.40 min after ramping at 20°C/min, hold at 215°C for 6.6 min

after ramping at 2°C/min, and hold at 250°C for 5 min after ramping at 5°C/min. Identities of

individual components were confirmed using prepared standards (NuCheck Prep, Elysian, MN)

and GCMS (Trace GC Ultra coupled to a Polar Q mass spectrometer, Thermo Electron

Corporation with X calibur software) (Suzanne Budge, Dalhousie University). Fatty acids and

fatty alcohols were described using the nomenclature of A:Bn-X, where A is the number of

carbon atoms, B is the number of double bonds, and X is the carbon position of the double bond

closest to the terminal methyl group. These results were integrated with Galaxie (version Varian

1.8.501.1) GC software.

Fatty Acid Stratification

To determine fatty acid (FA) stratification in the blubber a stratification index (SI) was

computed (Koopman, 2007). To calculate the SI, the absolute values of the differences between

deep and superficial layers in a subset of 16 select FA were added together. The 16 FA used

were iso5:0, 14:0; 14:1n-5; 16:0; 16:1n-7; 18:0; 18:1n-11; 18:n-9; 18:1n-7; 18:2n-6; 20:1n-11;

20:1n-9; 20:5n-3; 22:1n-11; 22:5n-3; and 22:6n-3 (Koopman, 2007). These FA were chosen to

compare the SI of this study to those calculated by Koopman (2007).

Statistics

To determine whether there were species differences, or differences associated with depth

of blubber layer in the variables measured, univariate analysis of variance (general linear model)

were conducted on all variables. Repeated measures tests were used to account for the fact that

each individual animal was sampled multiple times (three layers) (SPSS, 1997; Tabachnick and

Fidele, 2007). The within subject factors examined were percent vascularity, number of vascular

branches, vessel diameter, percent lipid, lipid class composition, and stratification index. If there

was significant interaction between depth and species effects, then repeated measures tests were

run separately on each layer to determine a species effect, and on each species to determine a

layer effect.

For each ANOVA, the data were tested for sphericity using Mauchly's test of sphericity.

If sphericity could not be assumed then the Greenhouse-Geisser correction factor, a conservative

value for low n-numbers, was applied to the F-statistic when testing for significance (SPSS,

1997; Tabachnick and Fidele, 2007). For each test, groups were evaluated for equality of

variance using Levene’s test. When within-subject effects were significant, post-hoc tests were

carried out to determine where differences existed using either a Tukey post-hoc (equal

variances) test or a Tamhane T-2 test (unequal variances).

All stastistical tests were carried out using SPSS (SPSS Inc., Chicago, IL). All means are

presented with SEM.

RESULTS

Histological Analysis of Vessels

Blubber microvasculature in Tursiops truncatus was stratified throughout the depth of the

blubber (p<0.001). On average, the superficial layer contained approximately 7% fewer

microvessels than the middle and deep layers (p<0.001), which were not significantly different

from each other (Figure 4; Table 2; p=0.073). This pattern was not found in either Kogia

breviceps or in Sus scrofa (pigs). Kogia showed an insignificant (p=0.160) trend of increasing

microvaculature density from superficial to deep blubber (Figure 5; Table 2). There was no

difference (p=0.898) in the density of the microvasculature of pig subcutaneous back fat based

on depth (Figure 6; Table 2). Two beaked whales that had been analyzed (M. densirostris, n=1;

Z. cavirostris, n=1) had similar microvasculature densities to each other and to the pigs, with no

obvious difference observed throughout the depth in either species (note that statistics were not

performed on beaked whales due to only single individuals being examined) (Table 2).

When comparing across species, Tursiops had greater microvasculature densities in their

middle and deep blubber layers compared to those layers in Kogia (p=0.005), pigs (p<0.001), or

the two individual beaked whales analyzed (Figure 7). Kogia had greater blubber

microvasculature density than either the pigs (p=0.017) or the beaked whales (Figure 7). The

blubber microvasculature densities of the two beaked whales mirrored the microvasculature

density found in pig fat (Figure 5).

There were no significant species (p=0.157) or depth (p=0.176) related differences in the

diameter of the microvessels (Table 2; Figure 8). The average range of vessel diameter size was

generally between 9 – 11 micrometers.

In Tursiops, there were significantly more branches per microvessel in the deep (5.55

branches/microvessel) and middle (5.76 branches/microvessel) layers than in the superficial

(2.75 branches/microvessel) layer (Figure 9; Table 2; p=0.006); this was the only species to

exhibit stratification in branching and the only species to differ from the others in these layers

(p=0.015). All layers of the remaining species contained an average of 2.92 branches/vessel

(Figure 9; Table 2) with no significant differences among Kogia or the pigs (p=0.761), and the

beaked whales followed a similar trend.

Lipid Analysis

In Tursiops, lipid content was stratified throughout the depth of the blubber (p=0.039).

The middle layer, consisting on average of 59.9% wet weight lipid, had more lipid than did the

superficial layer, 39.4% (p=0.001), and the deep layer, 41.9% (Figure 10; Table 3; p=0.001).

