MICROVASCULAR PATTERNS OF BLUBBER IN SHALLOW AND DEEP DIVINGODONTOCETES

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 1.75Âą0.22%, middle 1.87Âą0.22%, deep 1.88Âą0.46%). 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 cellâ&#x20AC;&#x2122;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 blubberâ&#x20AC;&#x2122;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 shortduration (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 (Blainvilleâ&#x20AC;&#x2122;s beaked whale, Mesoplodon densirostris and Cuvierâ&#x20AC;&#x2122;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 56-60째C 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 -27째C (temperature determined through preliminary trials with blubber samples). The samples were sectioned at 30 m thick and placed on a Superfrost Gold Plus slide (Fisher Scientific, Pittsburgh, PA). The slides were then stored in the freezer at -20째C in a slide box enclosed in a freezer bag until

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

Samples were rinsed in Sorensenâ&#x20AC;&#x2122;s phosphate-buffered saline (PBS) for 15 minutes (Presnell and Schreibman, 1997), and then incubated in a nitro blue tetrazolium chloride plus 5bromo-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: 65째C for 2 min, hold at 165째C for 0.40 min after ramping at 20째C/min, hold at 215째C for 6.6 min

after ramping at 2째C/min, and hold at 250째C for 5 min after ramping at 5째C/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 Leveneâ&#x20AC;&#x2122;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 â&#x20AC;&#x201C; 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 animalsâ&#x20AC;&#x2122; 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 blubberâ&#x20AC;&#x2122;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 â&#x20AC;&#x153;accessâ&#x20AC;? 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 deepestdiving 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 11Ë&#x161;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

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

Tta

Kbb

Ssb

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

Microvascular diameter

Microvascular branching

* 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

Kbb

Ssc

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

Kbb

Ssa

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

Kbb

Ssa

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.

SI a,b,c

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

Kba

Ssb

Md

Zc

29.568±2.838

21.455±3.572

5.455±0.727

40.072

23.097

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

Fatty acid iso 5 14:0 14:1n-5 16:0 16:1n-7 18:0 18:1n-11 18:1n-9 18:1n-7 18:2n-6 20:1n-11 20:1n-9 20:5n-3 22:1n-11 22:5n-3 22:6n-3

Kb

S

M

D

S

M

D

1.868±0.663 4.645±0.612 2.595±0.350 6.934±0.941 23.508±2.580 1.542±0.191 0.965±0.263 21.137±1.344 3.444±0.196 1.192±0.046 0.670±0.233 1.478±0.634 3.700±1.006 0.618±0.346 2.649±0.235 6.861±1.641

1.193±0.531 5.085±0.506 1.277±0.327 10.752±0.319 18.835±2.048 2.355±0.490 1.135±0.307 19.877±1.239 3.582±0.203 1.152±0.070 0.767±0.289 1.713±0.709 4.042±0.806 0.640±0.448 3.405±0.166 8.938±1.865

0.618±0.254 5.613±0.801 0.808±0.289 13.147±0.401 15.967±1.896 2.947±0.269 0.917±0.271 19.508±1.017 3.592±0.213 1.157±0.085 0.848±0.295 1.977±0.710 3.625±0.759 1.144±0.553 3.515±0.215 9.810±1.911

0.000 5.045±0.228 1.186±0.082 11.147±1.186 15.861±0.457 1.427±0.129 0.525±0.382 38.368±1.645 1.834±0.222 0.659±0.083 1.568±0.728 4.578±0.735 0.385±0.120 1.990±1.281 0.153±0.055 0.437±0.115

0.000 5.317±0.220 0.894±0.065 11.027±0.871 12.758±0.797 1.399±0.317 0.472±0.298 37.660±1.908 1.755±0.236 0.786±0.097 2.112±0.991 6.470±1.300 0.637±0.225 3.245±1.941 0.246±0.072 0.932±0.487

0.000 5.621±0.623 0.642±0.097 11.191±0.842 9.713±1.157 1.884±0.228 0.343±0.193 37.239±2.520 1.553±0.275 0.612±0.148 2.452±0.939 8.171±1.567 0.674±0.249 4.146±2.466 0.313±0.064 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 iso 5 14:0 14:1n-5 16:0 16:1n-7 18:0 18:1n-11 18:1n-9 18:1n-7 18:2n-6 20:1n-11 20:1n-9 20:5n-3 22:1n-11 22:5n-3 22:6n-3