Kogia blubber was also stratified in the amount of lipid per layer, with the superficial layer

(consisting of 33.7% lipid), having fewer lipids than the middle (75.1%; p=0.010) and deep

layers (69.2%; Figure 10; Table 3; p=0.030). There was no difference (p=0.683) in the amount

of lipid in pig fat based on depth (Figure 10; Table 3). There was also no obvious difference in

the amount of lipid throughout the depth of blubber in either of the beaked whales (Figure 10;

Table 3).

Tursiops blubber and pig fat contained only triacylglycerols (Table 3). The blubber of

beaked whales contained around 98% wax esters and less than 1% triacylglycerols (Figure 11;

Table 3). Kogia blubber was, on average, 75% wax esters and 22% triacylglycerols (Figure 11;

Table 3). There was no stratification of lipid type in any species (Figure 11; Table 3; all p>0.05).

Overall the FA Stratification Index (SI) showed considerable stratification in fatty acids

in Tursiops (p<0.0001), Kogia (p=0.001), and the beaked whales (see Table 4; Figures 12); there

was no difference in FA stratification between Tursiops and Kogia (p=0.147). There was less

stratification of fatty acids in pig adipose tissue than for Tursiops or Kogia (p= 0.009; see Table

4; figure 12). In all odontocetes examined, dietary fatty acids occurred in the highest

concentrations in the deep layers of the blubber, and decreased towards the superficial layers.

Fatty acids with an endogenous origin decreased from the superficial to the deep layers of

blubber.

DISCUSSION

This study provides the first quantitative description of microvessels in the blubber of any

marine mammal. Given the importance of this tissue for these animals, this is surprising.

Studies on the adipose tissue in rats, dogs, fetal pigs and humans have all shown that each

adipocyte is in contact with at least one capillary (Gersh and Still, 1945; Herd et al., 1968;

DiGiralamo et al., 1971; Rossell and Belfrage, 1979; Hausman and Richardson, 1983; Hausman

and Thomas, 1985; Anstrom et al., 2004; Hausman and Richardson, 2004). However, because

blubber is a highly specialized form of adipose tissue it cannot be assumed that blubber

vasculature is the same as that found in typical mammalian adipose tissue.

Vascular Distribution and Morphology

The microvasculature densities of pig adipose tissue found in this study were nearly

identical to those of other terrestrial mammals, mice and humans (Table 6; Lijnen et al., 2006;

Hemmeryckx et al., 2008; Lijnen et al., 2009; Pasarica et al., 2009). It therefore seems likely

that the microvasculature density of adipose tissue is conserved in terrestrial mammals.

Furthermore, this same relatively low density was found in the superficial layer across three

families of odontocetes (Delphinidae, Kogiidae, Ziphidae) suggesting some level of conservation

in the vascularity of fat that is located closest to the epidermis. The same vascular characteristics

(density, branching) observed in the superficial layer of fat were maintained throughout the depth

the blubber of both beaked whales, making this tissue fairly uniform in these species. However,

some deviations from this pattern were observed in Tursiops, and to a lesser degree in Kogia.

Tursiops showed the greatest divergence from the other species. The middle and deep

layers of blubber in Tursiops had significantly higher microvascular densities, and the

microvessels in these layers also had significantly more microvascular branching, than did all of

the other species. Thus, it is the middle and deep layers of Tursiops blubber that showed the

greatest departure from the other mammals in this study and in the literature.

In Kogia there was a slight but statistically insignificant increase in vascular density

towards the inner layers of blubber. One of the Kogia analyzed (Kb03) exhibited a dramatically

different pattern of vessel distribution than the other conspecifics (Figure 5). Excluding this

individual from the statistical analysis made the trend of greater vascular density in the inner and

deeper layers, compared to the superficial layer, significant (p=0.031). Thus, it is likely that

Kogia blubber does exhibit an increase in vascular density in the innermost blubber layers,

although not as pronounced as that observed in Tursiops.

Parry (1949) was unable to identify any capillaries in the blubber of a harbor porpoise

(Phocoena phocoena). Other studies that have examined the portion of the microvascular system

that lies close to, but not in, the blubber of marine mammals have concentrated on the vessels in

the appendages that are believed to contribute significantly to the animals’ thermoregulatory

abilities (Fawcett, 1942; Tomilin, 1957; Palmer and Weddell, 1964; Elsner, 1969; Elsner et al.,

1974; Bryden and Molyneux, 1978). However, the microvessels located within the blubber itself

have been largely overlooked.

The diameter of the microvessels was the same for all species in this study. The average

diameter was between 9 and 12 micrometers with no clear patterns throughout the depth in any

species. This result is not surprising, because the size of capillaries is likely constrained based

on the size of a red blood cell, which in humans falls between 6-9 m (Persons, 1929). This

result confirms that capillary size is conserved across species, at least in mammals (Hausman and

Richardson, 1983; Anstrom et al., 2004; Ross and Pawlina, 2006).

Relationship of Vascular Patterns to Biochemical Characteristics of Blubber

I hypothesized that I would see correlations between the blubber microvasculature and

blubber biochemical characteristics because the vascular system transports molecules, such as

fatty acids (Campbell et al., 1987), to and away from the blubber. Much more is known about

the biochemical characteristics of blubber, not only for the species in this study but for other

marine mammals in general, than on the vascular properties of blubber. In general, the

biochemical data obtained here agree with previous studies on the physiological aspects of

blubber of odontocetes (Struntz et al., 2004; Samuel and Worthy, 2004; Montie et al., 2008b;

Koopman, 2007).

In this study, the blubber of Tursiops was biochemically stratified among the superficial,

middle and deep layers in terms of total lipid content (see also Samuel and Worthy, 2004;

Montie et al., 2008b; Koopman, 2007), as well as in microvascularity. While the middle and

deep layers of blubber had higher microvascular densities than the superficial, the percent lipid

did not match this pattern. The middle had significantly more lipid than the superficial layer and

deep layers of blubber. Other studies have shown that the superficial layer of blubber in

Tursiops is structural, while the middle and deep layers are metabolic (Struntz et al., 2004;

Montie et al., 2008b). These studies have also suggested that the middle layer forms a more

stable lipid store, and that it is the adipocytes in the deep layer that are accessed first with

adipocytes in that layer showing more variety in cell size/volume (i.e. lots of small and large

cells) than in the middle layer (mostly large cells) and the superficial layer (mostly small cells)

(Struntz et al., 2004; Montie et al., 2008b). Thus in Tursiops the microvascular densities are

highest in the blubber layers where lipid thought to be primarily mobilized, and lowest in the

superficial layer of blubber where little or no lipid mobilization is believed to occur.

In agreement with past work, this study also showed that Kogia breviceps blubber was

stratified in terms of lipid content, with the superficial layer having fewer lipids than the middle

and deep layers (Koopman, 2007). In Kogia the microvascularity exhibited a slight increase

through the depth of the blubber. As mentioned above, higher microvascular densities in

Tursiops were found in the layers of blubber that are thought to be more metabolically active.

While much less is known about the blubber of Kogia, the slightly higher vascular densities in

the middle and deep layers may be due to lipid being mobilized more frequently in these layers

than the in the superficial layer. However the microvascular density differences are much closer

among all three layers in Kogia than in Tursiops, possibly suggesting that there are less

differences among the layers in how lipid is mobilized.

The blubber of M. densirostris and Z. cavirostris has not been previously analyzed for

differences in lipid content between outer and inner blubber layers. However, Koopman (2007)

analyzed the blubber of two other species of beaked whales, M. bidens and M. europaeus, and

found no stratification in lipid content between those layers. This study showed fairly uniform

lipid content among all layers in the blubber of M. densirostris and Z. cavirostris. It also showed

a uniform amount of microvascularity throughout the depth of the blubber in these two species.

There was, overall, an inverse relationship between lipid content and microvascular

densities of each species. The species with the highest lipid content (the beaked whales and

pigs) had the lowest microvascular densities. The species with the lowest lipid content

(Tursiops) had the highest microvascular densities; Kogia was intermediate in both categories.

This relationship was not due to the vasculature limiting the space that could be occupied by

lipid, as the small difference (~7.5%) in vascular densities cannot account for the larger

differences seen in lipid content (~49%).

Previous studies have shown that in odontocetes, lipid class composition of blubber is

strongly linked to phylogeny (e.g. Litchfield et al, 1975; Koopman, 2007). In agreement with

previous studies, the blubber of Kogia and both beaked whales was dominated by wax esters

(WE). The blubber of Tursiops and the adipose of pigs only contained triacylglycerols (TAG).

A prevalence of WE seemed to be associated with lower vascular densities. The blubber of

beaked whales and Kogia (both WE-dominated) had low vascular densities, but pigs, which

contained only TAG, had vascular densities similar to beaked whales and, in fact, lower than

Kogia. This comparison suggests that the correlation between vascularity and WE is more likely

a coincidence or an indirect relationship and that lipid class likely does not influence

microvascularity.

The final biochemical parameters I measured were fatty acid composition and fatty acid

stratification. The stratification indices I observed were slightly higher than those previously

reported by Koopman (2007). However, this result was not surprising because Koopman (2007)

separated the blubber into only two layers (thus providing a less extreme comparison), while this

study looked at the more dramatic differences between the deep and superficial blubber layers.

The blubber of all of the cetaceans was stratified to varying degrees, but the pig adipose showed

no stratification. The pig adipose tissue also contained many fewer fatty acids than any of the

cetaceans, which is not all that surprising given that the dietary intake of these animals was

constrained as they were not wild or free-ranging, and the fact that marine systems tend to

contain a diverse array of a larger number of fatty acids (Budge et al. 2006). There was no

obvious correlation between fatty acid stratification and microvascularity (data not shown). This

result was very surprising because initially I had thought that microvascular stratification may be

one cause of the fatty acid stratification. While it might be logical to assume that some aspect of

the vasculature was linked to controlling fatty acid stratification, it may be that the two are

unrelated and that there is another mechanism, or mechanisms (e.g. differential enzyme activity

or expression), controlling the fatty stratification found in blubber. At the current time our lack

of knowledge concerning the biochemical mechanisms of fatty acid deposition in adipose tissue,

as well as the limited number of animals and species analyzed in this study, precludes our ability

to determine the exact nature, if any, of a relationship between vascularity and fatty acid

stratification.