S

M

D

0.000 1.290±0.052 0.015±0.010 20.010±0.713 2.338±0.345 8.670±0.478 0.000 40.168±1.999 3.133±0.378 19.2125±2.678 0.000 0.928±0.045 0.000 0.000 0.060±0.014 0.000

0.000 1.258±0.048 0.003±0.003 20.723±0.787 2.140±0.303 9.928±0.625 0.000 39.790±2.011 2.948±0.328 18.295±2.501 0.000 0.945±0.026 0.000 0.000 0.063±0.014 0.000

0.000 1.238±0.055 0.010±0.010 20.950±0.896 2.030±0.323 10.533±0.723 0.000 38.870±2.044 2.835±0.303 18.595±2.733 0.000 0.940±0.023 0.000 0.000 0.063±0.014 0.000

0.000 4.051 0.712 4.102 11.712 0.763 5.407 32.949 3.271 0.814 9.746 9.780 0.153 0.000 0.068 0.186

0.000 3.959 0.312 4.337 5.881 1.725 4.107 30.623 3.023 0.904 11.549 14.523 0.230 9.364 0.181 0.444

0.000 3.429 0.177 6.373 4.489 2.237 2.635 28.084 2.723 0.913 10.686 17.604 0.795 8.611 0.471 1.428

0.000 4.378 0.564 7.780 12.226 2.890 0.821 44.237 3.676 0.393 0.718 3.061 0.000 0.000 0.000 0.000

0.000 5.560 0.347 8.859 8.322 3.623 0.468 47.313 3.589 0.520 0.971 5.010 0.000 0.607 0.503 0.121

0.000 6.085 0.262 6.748 5.772 4.621 0.192 45.405 3.121 0.610 2.755 9.468 0.000 0.453 0.122 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 subcutaneous

Percent microvascularity 0.918%

mouse

Source 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.

(b)

(a)

(c) Figure 2.

(d)

(e)

(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.

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

Blubber Depth S

M

D

(a)

(b)

(c)

Blubber Depth S

M

D

(d)

(e)

12

Microvasculature (% area)

10

8 Tt01 Tt02

6

Tt03 Tt04 Tt05

4

Tt06 2

0 Superficial

Middle Depth

Figure 4.

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

Deep

12

Microvasculature (% area)

10

8 Kb01 Kb02

6

Kb03 Kb04 Kb05

4

Kb06 2

0 Superficial

Middle

Deep

Depth

Figure 5.

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

12

Microvasculature (% area)

10

8

Ss01

6

Ss02 Ss03 Ss04

4

2

0 Superficial

Middle Depth

Figure 6.

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

Deep

12

Microvasculature (% area)

10

8 Tt 6

Kb Ss Md

4

Zc

2

0 Superficial

Middle

Deep

Depth

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)

11.5

11

Vessel diameter ( m)

10.5

10

Tt Kb

9.5

Ss Md Zc

9

8.5

8 Superficial

Middle

Deep

Depth

Figure 8.

Microvasculature diameter of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon densirostris and Ziphius cavirostris), and Sus scrofa (species mean +/- SEM)

7

Terminal branches per vessel

6

5

4

Tt Kb

3

Ss Md Zc

2

1

0 Superficial

Middle

Deep

Depth

Figure 9.

Microvascular branching of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon densirostris and Ziphius cavirostris), and Sus scrofa (species mean +/- SEM)

100 90

Percent lipid (wet weight)

80 70 60 Tt 50

Kb Ss

40

Md Zc

30 20 10 0 Superficial

Middle

Deep

Depth

Figure 10.

Percent lipid (wet weight) of Tursiops truncatus, Kogia breviceps, beaked whales (Mesoplodon densirostris and Ziphius cavirostris), and Sus scrofa (species mean +/- SEM)

100 90 80

Wax esters (% of lipid)

70 60 Tt 50

Kb Ss

40

Md Zc

30 20 10 0 Superficial

Middle

Deep

Depth

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)

45 40

Stratification Index

35 30 25 20 15 10 5 0 Tt

Kb

Ss

Md

Species

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)

Zc