Potential Influences on Blubber Vasculature Structure

We have a generally limited understanding of the biology, behavior and physiology of the

majority of odontocete species, particularly those with pelagic or deep-diving lifestyles. Without

adequate ancillary data it is difficult to make any strong inferences or conclusions about which

factors (environmental, biological, phylogenetic) might have the greatest influences on

microvascular characteristics of blubber. However, I do wish to explore some possible causes

and correlaries of the data, with the caveat that these are merely potential relationships that we

cannot fully evaluate at this time.

I had originally assumed that blubber’s microvascular patterns would be associated with

the diving characteristics (depth, length) of a given species. Given the constraints associated

with the dive response, I hypothesized that the deeper divers would have reduced microvascular

densities. At depth, diving mammals experience high pressure and consequently elevated

nitrogen partial pressures in their tissues (Serway and Faughn, 1992). Nitrogen is a lipophilic

molecule that is five times more soluble in lipid than blood and as such will move into fat (Ikels,

1964; Gerth, 1985). Therefore, I reasoned that having a reduced amount of microvasculature

could be a means to allow deep divers to limit the amount of nitrogen that could potentially enter

the blubber. Tursiops, the shallowest diver in the study, had the greatest amount of

microvasculature, and also the highest degree of microvascular stratification. Kogia, an

intermediate diver, had a lower amount of microvasculature. The deepest divers in the study, the

beaked whales, had the least amount of microvasculature. Even though only a limited number

of species have been analyzed to date, it would seem that microvascular densities seem to

correlate with dive depth, at least in odontocetes. However, after analyzing the microvascular

densities of adipose tissue in a terrestrial mammal, a pig, and comparing these with literature

values from other terrestrial mammals, this idea loses support (Table 6; Lijnen et al., 2006;

Hemmeryckx et al., 2008; Lijnen et al., 2009; Pasarica et al., 2009). The terrestrial mammals

had the same amount of microvasculature as the deepest divers (the beaked whales), suggesting

that lower microvascular densities may be the “ancestral” mammalian condition and remain

conserved in beaked whales. The "reduced vasculature" seen in the deep divers may be the more

conserved state, while the blubber microvasculaure of Tursiops seems to show the greatest

alteration from the typical mammalian pattern. These data may well indicate that rather than

thinking about Tursiops as a “model” ooontocete, we should really regard Tursiops as a species

showing significant departures from members of other families of odontocetes and other

mammals, at least in terms of its blubber vasculature. A logical extension is then to consider

possible reasons for the observed increased vasculature density and branching in Tursiops.

The coastal Tursiops utilized in this study, live in a coastal environment where they

experience many seasonal changes; water temperatures fall from around 30˚C in summer to

around 11˚C in winter and availability of prey also changes, making food supply less consistent

(Irvine et al. 1981; Wells et al., 1987; Thayer et al., 2003; Barros and Odell, 1990; Cockcroft and

Ross, 1990; Barros and Wells, 1998). Thus these animals must cope with variation in both

energy availability and thermoregulatory challenges imposed by seasonal fluctuations. This

higher degree of environmental variation is reflected in the seasonal mobilization of energy from

Tursiops truncatus blubber (Samuel and Worthy, 2004; Meagher et al., 2008). This study

showed that the microvascular density is amplified in the blubber of Tursiops, possibly

enhancing their ability to adapt to both the changing energy availability and temperature of their

coastal habitat. In summer, fish are abundant, Tursiops are able to consume more and increase

their blubber stores in preparation for winter (Barros and Odell, 1990; Cockcroft and Ross 1990;

Barros and Wells, 1998). In winter, a thick insulating layer is important in preventing heat loss,

the additional lipid stores can provide energy when food is unpredictable (Barros and Odell,

1990; Cockcroft and Ross 1990; Barros and Wells, 1998; Samuel and Worthy, 2004; Dunkin et

al., 2005; Meagher et al., 2008). By summer, lipid has been mobilized from the middle and deep

blubber layers decreasing the thickness of the blubber by 39% (Samuel and Worthy, 2004;

Meagher et al., 2008).

In contrast, Kogia live and forage in the deep waters of a pelagic environment (Caldwell

and Caldwell, 1989; Scott et al., 2001; Beatson, 2007). In this environment seasonal effects are

minimized, as the temperatures faced are more consistent at depth. In these more stable waters,

Kogia feed on pelagic cephalopods, which migrate vertically in the water column on a daily basis

and are available year round (Raun et al., 1970; Roper and Young, 1975; Martins et al., 1985).

In addition, there is no published evidence of seasonal changes in the blubber of Kogia, and

some researchers believe that the blubber of Kogia does not change seasonally (personal

correspondance with W.A. McLellan). This study found that Kogia had a lower microvascular

density that consisted of vessels with fewer branches than seen in Tursiops. Perhaps a more

energetically and thermally stable environment did not place the same selective pressure on

Kogia to have enhanced “access” to their blubber energy stores.

Beaked whales also live in the more energetically and thermally stable pelagic

environment and also feed on pelagic cephalopods (Heyning, 1989; Mead, 1989). As with

Kogia, there is no evidence in the literature or from whaling records that beaked whales use their

blubber lipids for energy. Although this study was limited to only two beaked whales, and those

of two different species, their blubber microvascular densities and branching patterns were not

significantly different than those found in terrestrial mammals (Lijnen et al., 2006; Hemmeryckx

et al., 2008; Lijnen et al., 2009; Pasarica et al., 2009), again lending support to the idea that they

have retained the typical mammalian characteristics of adipose vasculature in their blubber in the

absence of selection pressures for needing to mobilize blubber lipids as part of their life history

strategies.

Additional support for the idea that Kogia and the beaked whales may not make

significant use of their blubber for energy can be found in the lipid classes of the species in this

study. The blubber of Kogia and the beaked whales consisted predominantly of waxes. These

animals all had significantly less microvasculature than Tursiops, whose blubber consists solely

of TAG. While the specific reasons for the presence of waxes in the blubber of the deepest-

diving odontocetes have not yet been established, we do know that mammals are typically

incapable of digesting WE (Savory, 1971; Place, 1992; Pond, 1998). Thus, it is possible that

these animals may not be metabolizing the WE once they have been stored in the blubber,

supporting the hypothesis that Kogia and beaked whales are not mobilizing lipid from their

blubber to the same extent as Tursiops. The blubber of Kogia actually consisted of more TAG

than that of the beaked whales (see Table 3). If the idea that blubber mobilization occurs

primarily for TAG is true, then conceivably, Kogia may be mobilizing more lipid from their

blubber than the beaked whales. Such a concept is supported by the vascular data, which suggest

that, at least in the middle and deep layers of blubber, more deposition and mobilization of lipids

may be occurring in Kogia.

Despite the species differences described above, it is quite remarkable that all the

odontocetes in this study had the same microvascular densities, and number of vascular

branches, in the superficial layer of blubber. The conservative nature of vessels in the superficial

layer suggests that there may be constraints on vasculature in the outermost blubber layer, and I

hypothesize that this may be linked to thermoregulation. As a consequence of living in a coastal

habitat in temperate waters, Tursiops experiences significant fluctuations in water temperature.

During the winter, the water in the coastal habitat of Tursiops falls to about 11˚C (Irvine et al.

1981; Wells et al., 1987; Thayer et al., 2003). Kogia and the beaked whales live in a more

consistent environment, with greater exposure to colder water as a function of their deep diving

behavior (Caldwell and Caldwell, 1989; Scott et al., 2001; Beatson, 2007). A thick, lipid rich

layer of insulation is highly important for all species (Worthy and Edwards, 1990; Struntz et al.,

2004; Dunkin et al., 2005; Meagher et al., 2008). It could be that the thermoregulatory need to

prevent heat loss acts as physiological constraint for the amount of microvessels in the

superficial layer of blubber. The thermoregulatory consequences of increased vasculature in the

superficial blubber would be two-fold. First, blood travelling through vessels nearer the surface

of the body would lead to heat loss to the environment (Scholander and Schevill, 1955; Elsner et

al., 1974; Meagher et al., 2008). Second, if increasing the number of capillaries in the superficial

layer of blubber would cause the lipid in that layer to be mobilized, then an important layer of

insulation would be reduced allowing even more heat loss to the environment.

Another possible physiological constraint preventing increased microvascular densities

and metabolic use of the superficial layers of blubber might be the maintenance of a streamlined

body shape. By having a layer of blubber that is not metabolized surround the body, odontocetes

may maintain their streamlined form, which reduces the drag forces acting upon their bodies and

thus decreasing the energy needed when swimming (Ryg et al., 1988; Pabst, 1990; Koopman et

al., 2002; Pabst et al., 1999; Hamilton et al., 2004).

Conclusions and Future Directions

Here I have provided the first detailed, quantitative description of the microvasculature in

the blubber of marine mammals. Although the distribution and characteristics of blubber

microvasculature in this study did correlate with dive depth when only the marine mammals

were considered, the strong similarities across vasculature patterns in terrestrial mammals and all

odontocetes except Tursiops make it more likely that the microvascular densities are not directly

related or the consequence of dive depth. Rather, it seems plausible that the microvascular

densities observed here instead reflect varying degrees of lipid mobilization, an idea that begs

further investigation. Our limited understanding of the species analyzed and the limited number

of species used in this study prevents us from fully evaluating the relationships between the

microvasculature of blubber and the biology and life history of marine mammals.

This study has provided a good foundation for future work on vasculature in marine

mammals. In addition, it has raised many questions. To test the hypotheses formed in this study,

and to elucidate the relationships between blubber vasculature and food supply, lipid

mobilization, phylogeny, and other environmental conditions. The first goal of future studies

should be to analyze more species with consideration of the influence of phylogeny, life history

strategies, habitat, behavior, thermoregulation, and energy demands. In addition, analysis of a

series of life history stages within a species would allow us to evaluate how blubber vasculature

develops. Finally, placing these data within the broader mammalian context, including not only

the closely related baleen whales, but also the unrelated pinnipeds and sirenians, would provide

significant insight into evolutionary patterns and shared constraints across these convergent

species.

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Table 1. Specimen information for individual animals used in this study. Species abbreviations

are as follows: Tt, Tursiops truncatus; Kb, Kogia breviceps; Md, Mesoplodon

densirostris; Zc, Ziphius cavirostris; Ss, Sus scrofa.

Species Species ID Age Sex Stranding Location Stranding Date

Tt01 C163 Adult F San Luis Obispo, CA 9-22-2002

Tt02 WAM631 Adult F North Topsail Beach, NC 3-3-2007

Tt03 WAM633 Adult F Sneads Ferry, NC 7-7-2007

Tt04 BRF061 Adult M Nags Head, NC 4-5-2006

Tt05 WAM574 Adult F Shallote, NC 11-11-2001

Tt06 WAM573 Adult F Masonboro Inlet, NC 11-11-2001

Kb01 C71 Adult M Monterey County, CA 5-26-1997

Kb02 C67 Adult M San Mateo, CA 12-17-1996

Kb03 BRF092 Adult F Outer Banks, NC 9-1-2006

Kb04 ASF028 Adult F Masonboro Island, NC 9-13-1998

Kb05 KMS427 Adult F Outer Banks, NC 9-2-2006

Kb06 KMS429 Adult M Outer Banks, NC 9-2-2006

Ss01 Pig01 Adult Na Burgaw, NC 4-2009

Ss02 Pig02 Adult Na Burgaw, NC 4-2009

Ss03 Pig03 Adult Na Burgaw, NC 4-2009

Ss04 Pig05 Adult Na Burgaw, NC 4-2009

Md01 WAM593 Adult M Kure Beach, NC 1-28-2004

Zc02 WAM536 Adult F Long Beach, NC 5-13-1998

Table 2. Microvascular characteristics of blubber in the superficial (S), middle (M), and

deep (D) blubber layers within Tt, Tursiops truncatus; Kb, Kogia breviceps;

Md, Mesoplodon densirostris; Zc, Ziphius cavirostris; and subcutaneous fat of

Ss, Sus scrofa. All values represent means ± SEM.

Percent microvascularity

Tta

Kbb

Ssc

Md Zc

S 3.260±0.391* 3.257±0.526 1.758±0.225 2.770 1.610

M 8.398±0.647 4.377±0.551 1.873±0.223 2.320 1.530

D 9.314±0.723 4.500±0.543 1.883±0.458 1.790 1.990

Microvascular diameter

Tta

Kba

Ssa

Md Zc

S 10.910±0.300 10.655±0.300 10.020±0.280 9.238 10.191

M 9.557±0.171 10.924±0.171 9.825± 0.156 9.091 9.604

D 10.143±0.522 10.534±0.522 9.783± 0.477 9.018 9.824

Microvascular branching

Tta Kb

b Ss

b Md Zc

S 2.753±0.112* 2.917±0.133 2.810±0.059 2.920 2.580

M 5.763±0.639 2.927±0.301 2.635±0.043 2.800 2.540

D 5.547±0.543 2.777±0.148 2.900±0.160 2.440 2.460

* Denotes significant difference among blubber layers/depths. a,b,c

Denotes significant difference among species.

Table 3. Biochemical characteristics of blubber in the superficial (S), middle (M), and

deep (D) blubber layers within Tt, Tursiops truncatus; Kb, Kogia breviceps;

Md, Mesoplodon densirostris; Zc, Ziphius cavirostris; and subcutaneous fat of

Ss, Sus scrofa. All values represent means ± SEM.

Percent lipid (wet weight)

Tta Kb

b Ss

c Md Zc

S 39.4±3.5 33.7±4.1* 79.0±2.3 79.7 78.3

M 59.9±3.7* 75.1±2.6 76.8±4.2 81.8 83.0

D 42.0±7.2 69.2±3.1 79.2±3.0 83.9 78.7

Percent lipid composed of triacylglycerols

Tta Kb

b Ss

a Md Zc

S 99.3±0.2 24.2±10.3 99.4±0.3 0.6 0.0

M 97.8±1.1 18.9±7.8 99.6±0.2 1.7 0.0

D 97.1±2.4 23.3±10.6 99.8±0.1 10.8 0.4

Percent lipid composed of wax esters

Tta Kb

b Ss

a Md Zc

S 0.0 74.4±6.7 0.0 98.6 97.7

M 0.0 80.6±5.0 0.0 98.3 97.1

D 0.0 74.2±6.7 0.0 85.3 99.1

* Denotes significant difference among blubber layers/depths. a,b,c

Denotes significant difference among species.

Table 4. Average Stratification Index (SI) for fatty acids in the blubber/adipose tissue within

Tt, Tursiops truncatus; Kb, Kogia breviceps; Md, Mesoplodon densirostris;

Zc, Ziphius cavirostris; Ss, Sus scrofa. All values represent means ± SEM.

Tta Kb

a Ss

b Md Zc

SI 29.568±2.838 21.455±3.572 5.455±0.727 40.072 23.097

a,b,c Denotes significant difference among species.

Table 5. Select fatty acids and fatty alcohols found in the superficial (S), middle (M), and deep (D) layers of blubber/adipose tissue

in Tt, Tursiops truncatus; Kb, Kogia breviceps; Md, Mesoplodon densirostris; Zc, Ziphius cavirostris; Ss, Sus scrofa.

All values represent means ± SEM.

Tt Kb

Fatty acid S M D S M D

iso 5 1.868±0.663 1.193±0.531 0.618±0.254 0.000 0.000 0.000

14:0 4.645±0.612 5.085±0.506 5.613±0.801 5.045±0.228 5.317±0.220 5.621±0.623

14:1n-5 2.595±0.350 1.277±0.327 0.808±0.289 1.186±0.082 0.894±0.065 0.642±0.097

16:0 6.934±0.941 10.752±0.319 13.147±0.401 11.147±1.186 11.027±0.871 11.191±0.842

16:1n-7 23.508±2.580 18.835±2.048 15.967±1.896 15.861±0.457 12.758±0.797 9.713±1.157

18:0 1.542±0.191 2.355±0.490 2.947±0.269 1.427±0.129 1.399±0.317 1.884±0.228

18:1n-11 0.965±0.263 1.135±0.307 0.917±0.271 0.525±0.382 0.472±0.298 0.343±0.193

18:1n-9 21.137±1.344 19.877±1.239 19.508±1.017 38.368±1.645 37.660±1.908 37.239±2.520

18:1n-7 3.444±0.196 3.582±0.203 3.592±0.213 1.834±0.222 1.755±0.236 1.553±0.275

18:2n-6 1.192±0.046 1.152±0.070 1.157±0.085 0.659±0.083 0.786±0.097 0.612±0.148

20:1n-11 0.670±0.233 0.767±0.289 0.848±0.295 1.568±0.728 2.112±0.991 2.452±0.939

20:1n-9 1.478±0.634 1.713±0.709 1.977±0.710 4.578±0.735 6.470±1.300 8.171±1.567

20:5n-3 3.700±1.006 4.042±0.806 3.625±0.759 0.385±0.120 0.637±0.225 0.674±0.249

22:1n-11 0.618±0.346 0.640±0.448 1.144±0.553 1.990±1.281 3.245±1.941 4.146±2.466

22:5n-3 2.649±0.235 3.405±0.166 3.515±0.215 0.153±0.055 0.246±0.072 0.313±0.064

22:6n-3 6.861±1.641 8.938±1.865 9.810±1.911 0.437±0.115 0.932±0.487 1.209±0.606

Abundant fatty alcohol

alc 16:0 0.000 0.000 0.000 7.587±1.047 9.882±0.999 10.362±1.449

alc 18:1n-9 0.000 0.000 0.000 14.872±2.528 14.622±1.921 13.177±2.065

Table 5 cont.

Ss

Fatty acid S M D

iso 5 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

14:0 1.290±0.052 1.258±0.048 1.238±0.055 4.051 3.959 3.429 4.378 5.560 6.085

14:1n-5 0.015±0.010 0.003±0.003 0.010±0.010 0.712 0.312 0.177 0.564 0.347 0.262

16:0 20.010±0.713 20.723±0.787 20.950±0.896 4.102 4.337 6.373 7.780 8.859 6.748

16:1n-7 2.338±0.345 2.140±0.303 2.030±0.323 11.712 5.881 4.489 12.226 8.322 5.772

18:0 8.670±0.478 9.928±0.625 10.533±0.723 0.763 1.725 2.237 2.890 3.623 4.621

18:1n-11 0.000 0.000 0.000 5.407 4.107 2.635 0.821 0.468 0.192

18:1n-9 40.168±1.999 39.790±2.011 38.870±2.044 32.949 30.623 28.084 44.237 47.313 45.405

18:1n-7 3.133±0.378 2.948±0.328 2.835±0.303 3.271 3.023 2.723 3.676 3.589 3.121

18:2n-6 19.2125±2.678 18.295±2.501 18.595±2.733 0.814 0.904 0.913 0.393 0.520 0.610

20:1n-11 0.000 0.000 0.000 9.746 11.549 10.686 0.718 0.971 2.755

20:1n-9 0.928±0.045 0.945±0.026 0.940±0.023 9.780 14.523 17.604 3.061 5.010 9.468

20:5n-3 0.000 0.000 0.000 0.153 0.230 0.795 0.000 0.000 0.000

22:1n-11 0.000 0.000 0.000 0.000 9.364 8.611 0.000 0.607 0.453

22:5n-3 0.060±0.014 0.063±0.014 0.063±0.014 0.068 0.181 0.471 0.000 0.503 0.122

22:6n-3 0.000 0.000 0.000 0.186 0.444 1.428 0.000 0.121 0.279

Abundant fatty alcohol

alc 16:0 0.000 0.000 0.000 9.660 10.060 8.330 9.480 11.980 11.060

alc 18:1n-9 0.000 0.000 0.000 13.250 18.220 21.700 22.110 18.670 20.970

Table 6. Mean percent area consisting of microvascularity in a diversity of adipose tissues of

terrestrial mammals.

Species Adipose depot Percent

microvascularity

Source

mouse subcutaneous 0.918% Lijnen et al., 2006

mouse gonadal 0.999% Lijnen et al., 2006

mouse inguinal 2.46% Hemmeryckx et al., 2008

mouse gonadal 2.04% Hemmeryckx et al., 2008

mouse subcutaneous 1.67% Lijnen et al., 2009

human abdominal subcutaneous 1.95-3.48% Pasarica et al., 2009

Figure 1. (a) Sampling site of blubber for all analyses, except those of the pig, Sus scrofa. Image reprinted with permission from

Dr. Sentiel Rommel. (b) Sampling site for adipose tissue of the pig, Sus Scrofa. Image printed with permission from

Jeffrey McClelland. (c) The skin was removed from blubber and adipose tissue samples. These samples were then cut

into thirds and the middle of each third was used for the corresponding depth (superficial, middle, deep). Fat was cut this

way to obtain the different blubber depths for al analyses in this study.

Figure 2. (a) Microvessels in the blubber of Tursiops truncatus (10x). (b) Microvessels in the blubber of Kogia breviceps

blubber (10x). (c) Microvessel in blubber with two terminal branches (d) Microvessel in blubber with three terminal

branches. (e) Microvessel in blubber with five terminal branches. (c,d,e,) Arrows denote terminal branches.

(a) (b)

(d) (c) (e)

Figure 3. Microvasculture in the superficial (S), middle (M), and deep (D) layers of blubber (40x): (a) Tursiops truncatus

(b) Kogia breviceps (c) Sus scrofa (d) Mesoplodon densirostris (e) Ziphius cavirostris

(a) (b) (c)

Blubber

Depth

S

M

D

(d) (e)

Blubber

Depth

S

M

D

Figure 4. Percent area of microvessels analyzed by blubber depth (Tursiops truncatus)

0

2

4

6

8

10

12

Superficial Middle Deep

Mic

rov

ascu

latu

re (

% a

rea)

Depth

Tt01

Tt02

Tt03

Tt04

Tt05

Tt06

Figure 5. Percent area consisting of microvessels analyzed by blubber depth (Kogia breviceps)

0

2

4

6

8

10

12

Superficial Middle Deep

Mic

rov

ascu

latu

re (

% a

rea)

Depth

Kb01

Kb02

Kb03

Kb04

Kb05

Kb06

Figure 6. Percent area consisting of microvessels analyzed by blubber depth (Sus scrofa)

0

2

4

6

8

10

12

Superficial Middle Deep

Mic

rov

ascu

latu

re (

% a

rea)

Depth

Ss01

Ss02

Ss03

Ss04

Figure 7. Comparison of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon densirostris and Ziphius

cavirostris), and Sus scrofa percent area of microvessels analyzed by blubber depth (species mean +/- SEM)

0

2

4

6

8

10

12

Superficial Middle Deep

Mic

rov

ascu

latu

re (

% a

rea)

Depth

Tt

Kb

Ss

Md

Zc

Figure 8. Microvasculature diameter of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon densirostris and

Ziphius cavirostris), and Sus scrofa (species mean +/- SEM)

8

8.5

9

9.5

10

10.5

11

11.5

Superficial Middle Deep

Ves

sel

dia

met

er (

m)

Depth

Tt

Kb

Ss

Md

Zc

Figure 9. Microvascular branching of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon densirostris and

Ziphius cavirostris), and Sus scrofa (species mean +/- SEM)

0

1

2

3

4

5

6

7

Superficial Middle Deep

Ter

min

al b

ran

ches

per

ves

sel

Depth

Tt

Kb

Ss

Md

Zc

Figure 10. Percent lipid (wet weight) of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon densirostris and Ziphius

cavirostris), and Sus scrofa (species mean +/- SEM)

0

10

20

30

40

50

60

70

80

90

100

Superficial Middle Deep

Per

cen

t li

pid

(w

et w

eig

ht)

Depth

Tt

Kb

Ss

Md

Zc

Figure 11. Percent of wax esters in the adipose of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon

densirostris and Ziphius cavirostris), and Sus scrofa (species mean +/- SEM)

0

10

20

30

40

50

60

70

80

90

100

Superficial Middle Deep

Wax

est

ers

(% o

f li

pid

)

Depth

Tt

Kb

Ss

Md

Zc

Figure 12. Fatty acid Stratification Index of Tursiops truncatus (Tt), Kogia breviceps (Kb), Sus scrofa (Ss),

Mesoplodon densirostris (Md), and Ziphius cavirostris (Zc) (avg +/- SEM)

0

5

10

15

20

25

30

35

40

45

Tt Kb Ss Md Zc

Str

atif

icat

ion

In

dex

Species